New classes of products are being tested for use in humans and animals, all sharing genes as common targets. Products based on antisense technology directed toward neutralizing messenger RNA are probably being pursued most vigorously; gene therapy through permanent alteration of chromosomes might hold the greatest potential for treatment of diseases like cancer and for correction of genetic disease. The products depend either on classes of compounds that are related to nucleic acids (oligonucleotides and oligonucleotide analogues), on cells that have been genetically altered, of on viruses that bear appropriate nucleic acids.
For the large-scale production of nucleotides and nucleotide analogues, new molecular techniques must be developed. There are now no procedures for making substantial quantities of these types of materials in high purity and with appropriate chirality. Basic chemical and biochemical techniques must be developed for their preparation; new techniques (probably based on high-pressure chromatography) will be required for large-scale purification, and biological methods might be required for preparation of precursors and perhaps for formation of bonds.
For genetically modified cells and viruses, the usual techniques for mammalian-cell culture and molecular biology will be required, as will additional measures for safety and for economical, patient-specific production.
Microencapsulation for Cell Delivery
Microencapsulation is currently the most widely used form of cell delivery with preparation methods including:
1. Gelation and polyelectrolyte complexation,
2. Interfacial polymerization/phase inversion and
3. Conformal coating.
Microencapsulation involves surrounding a collection of cells with a thin generally micrometer sized, semipermeable membrane. Its primary purpose is to protect the encapsulated cells from the host’s immune system, while allowing the exchange of small molecules and thereby ensuring cell survival and function. There are several requirements for polymer capsules or hydrogels used as components of microcapsules:
# Noncytotoxicity to the encapsulated cells
# Biocompatibility with the surrounding environment where capsules are to be implanted (e.g., minimal fibrotic response)
# Adequate permeability for diffusion of essential nutrients (e.g., oxygen and glucose for islets of Langerhans) and cell secretory products (such as insulin, metabolic waste)
# Impermeability to secreted antibodies of the host’s immune system (e.g., immunoglobulins and glycoproteins after complement activation
# Chemical and mechanical stability
From the technological point of view, the requirements for microencapsulation include:
# Small capsule diameters to ensure sufficient diffusion and internal organ transplantability (depending on application, <>
# Minimum shrinking/swelling due to changes in osmotic conditions upon transplantation
# Uniform wall thickness for optimum transport of molecules across the membrane and effective immunoprotection.
In addition, the technology used for encapsulation must be nontraumatic to the encapsulated cells. This includes minimizing the mechanical stress during encapsulation and solvent toxicity (if any), as well as optimizing temperature, viscosity, pH and ionic strength. This, in turn, limits the concentration and molecular mass which can be employed. In addition, the ionic content of the polymer backbone (density distribution of charges in the polymer chain), the chemistry and location of functional group attachment, the chain rigidity, aromaticity, conformation and extent of branching were identified as important variables in the type of complex produced. The presence of secondary hydrogen bonding interactions was also found to be significant.
Several problems may prevent wide scale application of microcapsules in the clinic. The capsules can clump together, in which case the cells towards the center may suffer severely from limited diffusion of oxygen and nutrients. A substantial fraction of the capsules may also adhere to tissue. If the capsules degrade, the liberated islet cells, even if nonviable, would greatly increase the antigenic burden on the patient. Semipermeable polymeric membranes have been developed with the aim of permitting the transplantation of xenogenic cells thus removing the need for immunosuppression therapy. However, early clinical implementations is not likely to involve xenografts or genetically modified cells but rather auto- and allografts supplemented by immunosuppression when necessary.
1. Gelation and polyelectrolyte complexation,
2. Interfacial polymerization/phase inversion and
3. Conformal coating.
Microencapsulation involves surrounding a collection of cells with a thin generally micrometer sized, semipermeable membrane. Its primary purpose is to protect the encapsulated cells from the host’s immune system, while allowing the exchange of small molecules and thereby ensuring cell survival and function. There are several requirements for polymer capsules or hydrogels used as components of microcapsules:
# Noncytotoxicity to the encapsulated cells
# Biocompatibility with the surrounding environment where capsules are to be implanted (e.g., minimal fibrotic response)
# Adequate permeability for diffusion of essential nutrients (e.g., oxygen and glucose for islets of Langerhans) and cell secretory products (such as insulin, metabolic waste)
# Impermeability to secreted antibodies of the host’s immune system (e.g., immunoglobulins and glycoproteins after complement activation
# Chemical and mechanical stability
From the technological point of view, the requirements for microencapsulation include:
# Small capsule diameters to ensure sufficient diffusion and internal organ transplantability (depending on application, <>
# Minimum shrinking/swelling due to changes in osmotic conditions upon transplantation
# Uniform wall thickness for optimum transport of molecules across the membrane and effective immunoprotection.
In addition, the technology used for encapsulation must be nontraumatic to the encapsulated cells. This includes minimizing the mechanical stress during encapsulation and solvent toxicity (if any), as well as optimizing temperature, viscosity, pH and ionic strength. This, in turn, limits the concentration and molecular mass which can be employed. In addition, the ionic content of the polymer backbone (density distribution of charges in the polymer chain), the chemistry and location of functional group attachment, the chain rigidity, aromaticity, conformation and extent of branching were identified as important variables in the type of complex produced. The presence of secondary hydrogen bonding interactions was also found to be significant.
Several problems may prevent wide scale application of microcapsules in the clinic. The capsules can clump together, in which case the cells towards the center may suffer severely from limited diffusion of oxygen and nutrients. A substantial fraction of the capsules may also adhere to tissue. If the capsules degrade, the liberated islet cells, even if nonviable, would greatly increase the antigenic burden on the patient. Semipermeable polymeric membranes have been developed with the aim of permitting the transplantation of xenogenic cells thus removing the need for immunosuppression therapy. However, early clinical implementations is not likely to involve xenografts or genetically modified cells but rather auto- and allografts supplemented by immunosuppression when necessary.
Role of Bio-Process engineering in agriculture and food
Bioprocess engineering in agriculture and the food industry involves the application of biocatalysts (living cells or their components) to produce useful and value-added products, and it offers opportunities to design and produce new or improved agricultural and food products and their manufacturing processes. This will likely have a great impact on the food-processing industry. In the increasingly health-conscious society, genetically engineered microorganisms and specialty enzymes will find increased use in improving the nutritional, flavoring, and storage characteristics and safety of food products. Products under development range from genetically improved strains of freeze-resistant yeast used in frozen bakery products to phage-resistant dairy (yogurt) starter cultures. Chymosin, a product of recombinant E. coli, is already used in the milk-clotting step of cheese manufacture, and a recombinant maltogenic amylase is being used as an antistaling agent. Enzyme-based immunoassays could develop into a widely used method for detecting pesticides in foods at parts-per-billion concentrations. Challenges that must be addressed include the economics of production and regulatory issues.
The most important applications of bioprocess-engineering research and development related to agriculture and food involve production of agricultural chemicals for control of animal and plant diseases, growth-stimulating agents for improved yield, and biological insecticides and herbicides; increasing bioprocess efficiencies for fermented foods, natural food additives, food enzymes as processing aids, and separation and purification of the products; use of plant-cell culture systems to produce secondary metabolites or chemical substances of economic importance; and efficient use of renewable biomass resources for production of liquid fuel and chemical feedstocks and efficient treatment and management of agricultural wastes and wastes from food-processing industries.
The most important applications of bioprocess-engineering research and development related to agriculture and food involve production of agricultural chemicals for control of animal and plant diseases, growth-stimulating agents for improved yield, and biological insecticides and herbicides; increasing bioprocess efficiencies for fermented foods, natural food additives, food enzymes as processing aids, and separation and purification of the products; use of plant-cell culture systems to produce secondary metabolites or chemical substances of economic importance; and efficient use of renewable biomass resources for production of liquid fuel and chemical feedstocks and efficient treatment and management of agricultural wastes and wastes from food-processing industries.
Adhesion based Immobilization Techniques
Each immobilization method has specific properties and advantages. Therefore, the selection of a cell delivery technique depends heavily on the intended application.
Adhesion : Adhesion to a three-dimensional structure is used to immobilize cells for culture or analytical procedures as well as to provide a structural template directing cell growth and differentiation. Adhesion alone does not offer immunoisolation. For in vivo investigations, adhesion-based immobilization must be used in conjunction with either a polymeric membrane or matrix entrapment methods. This method is effective for surface binding, either on top of gel films or within hydrogel foams. Several hydrogels can be engineered with bioadhesive properties by methods which include interfacial polymerization, phase separation, interfacial precipitation and polyelectrolyte complexation. Factors affecting cell affinity and behavior on hydrogels include the general chemistry of the monomers and the crosslinks, hydrophilic and hydrophobic properties, and the surface charge and functionality. One method to enhance cell adhesion is by adding immobilized cell-adhesive proteins or oligopeptides, such as the arginine-glycine-aspartic acid sequence, in the hydrogel. The physical characteristics of the hydrogel also govern the adhesion affinity. Therefore, altering the pore size and network structure can modify cell adhesion as well as morphology and function. For some adhesion applications the mechanical strength is also important with a lower fractional porositygenerally creating stronger networks. Furthermore, closed pore systems make stronger hydrogels than open pore ones. With the adhesion approach, cells are generally plated onto the hydrogel and allowed to attach and migrate. Supplemented culture media provide the cells with essential nutrients for growth and development as well as a means of oxygen and metaboli product transport while in vitro.
Macroporous hydrogel membranes are manufactured by several techniques. One method of constructing pores large enough for cell growth is by phase separation in the polymer and solvent mixture. The “freeze thaw” and the porosigen techniques are two other approaches. The hydrogel is polymerized around a crystalline matrix made from freezing the aqueous solvent (freeze-thaw technique) or around a porosigen of desired size (porosigen technique). With the “freeze-thaw” method, the ice-based crystalline matrix is then thawed after UV polymerization, leaving a macroporous foam. The porosigen technique also requires removal of the crystalline porosigens, in this case usually by leaching or dispersion after polymerizing of the hydrogel with free-radical initiators has taken place. Another method for constructing hydrogel foams uses gas bubbles from sodium bicarbonate to create the macroporous network. Bubbles are trapped during the gelation stage. Thus, the foam morphology is dependent on the polymerization kineics and varies for different hydrogel compositions.
Adhesion : Adhesion to a three-dimensional structure is used to immobilize cells for culture or analytical procedures as well as to provide a structural template directing cell growth and differentiation. Adhesion alone does not offer immunoisolation. For in vivo investigations, adhesion-based immobilization must be used in conjunction with either a polymeric membrane or matrix entrapment methods. This method is effective for surface binding, either on top of gel films or within hydrogel foams. Several hydrogels can be engineered with bioadhesive properties by methods which include interfacial polymerization, phase separation, interfacial precipitation and polyelectrolyte complexation. Factors affecting cell affinity and behavior on hydrogels include the general chemistry of the monomers and the crosslinks, hydrophilic and hydrophobic properties, and the surface charge and functionality. One method to enhance cell adhesion is by adding immobilized cell-adhesive proteins or oligopeptides, such as the arginine-glycine-aspartic acid sequence, in the hydrogel. The physical characteristics of the hydrogel also govern the adhesion affinity. Therefore, altering the pore size and network structure can modify cell adhesion as well as morphology and function. For some adhesion applications the mechanical strength is also important with a lower fractional porositygenerally creating stronger networks. Furthermore, closed pore systems make stronger hydrogels than open pore ones. With the adhesion approach, cells are generally plated onto the hydrogel and allowed to attach and migrate. Supplemented culture media provide the cells with essential nutrients for growth and development as well as a means of oxygen and metaboli product transport while in vitro.
Macroporous hydrogel membranes are manufactured by several techniques. One method of constructing pores large enough for cell growth is by phase separation in the polymer and solvent mixture. The “freeze thaw” and the porosigen techniques are two other approaches. The hydrogel is polymerized around a crystalline matrix made from freezing the aqueous solvent (freeze-thaw technique) or around a porosigen of desired size (porosigen technique). With the “freeze-thaw” method, the ice-based crystalline matrix is then thawed after UV polymerization, leaving a macroporous foam. The porosigen technique also requires removal of the crystalline porosigens, in this case usually by leaching or dispersion after polymerizing of the hydrogel with free-radical initiators has taken place. Another method for constructing hydrogel foams uses gas bubbles from sodium bicarbonate to create the macroporous network. Bubbles are trapped during the gelation stage. Thus, the foam morphology is dependent on the polymerization kineics and varies for different hydrogel compositions.
Future of Plant-cell Tissue Culture
The commercial potential of plant-cell tissue culture has not yet been fully recognized and is underexploited. Plant-cell tissue culture has two primary products: plant tissue for efficient micropropagation of plants and the use of plant-tissue culture to produce specialty chemicals.
Plant-cell, tissue, and organ cultures can be used in processes analogous to traditional fermentation processes for producing chemicals. Although less than 5% of the world's plants have even been identified taxonomically, from among the known plants over 20,000 chemicals are produced - about 4 times as many as from all microorganisms. Very few of the chemicals in pure or semipure form have been tested for their pharmacological activity for other uses. The enzymatic systems in plants can be used to generate completely new compounds when supplied with analogues of natural substrates; thus, plants contain an underused biochemical diversity. Even the limited use of this vast biochemical potential has had important impacts on mankind; in western countries, about one-fourth of all medicines are derived from compounds extracted from plants. Other plant products are used as flavors, fragrances, or pesticides.
Plant-cell tissue culture to produce chemicals commercially has been exploited in Japan, although regulatory approval for medicinal uses has proved difficult and commercial production is restricted to food uses and pigment production. In Japan, a government-sponsored consortium of universities and corporations was recently developed to establish a foundation for plant-cell culture exploitation (i.e., a precompetitive research thrust). In the US plant-cell tissue is not being exploited for chemical production, although some companies are developing processes for the production of the chemotherapeutic agent taxol.
The major technical barriers to the commercial exploitation of plant-cell tissue culture are low growth rates and relatively low product yields. To mitigate those problems, research is needed in subjects as diverse as bioreactor strategies to maintain high-density cultures and enable large-scale production of chemicals through organ cultures and a mechanistic understanding of the role of elicitors in activating pathways for secondary metabolites that could lead to higher productivities of compounds with therapeutic value.
Plant-cell, tissue, and organ cultures can be used in processes analogous to traditional fermentation processes for producing chemicals. Although less than 5% of the world's plants have even been identified taxonomically, from among the known plants over 20,000 chemicals are produced - about 4 times as many as from all microorganisms. Very few of the chemicals in pure or semipure form have been tested for their pharmacological activity for other uses. The enzymatic systems in plants can be used to generate completely new compounds when supplied with analogues of natural substrates; thus, plants contain an underused biochemical diversity. Even the limited use of this vast biochemical potential has had important impacts on mankind; in western countries, about one-fourth of all medicines are derived from compounds extracted from plants. Other plant products are used as flavors, fragrances, or pesticides.
Plant-cell tissue culture to produce chemicals commercially has been exploited in Japan, although regulatory approval for medicinal uses has proved difficult and commercial production is restricted to food uses and pigment production. In Japan, a government-sponsored consortium of universities and corporations was recently developed to establish a foundation for plant-cell culture exploitation (i.e., a precompetitive research thrust). In the US plant-cell tissue is not being exploited for chemical production, although some companies are developing processes for the production of the chemotherapeutic agent taxol.
The major technical barriers to the commercial exploitation of plant-cell tissue culture are low growth rates and relatively low product yields. To mitigate those problems, research is needed in subjects as diverse as bioreactor strategies to maintain high-density cultures and enable large-scale production of chemicals through organ cultures and a mechanistic understanding of the role of elicitors in activating pathways for secondary metabolites that could lead to higher productivities of compounds with therapeutic value.
Understanding of Immunoisolation
A variety of systems can be employed for cell or enzyme immobilization. These include, for example, microcarriers, gel entrapment, hollow fibers, encapsulation and conformal coatings. The latter three have been extensively tested in small animal models over the last 20 years, particularly in the area of diabetes therapy. The polymeric materials used in bioartificial endocrine devices (the terms bioartificial and endocrine device are often distinguished from ‘artificial organs’ due to the presence of tissue in the former two) serve two major purposes:
1. As a scaffold and an extracellular matrix they favor the attachment and differentiation of functional cells or cell clusters and keep them separate from one another;
2. As permselective envelopes which provide immunoisolation of the transplant from the host.
The central concept of immunoisolation is the placement of a semipermeable barrier between the host and the transplanted tissue. The properties required for the semipermeable membranes used in cell transplantation depend strongly on the source of cells. An allograft is a transplant between individuals within one species, while a xenograft is a graft between individuals from different species.
Immunoisolation of transplanted cells by artificial barriers that permit crossover of low molecular weight substances, such as nutrients, electrolytes, oxygen, and bioactive secretory products, though not of immune cells and high molecular weight proteins such as antibodies (IgG, IgM), provides great promise for developing new technologies to overcome these problems in a reasonable time frame.
Device Geometry Considerations
The immunoisolation of allogeneic or xenogeneic islets can be achieved via two main classes of technology: macroencapsulation and microencapsulation. Macroencapsulation refers to the reliance on larger, prefabricated “envelopes” in which a slurry of islets or cell clusters is slowly introduced and sealed prior to implantation. An intravascular device usually consists of a tube through which blood flows, on the outside of which is the implanted tissue contained within a housing. The device is then implanted as a shunt in the cardiovascular system. Extravascular devices are implanted directly into tissue in a body space such as the peritoneal cavity, though some have also been vascularized into a major artery such as in Calafiore’s clinical trial. Geometrical alternatives include cylindrical tubular membranes containing tissue within the lumen and planar diffusion chambers comprised of parallel flat sheet membranes between which the implanted tissue is placed.
Microencapsulation refers to the formation of a spherical gel around each group of islets, cell cluster or tissue fragment. Microcapsules based on natural or synthetic polymers have been used for the encapsulation of both mammalian and microbial cells as well as various bioactive substances such as enzymes, proteins and drugs. A review of alternative semipermeable microcapsules prepared from oppositely charged water soluble polyelectrolyte pairs has been presented in recent papers. The main advantage of this approach is that cells, or bioactive agents, are isolated from the body by a microporous semipermeable membrane and the encapsulated material is thus protected against the attack of the immune system. In the case of microencapsulated pancreas islets, a suspension of microcapsules is typically introduced in the peritoneal cavity to deliver insulin to the portal circulation.
1. As a scaffold and an extracellular matrix they favor the attachment and differentiation of functional cells or cell clusters and keep them separate from one another;
2. As permselective envelopes which provide immunoisolation of the transplant from the host.
The central concept of immunoisolation is the placement of a semipermeable barrier between the host and the transplanted tissue. The properties required for the semipermeable membranes used in cell transplantation depend strongly on the source of cells. An allograft is a transplant between individuals within one species, while a xenograft is a graft between individuals from different species.
Immunoisolation of transplanted cells by artificial barriers that permit crossover of low molecular weight substances, such as nutrients, electrolytes, oxygen, and bioactive secretory products, though not of immune cells and high molecular weight proteins such as antibodies (IgG, IgM), provides great promise for developing new technologies to overcome these problems in a reasonable time frame.
Device Geometry Considerations
The immunoisolation of allogeneic or xenogeneic islets can be achieved via two main classes of technology: macroencapsulation and microencapsulation. Macroencapsulation refers to the reliance on larger, prefabricated “envelopes” in which a slurry of islets or cell clusters is slowly introduced and sealed prior to implantation. An intravascular device usually consists of a tube through which blood flows, on the outside of which is the implanted tissue contained within a housing. The device is then implanted as a shunt in the cardiovascular system. Extravascular devices are implanted directly into tissue in a body space such as the peritoneal cavity, though some have also been vascularized into a major artery such as in Calafiore’s clinical trial. Geometrical alternatives include cylindrical tubular membranes containing tissue within the lumen and planar diffusion chambers comprised of parallel flat sheet membranes between which the implanted tissue is placed.
Microencapsulation refers to the formation of a spherical gel around each group of islets, cell cluster or tissue fragment. Microcapsules based on natural or synthetic polymers have been used for the encapsulation of both mammalian and microbial cells as well as various bioactive substances such as enzymes, proteins and drugs. A review of alternative semipermeable microcapsules prepared from oppositely charged water soluble polyelectrolyte pairs has been presented in recent papers. The main advantage of this approach is that cells, or bioactive agents, are isolated from the body by a microporous semipermeable membrane and the encapsulated material is thus protected against the attack of the immune system. In the case of microencapsulated pancreas islets, a suspension of microcapsules is typically introduced in the peritoneal cavity to deliver insulin to the portal circulation.
Understanding the Bio-remediation Process
Bioremediation refers to the use of entire organisms (mostly soil microorganisms) or selected constituents of microbial cells (mostly enzymes) for chemical transformations. Bioremediation transforms a toxic substance into a harmless or less toxic substance. Ideally, the toxic substance is transformed into carbon dioxide and water. If the toxic substance contains a metal or a halogen, such as chlorine or fluorine, there will be additional side-products (perhaps the free metal atom or its ion or a halide ion). Mineralization is the term used to describe the complete degradation of a chemical substance to water and carbon dioxide. Bioaugmentation, another frequently used term, involves the deliberate addition of microorganisms that have been cultured, adapted, and enhanced for specific contaminants and conditions at the site.
Microorganisms used in bioremediation include aerobic (which use free oxygen) and anaerobic (which live only in the absence of free oxygen). Aerobic microbes have been the organisms of choice for degrading hazardous wastes.
Bioremediation is practiced in two modes - in situ and ex situ. In situ bioremediation involves the use of microorganisms to degrade wastes at the site (both on and below the surface) and avoid excavation of contaminated soil and transfer to different locations. Surface remediation is used to treat the top parts of the soil through aeration by the addition of microorganisms, nutrients, and water. Subsurface bioremediation uses microorganisms already in the soil and groundwater and adds oxygen and nutrients. Ex situ treatment involves the excavation of contaminated soil and its transfer to appropriate treatment sites, i.e., bioreactors. The contaminated soil is aerated and treated with nutrients to provide an active environment for the microorganisms of choice. Treatment continues until the soil is sufficiently clean and can be returned to the site. Ex situ techniques are varied but can involve slurry-phase treatments that combine contaminated soil or sludge in bioreactors or solid-phase treatments that involve placing contaminated soils in lined treatment beds. Bioremediation of water or leachate includes treatment with special bioreactors or filters that contain an active film of microorganisms. The choice of method involves many factors, including the contaminant, the site, and the costs that can be borne. Ex situ treatment is usually very expensive. Most often, the microorganisms are expected to reproduce in situ.
Microorganisms used in bioremediation include aerobic (which use free oxygen) and anaerobic (which live only in the absence of free oxygen). Aerobic microbes have been the organisms of choice for degrading hazardous wastes.
Bioremediation is practiced in two modes - in situ and ex situ. In situ bioremediation involves the use of microorganisms to degrade wastes at the site (both on and below the surface) and avoid excavation of contaminated soil and transfer to different locations. Surface remediation is used to treat the top parts of the soil through aeration by the addition of microorganisms, nutrients, and water. Subsurface bioremediation uses microorganisms already in the soil and groundwater and adds oxygen and nutrients. Ex situ treatment involves the excavation of contaminated soil and its transfer to appropriate treatment sites, i.e., bioreactors. The contaminated soil is aerated and treated with nutrients to provide an active environment for the microorganisms of choice. Treatment continues until the soil is sufficiently clean and can be returned to the site. Ex situ techniques are varied but can involve slurry-phase treatments that combine contaminated soil or sludge in bioreactors or solid-phase treatments that involve placing contaminated soils in lined treatment beds. Bioremediation of water or leachate includes treatment with special bioreactors or filters that contain an active film of microorganisms. The choice of method involves many factors, including the contaminant, the site, and the costs that can be borne. Ex situ treatment is usually very expensive. Most often, the microorganisms are expected to reproduce in situ.
Futuristic use of Transgenic Animals & Transgenic Plants
Transgenic Animals:Transgenic animals are being developed for a wide variety of applications. In the near future, transgenic animals will be used increasingly in safety evaluation of new pharmaceuticals and accelerating their regulatory approval. The feasibility of producing human pharmaceutical proteins in the milk of transgenic livestock has been established.
As an alternative to cell-culture systems, such livestock appear to be appealing because of high volumetric productivity, low operating costs, capability of posttranslation modification of proteins, and potential for expansion of the producing organism. Bioprocess engineers face numerous technical challenges in converting a transgenic mammary gland system into a commercial prototype for large-scale manufacture of high-market-volume proteins, including the following:
1) Purification techniques for obtaining high-purity proteins that must be recovered and fractionated from a complex mixture of fats, proteins, sugars, and ions, some of which are in colloidal form.
2) Optimization of product stability during recovery.
3) Instrumentation to characterize posttranslation modifications made by the mammary gland "bioreactor."
4) Development of on-line sensors to monitor changes in bioactivity of products during purification.
5) Bioseparations of milk proteins.
In the longer term, transgenic animals might provide a source of tissues and organs for use in transplantation patients. Bioprocess engineering will be needed to design novel equipment to maintain, purify, and store the living tissues without affecting viability or graft response.
The hurdles to be surmounted in developing the necessary genetic tools for systematic pathway engineering are substantial, but basic research at the molecular level will continue to provide improved production strains and novel products, and continued interest in the fundamentals of bioprocessing of milk will help to define separation strategies for this complex biological fluid.
Transgenic Plants:Transgenic plants are capable of generating specialty chemicals or other bioproducts. Special bioprocessing capabilities will then also need to be developed for extracting, concentrating, and purifying such products from plant tissue. This sector of bioprocess engineering might also be important to the prospects of expanding crops or developing new varieties that are rich in fermentable carbohydrates, which are readily used as feedstocks for large-scale manufacturing of specialty and industrial chemicals.
Transgenic tobacco plants have been developed to produce monoclonal antibodies identical in function with the original mouse antibody. Other proteins produced in plants are human serum albumin and enkephalins. Processes to recover and purify proteins from plant-cell extracts will be needed if such systems are commercialized.
As an alternative to cell-culture systems, such livestock appear to be appealing because of high volumetric productivity, low operating costs, capability of posttranslation modification of proteins, and potential for expansion of the producing organism. Bioprocess engineers face numerous technical challenges in converting a transgenic mammary gland system into a commercial prototype for large-scale manufacture of high-market-volume proteins, including the following:
1) Purification techniques for obtaining high-purity proteins that must be recovered and fractionated from a complex mixture of fats, proteins, sugars, and ions, some of which are in colloidal form.
2) Optimization of product stability during recovery.
3) Instrumentation to characterize posttranslation modifications made by the mammary gland "bioreactor."
4) Development of on-line sensors to monitor changes in bioactivity of products during purification.
5) Bioseparations of milk proteins.
In the longer term, transgenic animals might provide a source of tissues and organs for use in transplantation patients. Bioprocess engineering will be needed to design novel equipment to maintain, purify, and store the living tissues without affecting viability or graft response.
The hurdles to be surmounted in developing the necessary genetic tools for systematic pathway engineering are substantial, but basic research at the molecular level will continue to provide improved production strains and novel products, and continued interest in the fundamentals of bioprocessing of milk will help to define separation strategies for this complex biological fluid.
Transgenic Plants:Transgenic plants are capable of generating specialty chemicals or other bioproducts. Special bioprocessing capabilities will then also need to be developed for extracting, concentrating, and purifying such products from plant tissue. This sector of bioprocess engineering might also be important to the prospects of expanding crops or developing new varieties that are rich in fermentable carbohydrates, which are readily used as feedstocks for large-scale manufacturing of specialty and industrial chemicals.
Transgenic tobacco plants have been developed to produce monoclonal antibodies identical in function with the original mouse antibody. Other proteins produced in plants are human serum albumin and enkephalins. Processes to recover and purify proteins from plant-cell extracts will be needed if such systems are commercialized.
Understand the Basics of Metabolic Engineering
A powerful new approach to product development is the creative application of fermentation technology and molecular biology for "metabolic engineering." Examples of metabolic engineering for heterologous-protein production include deletion of proteases that eliminate product and production of factors that facilitate product maturation and secretion. For protein production on an industrial scale, metabolic engineering could be useful in shifting metabolic flow toward a desired product, creating arrays of enzymatic activities for synthesis of novel structures, and accelerating rate-limiting steps. Metabolic engineering has recently been used to increase the efficiency of nutrient assimilation (increasing the growth rate), improve the efficiency of ATP production (decreasing nutrient demands), and reduce the production of inhibitory end products (increasing final cell densities).
Central to molecular modification of multigene pathways, such as those involved in antibiotic production, is the development of new vectors and transformation procedures and other tools of molecular biology. Another important discovery in metabolic engineering is the isolation of positive-control genes that regulate production of secondary metabolites. Positive regulators have been found in biosynthetic gene clusters for actinorhodin, bialophos, streptomycin, and undecylprodigiosin, all of which are Streptomyces products.
Genes encoding the converting enzymes D-amino acid oxidase and cephalosporin acylase were cloned from Fusarium and Pseudomonas, respectively, into the fungus Acremonium chrysogenum. Expression of this "artificial" antibiotic biosynthetic pathway was confirmed by analysis of transformants that synthesized and secreted detectable amounts of 7- aminocephalosporanic acid.
In addition to classical mutation, new tools have become available for genetic manipulation of important producers of natural products, such as Streptomyces. The ability to clone and manipulate biosynthetic genes for antibiotic production, regulatory genes for improved synthesis, and genes from primary metabolic pathways that contribute to secondary biosynthetic pathways can facilitate construction of strains that have substantially altered metabolic properties. In addition, the cloning of heterologous genes into bacterial hosts has generated strains that can produce compounds that are foreign and even
Deleterious to cell physiology.
Central to molecular modification of multigene pathways, such as those involved in antibiotic production, is the development of new vectors and transformation procedures and other tools of molecular biology. Another important discovery in metabolic engineering is the isolation of positive-control genes that regulate production of secondary metabolites. Positive regulators have been found in biosynthetic gene clusters for actinorhodin, bialophos, streptomycin, and undecylprodigiosin, all of which are Streptomyces products.
Genes encoding the converting enzymes D-amino acid oxidase and cephalosporin acylase were cloned from Fusarium and Pseudomonas, respectively, into the fungus Acremonium chrysogenum. Expression of this "artificial" antibiotic biosynthetic pathway was confirmed by analysis of transformants that synthesized and secreted detectable amounts of 7- aminocephalosporanic acid.
In addition to classical mutation, new tools have become available for genetic manipulation of important producers of natural products, such as Streptomyces. The ability to clone and manipulate biosynthetic genes for antibiotic production, regulatory genes for improved synthesis, and genes from primary metabolic pathways that contribute to secondary biosynthetic pathways can facilitate construction of strains that have substantially altered metabolic properties. In addition, the cloning of heterologous genes into bacterial hosts has generated strains that can produce compounds that are foreign and even
Deleterious to cell physiology.
Know the Basics of Protein Engineering
Advances in molecular biology have provided researchers with the opportunity to develop increasingly rational approaches to the design of therapeutic drugs. This technology, when used with computer-assisted molecular modeling, is called protein engineering.
Protein engineering combines many techniques, including gene cloning, site-directed mutagenesis, protein expression, structural characterization of the product, and bioactivity analyses; it can be used to modify the primary sequence of a protein at selected sites to improve stability, pharmacokinetics, bioactivity, and serum half-life. A second application of protein engineering is the design of hybrid proteins that contain regions that aid separation and purification. That is achieved by introducing, next to the structural gene for the desired product, a DNA sequence that encodes for a specific polypeptide "tail."
The tails can be inserted at the N or C terminal of the protein to yield a fusion protein with special properties that facilitate separation. Such genetic modifications can be designed to take advantage of affinity, ion-exchange, hydrophobic, metal chelate, and covalent separations. The special properties of fusion proteins allow crude microbial extracts to be passed over an adsorbent that binds specifically to the tail, so that the desired product is retained and contaminants pass through. After elution and treatment to remove the tail, the product is purified further by standard methods, such as size-exclusion chromatography or high-performance liquid chromatography (HPLC).
Protein engineering combines many techniques, including gene cloning, site-directed mutagenesis, protein expression, structural characterization of the product, and bioactivity analyses; it can be used to modify the primary sequence of a protein at selected sites to improve stability, pharmacokinetics, bioactivity, and serum half-life. A second application of protein engineering is the design of hybrid proteins that contain regions that aid separation and purification. That is achieved by introducing, next to the structural gene for the desired product, a DNA sequence that encodes for a specific polypeptide "tail."
The tails can be inserted at the N or C terminal of the protein to yield a fusion protein with special properties that facilitate separation. Such genetic modifications can be designed to take advantage of affinity, ion-exchange, hydrophobic, metal chelate, and covalent separations. The special properties of fusion proteins allow crude microbial extracts to be passed over an adsorbent that binds specifically to the tail, so that the desired product is retained and contaminants pass through. After elution and treatment to remove the tail, the product is purified further by standard methods, such as size-exclusion chromatography or high-performance liquid chromatography (HPLC).
Challenges to Isolation and Purification of Proteins
Isolation generally denotes the separation of the product from the bulk of the producing organism. The disposition and state of the expressed protein affect the isolation procedure. For mammalian cells and some E. coli, Streptomyces, Bacillus, and yeast products, the protein is released from the cell into the surrounding medium, and isolation is effected by a solid-liquid separation step, usually centrifugation or microfiltration or ultrafiltration. If the product has aggregated either in the cytoplasmic or periplasmic space, isolation is more involved. Generally, the cell is first lysed by mechanical, chemical, or enzymatic treatment (or a combination). In some cases, the more dense aggregate can be separated by centrifugation from most of the soluble and insoluble cell components; in other cases, the aggregate is first solubilized while still in the soluble protein mixture.
Purification of the protein is a critical and often expensive part of the process. It might account for 50% or more of the total production cost. Purification has several objectives: to remove contaminating components from the host organism, i.e., other proteins, DNA, and lipids; to separate the desired protein (or family of proteins) from undesired variants of the desired protein; to remove and avoid the introduction of endotoxin; to inactivate viruses; to obtain required yields at acceptable cost; to avoid chemical or biochemical modification of the protein; and to make the process consistent and reliable. In some cases, the first and additional objective is to fold the protein into its desired conformation.
The most common individual operations are centrifugation, filtration, membrane separation, adsorption separation, and chromatography.
The difficulty of separation can often be decreased by changing the organism or culture conditions to produce a more uniform protein. However, it is still necessary to combine a series of purification steps each of which separates according to a different principle. Ultrafiltration steps are often used between separation steps to concentrate the protein solution or to make the buffer solution compatible with the next separation step. The final steps are designed to place the purified protein in the solution used for the product form.
The complexity of the individual purification steps and the need to be able to integrate them into a manufacturing system translate into a major opportunity for bio-processing engineering as the process moves from the bench to the plant. Research and development in purification, scaleup integration, and system design will continue to have high priority.
Purification of the protein is a critical and often expensive part of the process. It might account for 50% or more of the total production cost. Purification has several objectives: to remove contaminating components from the host organism, i.e., other proteins, DNA, and lipids; to separate the desired protein (or family of proteins) from undesired variants of the desired protein; to remove and avoid the introduction of endotoxin; to inactivate viruses; to obtain required yields at acceptable cost; to avoid chemical or biochemical modification of the protein; and to make the process consistent and reliable. In some cases, the first and additional objective is to fold the protein into its desired conformation.
The most common individual operations are centrifugation, filtration, membrane separation, adsorption separation, and chromatography.
The difficulty of separation can often be decreased by changing the organism or culture conditions to produce a more uniform protein. However, it is still necessary to combine a series of purification steps each of which separates according to a different principle. Ultrafiltration steps are often used between separation steps to concentrate the protein solution or to make the buffer solution compatible with the next separation step. The final steps are designed to place the purified protein in the solution used for the product form.
The complexity of the individual purification steps and the need to be able to integrate them into a manufacturing system translate into a major opportunity for bio-processing engineering as the process moves from the bench to the plant. Research and development in purification, scaleup integration, and system design will continue to have high priority.
Understanding of Bioartificial Organs
Tissue engineering involves the in vitro or in vivo generation of organoids such as cartilage, skin or nerves. More ambitious projects seek to ameliorate the quality of life of diseased or injured patients and reduce the economic burden of treatment. Bioartificial organs involve an in vitro prepared tissue-material interface fabricated into a durable device. A typical example is the bioartificial pancreas, which will be discussed in the following section as a case study. The extra-corporeal bioartificial liver and more recently the bioartificial kidney14 are examples of the transient replacement of organ functions, the former intended as a bridge to stabilize comatose patients until a whole organ can be procured. As the bioartificial pancreas is often microcapsule based, a specific section will be dedicated to review encapsulation technology prior to the application of this bioartificial organ for in situ insulin production.
Bioartificial organs require the combination of several research areas. The understanding of cellular differentiation and growth and how extracellular matrix components affect cell function comes under the umbrella of cell biology. Immunology and molecular genetics will also be needed to contribute to the design of cells or cell transplant systems that are not rejected by the immune system. Cell source and cell preservation are other important issues. The transplanted cells may come from cell lines or primary tissues—from the patients themselves, other human donors, animal sources or fetal tissue. In choosing the cell source, a balance must be struck between ethical issues, safety issues and efficacy. The sterilization and depyrogenation of the polymers involved in transplants is also critical. The materials used in tissue engineering and polymer processing are other key issues. The development of controlled release systems, which deliver molecules over long time periods, will be important in administering numerous tissue controlling factors, growth factors and angiogenesis stimulators. Finally, it will be useful to develop methods of surface analysis for studying interfaces between cell and materials and mathematical models and in vitro systems that can predict in vivo cellular events.
Bioartificial organs require the combination of several research areas. The understanding of cellular differentiation and growth and how extracellular matrix components affect cell function comes under the umbrella of cell biology. Immunology and molecular genetics will also be needed to contribute to the design of cells or cell transplant systems that are not rejected by the immune system. Cell source and cell preservation are other important issues. The transplanted cells may come from cell lines or primary tissues—from the patients themselves, other human donors, animal sources or fetal tissue. In choosing the cell source, a balance must be struck between ethical issues, safety issues and efficacy. The sterilization and depyrogenation of the polymers involved in transplants is also critical. The materials used in tissue engineering and polymer processing are other key issues. The development of controlled release systems, which deliver molecules over long time periods, will be important in administering numerous tissue controlling factors, growth factors and angiogenesis stimulators. Finally, it will be useful to develop methods of surface analysis for studying interfaces between cell and materials and mathematical models and in vitro systems that can predict in vivo cellular events.
Methods adopted for Micro-capsule Formation
The most widely used procedure for micro-capsule formation involves the gelation of charged poly-electrolytes around the cell core. The popular alginate-L-polylysine micro-capsules, for example, are obtained in the following sequence:
1) The cells are embedded in alginate droplets with the aid of a droplet generator (air / liquid jet or an electrostatic generator);
2) The droplets are transformed into rigid beads by inducing cross-linking with calcium ions;
3) The beads are coated with polylysine and alginate, thereby forming the semi-permeable capsule; and
4) The alginate core is liquefied with a chelating agent.
Micro-capsules surrounding individual cells or clusters such as islets should be physically durable, smooth and spherical for optimal bio-compatibility. Smoothness is one factor, which, in addition to the interfacial composition, reduces tissue irritation, which decreases the probability of cell overgrowth on the capsule surface if aggregated tissue such as beta-cell clusters (beta cells transform blood glucose concentration stimuli into a regulated, pulsatile, insulin secretion) is employed. The capsules should be as small as possible in relation to the islet size to optimize nutrient ingress and hormone egress.
The poly-electrolyte complexation technique used to make alginate-polylysine capsules is advantageous since the capsules are formed under very mild conditions. A disadvantage, however, is the impurities and batch to batch irreproducibility of the alginate, a naturally derived polysaccharide.The high mannuronic acid content of alginate was shown to be responsible for fibrotic tissue response. Fibrosis was reduced and a more resistant micro-capsule was fabricated by decreasing the mannuronic acid level of the alginate at the expense of the guluronic acid content, although these conclusions have been questioned by some authors. Another disadvantage of alginate-polylysine micro-capsules is that the alginate-polylysine membrane, a weak polyelectrolyte complex, gives the micro-capsules relatively poor mechanical properties.
Local changes in pH or ionic concentration may have influence on the integrity of these microcapsules drastically.
Several different hydrogels have been investigated to determine the efficacy of encapsulation therapy as treatment for multiple diseases in a variety of animal models. For instance, alginate-polylysine-alginate micro-capsules have been employed to encapsulate islets and to reverse the effects of diabetes in rats and mice. The mild encapsulation procedure preserved the integrity of the islet’s secretory function with long term viability maintained. Modified alginate-polylysine micro-capsules, which are smaller and stronger than the previous versions, improved the survival of the xenographic tissue grafts. Coating alginate-polylysine capsules with a poly(ethylene glycol)hydrogel or incorporating monomethoxy poly(ethylene glycol) pendant chains to the polylysine polymer backbone has led to improved biocompatibility compared to unmodified capsules. In an attempt to simultaneously control biocompatibility and permeability, polymer blends have been selected that were optimal with respect to islet cytotoxicity (as measured by in vivo tests or) as well as thermodynamic (swelling / shrinking) and mechanical parameters.
1) The cells are embedded in alginate droplets with the aid of a droplet generator (air / liquid jet or an electrostatic generator);
2) The droplets are transformed into rigid beads by inducing cross-linking with calcium ions;
3) The beads are coated with polylysine and alginate, thereby forming the semi-permeable capsule; and
4) The alginate core is liquefied with a chelating agent.
Micro-capsules surrounding individual cells or clusters such as islets should be physically durable, smooth and spherical for optimal bio-compatibility. Smoothness is one factor, which, in addition to the interfacial composition, reduces tissue irritation, which decreases the probability of cell overgrowth on the capsule surface if aggregated tissue such as beta-cell clusters (beta cells transform blood glucose concentration stimuli into a regulated, pulsatile, insulin secretion) is employed. The capsules should be as small as possible in relation to the islet size to optimize nutrient ingress and hormone egress.
The poly-electrolyte complexation technique used to make alginate-polylysine capsules is advantageous since the capsules are formed under very mild conditions. A disadvantage, however, is the impurities and batch to batch irreproducibility of the alginate, a naturally derived polysaccharide.The high mannuronic acid content of alginate was shown to be responsible for fibrotic tissue response. Fibrosis was reduced and a more resistant micro-capsule was fabricated by decreasing the mannuronic acid level of the alginate at the expense of the guluronic acid content, although these conclusions have been questioned by some authors. Another disadvantage of alginate-polylysine micro-capsules is that the alginate-polylysine membrane, a weak polyelectrolyte complex, gives the micro-capsules relatively poor mechanical properties.
Local changes in pH or ionic concentration may have influence on the integrity of these microcapsules drastically.
Several different hydrogels have been investigated to determine the efficacy of encapsulation therapy as treatment for multiple diseases in a variety of animal models. For instance, alginate-polylysine-alginate micro-capsules have been employed to encapsulate islets and to reverse the effects of diabetes in rats and mice. The mild encapsulation procedure preserved the integrity of the islet’s secretory function with long term viability maintained. Modified alginate-polylysine micro-capsules, which are smaller and stronger than the previous versions, improved the survival of the xenographic tissue grafts. Coating alginate-polylysine capsules with a poly(ethylene glycol)hydrogel or incorporating monomethoxy poly(ethylene glycol) pendant chains to the polylysine polymer backbone has led to improved biocompatibility compared to unmodified capsules. In an attempt to simultaneously control biocompatibility and permeability, polymer blends have been selected that were optimal with respect to islet cytotoxicity (as measured by in vivo tests or) as well as thermodynamic (swelling / shrinking) and mechanical parameters.
What are the techniques of Interfacial Polymerization & Photo Polymerization ?
Interfacial polymerization is a method developed for encapsulation of mammalian cells. Cells are coextruded with a generally hydrophobic polymer solution through a coaxial needle assembly. Shear and mechanical forces due to a coaxial air/liquid stream flowing past the tip of the needle assembly causes the hydrogel to envelop the cells and fall off. The encapsulated cells fall subsequently through a series of oil phases, which cause precipitation of the hydrogel around the cell. This process, based on membrane phase inversion, is used primarily when encapsulating cells with hydrogels from the polyacrylate family. Polyacrylates are well tolerated by the host’s immune system and have exceptional hydrolytic stability. A potential disadvantage of this technique is that organic solvents, which may be harmful to living cells, are used to precipitate the hydrogel. To eliminate the use of organic solvents, complex coacervation was developed using acidic and basic water-soluble polymers. Briefly, a droplet containing one of these polymers and cells is added to the other polymer. A thin membrane encapsulates the droplet due to ionic interactions of the two polymers. The major disadvantage of this method is that the capsules may be unstable due to high water uptake in the capsule wall. Modifications have been made to better control permeability and stability of the hydrogel capsules.
Photopolymerization has also been used to conformally coat hydrogel capsules to:
1) Improve their biocompatibility and
2) Reduce the volume to a minimum in order to reduce implant size, a critical issue if an internal organ is the intended transplantation site.
Photopolymerization permits gelation of the polymer membrane in the presence of dissolved oxygen, which is helpful for cell survival during the encapsulation process. The advantage of this technique is that the membrane is directly in contact with the encapsulated cells. Minimizing diffusion distance for oxygen, nutrients, and cell products is important for eliminating necrosis at the center of the capsule12 and for improving therapeutic efficiency.
Photopolymerization has also been used to conformally coat hydrogel capsules to:
1) Improve their biocompatibility and
2) Reduce the volume to a minimum in order to reduce implant size, a critical issue if an internal organ is the intended transplantation site.
Photopolymerization permits gelation of the polymer membrane in the presence of dissolved oxygen, which is helpful for cell survival during the encapsulation process. The advantage of this technique is that the membrane is directly in contact with the encapsulated cells. Minimizing diffusion distance for oxygen, nutrients, and cell products is important for eliminating necrosis at the center of the capsule12 and for improving therapeutic efficiency.
What is Tissue Sourcing ?
Organs and cells of animal origin are being considered as a source of tissue for xenotransplantation. If islet transplantation is to become a widespread treatment for type 1 diabetics, solutions must be found for increasing the availability of insulin-producing tissue and for overcoming the need for continuous immunosuppression. Insulin-producing cells being considered for clinical transplantation include porcine and bovine islets, fish-Brockman bodies, genetically engineered insulin-secreting cell lines and in vitro produced “human” beta-cells.
Both primary tissue and cultured cell lines have been employed in small animal xenotransplantation, including cells that have been genetically modified. Substantial efforts have also been made in the isolation of primary tissue, especially for pancreatic islets, though further improvements are necessary for practical, large-scale processing. The most urgent problem in transplantation is the shortage of donor organs and tissue.
Xenotransplantation could offer some advantages over the use of human organs. Xenotransplantation could be planned in advance, the organ would be transplanted while it was still fresh and undamaged. In addition, a planned transplantation allows the administration of therapeutic regimens that call for the pretreatment of the recipient. Another advantage is the possibility that animal sources could be genetically engineered in order to lower the risk of rejection by expressing specific genes for the benefit of the patient. However, the concern over retroviruses has led to political moratoriums on the clinical use of xenotransplantation. It has yet to be established in nonrodent models as a viable alternative.
Alternative Tissue Sources
The optimal source of xenogeneic islets remains controversial. Islets have been isolated from primates and xenografted into immunosuppressed, diabetic rodents, with short-term reversal of diabetes. However, there are ethical issues surrounding the use of primates for these studies. Other promising islet sources are porcine, bovine and rabbit islets, all of which function remarkably well in diabetic rodents. Long-term human, bovine and porcine islet xenograft survival has been documented in nude mice and rats, suggesting that, in the absence of an immune response, sufficient islet-specific growth factors are present in xenogeneic recipients.
Porcine islets are at present receiving the greatest attention since pigs produce an insulin which is structurally very similar to human insulin and pigs are, on the other hand, the only large animals slaughtered in sufficient quantities to supply the estimated demand from type 1 diabetics. In addition, porcine islets within microcapsules have been reported to correct diabetes in cynomologus monkeys. Elaborate studies are in progress to engineer a “perfect pig”, having adequate levels of complement-inhibiting factors. Thus, porcine sources are
perhaps most likely to provide islets for an inaugural human xeno-islet trial. However, porcine islets are fragile and have poor long-term stability. The in vitro glucose-stimulated insulin secretion rate per unit islet volume appears to be substantially smaller for porcine islets than for other species including human. Lastly, there is significant current concern regarding the potential for transmission of infectious agents from porcine organ sources to human xenograft recipients, and to the population at large. None of these characteristics bode well for their practical large-scale use, and serious consideration and investigation is being given to alternate animal sources. There is also speculation that neonatal porcine islets, which culture better and present minimal infrastructure problems, would be an ultimate substitute. Isolation of bovine islets is technically easier and calf islets are glucose-responsive. However, adult bovine islets are relatively insensitive to glucose. The rabbit pancreas is also an attractive source of islets since rabbit insulin differs from human insulin at only one amino acid and rabbit islets are glucose responsive.
Both primary tissue and cultured cell lines have been employed in small animal xenotransplantation, including cells that have been genetically modified. Substantial efforts have also been made in the isolation of primary tissue, especially for pancreatic islets, though further improvements are necessary for practical, large-scale processing. The most urgent problem in transplantation is the shortage of donor organs and tissue.
Xenotransplantation could offer some advantages over the use of human organs. Xenotransplantation could be planned in advance, the organ would be transplanted while it was still fresh and undamaged. In addition, a planned transplantation allows the administration of therapeutic regimens that call for the pretreatment of the recipient. Another advantage is the possibility that animal sources could be genetically engineered in order to lower the risk of rejection by expressing specific genes for the benefit of the patient. However, the concern over retroviruses has led to political moratoriums on the clinical use of xenotransplantation. It has yet to be established in nonrodent models as a viable alternative.
Alternative Tissue Sources
The optimal source of xenogeneic islets remains controversial. Islets have been isolated from primates and xenografted into immunosuppressed, diabetic rodents, with short-term reversal of diabetes. However, there are ethical issues surrounding the use of primates for these studies. Other promising islet sources are porcine, bovine and rabbit islets, all of which function remarkably well in diabetic rodents. Long-term human, bovine and porcine islet xenograft survival has been documented in nude mice and rats, suggesting that, in the absence of an immune response, sufficient islet-specific growth factors are present in xenogeneic recipients.
Porcine islets are at present receiving the greatest attention since pigs produce an insulin which is structurally very similar to human insulin and pigs are, on the other hand, the only large animals slaughtered in sufficient quantities to supply the estimated demand from type 1 diabetics. In addition, porcine islets within microcapsules have been reported to correct diabetes in cynomologus monkeys. Elaborate studies are in progress to engineer a “perfect pig”, having adequate levels of complement-inhibiting factors. Thus, porcine sources are
perhaps most likely to provide islets for an inaugural human xeno-islet trial. However, porcine islets are fragile and have poor long-term stability. The in vitro glucose-stimulated insulin secretion rate per unit islet volume appears to be substantially smaller for porcine islets than for other species including human. Lastly, there is significant current concern regarding the potential for transmission of infectious agents from porcine organ sources to human xenograft recipients, and to the population at large. None of these characteristics bode well for their practical large-scale use, and serious consideration and investigation is being given to alternate animal sources. There is also speculation that neonatal porcine islets, which culture better and present minimal infrastructure problems, would be an ultimate substitute. Isolation of bovine islets is technically easier and calf islets are glucose-responsive. However, adult bovine islets are relatively insensitive to glucose. The rabbit pancreas is also an attractive source of islets since rabbit insulin differs from human insulin at only one amino acid and rabbit islets are glucose responsive.
The language of Molecular Cloning
cDNA (Complementary DNA):
* A synthetic DNA copied from messenger RNA (mRNA) by the enzyme reverse transcriptase. Used to refer to either a single-stranded copy or its double-stranded derivative. Usage: "a cDNA clone", "a cDNA library" or "to isolate a cDNA".
Clone:
* A recombinant DNA molecule containing a gene or other DNA sequence of interest. Also, the act of generating such a molecule. Usage: "to isolate a clone" or "to clone a gene"
Host:
* The organism used to isolate and propagate a recombinant DNA molecule. Usually a strain of the bacterium Escherichia coli or the yeast Saccharomyces cerevisiae. Usage: "What host did they use?"
Hybridization:
* The act of two complementary single-stranded nucleic acid molecules forming bonds and becoming a double-stranded molecule. Usage: "The probe hybridized to a gene".
Insert:
* A fragment of human DNA cloned into a particular vector. Usage:"They purified the insert"
Library:
* A collection of recombinant clones from a source known to contain the gene, cDNA, or other DNA sequences of interest. In principle, a library may contain all the DNA sequences represented in the original cell, tissue, or chromosome. Usage: "a muscle cDNA library" or "a human genomic library"
Ligation:
* The act of forming phosphodiester bonds to join two double-stranded DNA molecules with the enzyme DNA ligase. Ligation is the essential step in creating recombinant DNA molecules. Usage: "The fragments were ligated together"
Probe:
* A cloned DNA or RNA molecule, labeled with radioactivity or another detectable tracer, used to identify its complementary sequences by molecular hybridization; also, the act of using such a molecule. Usage: "the beta-globin probe" or "to probe a patient's DNA"
Restriction endonucleases (restriction enzymes):
* Enzymes that recognize specific double-stranded DNA sequences and cleve the DNA at or near the recognition site. Usage: "a restriction enzyme digest" (or just "a restriction digest") or "the restriction enzyme EcoRI".
Southern Blot:
* A filter to which DNA has been transferred, usually after restriction enzyme digestion and gel electrophoresis to separate DNA molecules by size (named after the developer of the technique, Ed Southern); also, the act of generating such a filter and hybridizing it to a specific probe. Usage: "to probe a Southern blot" or "they did a Southern".
Vector:
* the DNA molecule into which the gene or other DNA fragment of interest is cloned, capable of replicating in a particular host. Examples include plasmids, bacteriophage lambda, cosmids, and yeast artificial chromosomes. Usage: "a cloning vector" or "the cosmid vector".
* A synthetic DNA copied from messenger RNA (mRNA) by the enzyme reverse transcriptase. Used to refer to either a single-stranded copy or its double-stranded derivative. Usage: "a cDNA clone", "a cDNA library" or "to isolate a cDNA".
Clone:
* A recombinant DNA molecule containing a gene or other DNA sequence of interest. Also, the act of generating such a molecule. Usage: "to isolate a clone" or "to clone a gene"
Host:
* The organism used to isolate and propagate a recombinant DNA molecule. Usually a strain of the bacterium Escherichia coli or the yeast Saccharomyces cerevisiae. Usage: "What host did they use?"
Hybridization:
* The act of two complementary single-stranded nucleic acid molecules forming bonds and becoming a double-stranded molecule. Usage: "The probe hybridized to a gene".
Insert:
* A fragment of human DNA cloned into a particular vector. Usage:"They purified the insert"
Library:
* A collection of recombinant clones from a source known to contain the gene, cDNA, or other DNA sequences of interest. In principle, a library may contain all the DNA sequences represented in the original cell, tissue, or chromosome. Usage: "a muscle cDNA library" or "a human genomic library"
Ligation:
* The act of forming phosphodiester bonds to join two double-stranded DNA molecules with the enzyme DNA ligase. Ligation is the essential step in creating recombinant DNA molecules. Usage: "The fragments were ligated together"
Probe:
* A cloned DNA or RNA molecule, labeled with radioactivity or another detectable tracer, used to identify its complementary sequences by molecular hybridization; also, the act of using such a molecule. Usage: "the beta-globin probe" or "to probe a patient's DNA"
Restriction endonucleases (restriction enzymes):
* Enzymes that recognize specific double-stranded DNA sequences and cleve the DNA at or near the recognition site. Usage: "a restriction enzyme digest" (or just "a restriction digest") or "the restriction enzyme EcoRI".
Southern Blot:
* A filter to which DNA has been transferred, usually after restriction enzyme digestion and gel electrophoresis to separate DNA molecules by size (named after the developer of the technique, Ed Southern); also, the act of generating such a filter and hybridizing it to a specific probe. Usage: "to probe a Southern blot" or "they did a Southern".
Vector:
* the DNA molecule into which the gene or other DNA fragment of interest is cloned, capable of replicating in a particular host. Examples include plasmids, bacteriophage lambda, cosmids, and yeast artificial chromosomes. Usage: "a cloning vector" or "the cosmid vector".
The Molecular Analysis of a Human Mutation
How does one proceed when one has a patient with a genetic disorder known or suspected to be due to a mutation in a particular gene? For example, consider a patient with a diagnosis of ß-thalassemia, an autosomal recessive defect in the ß-globin gene. The initial diagnosis is generally made on the basis of clinical and hematological findings alone. However, it is important to examine the gene itself, first to confirm the clinical diagnosis and second, to determine the specific mutation in the ß-globin locus both for future use in carrier testing and possible prenatal diagnosis in the patient's family, and for increasing the understanding of the relationship between specific mutations in the gene and the resulting patho-psychology.
Several tests can be used initially to examine the gross integrity of the ß-globin gene itself and its mRNA. Is the gene present in the patient in both normal amount (i.e., two copies) and structure? Or is one or both copies of the gene deleted or structurally rearranged, as has been described in some cases of ß-thalassemia? If the gene is present, is it transcribed? The Southern blotting technique is now a standard method for using a cloned gene probe (in this case, for the ß-globin gene) to examine the integrity of a DNA sample. Southern blotting can address the question of whether it is grossly normal in structure. By this method one can detect large molecular defects that are well below the level of sensitivity of chromosome analysis. However, as currently used in diagnostic laboratories, it cannot reveal the presence of single mutations, such as base-pair changes or very small deletions of only a few base pairs. In order to examine whether mRNA is present, a technique called Northern blotting is used. This approach also enables one to detect major changes in mRNA levels or structure for a specific gene, but not to detect minor alterations.
Having asked whether there are gross changes in the gene or in its mRNA, one can proceed to a number of methods developed to examine gene structure and expressions at increasingly finer levels of analysis. In ß-thalassemia, as in many other genetic disorders, particular mutations responsible for the disease in other patients. To determine whether one of these already known mutations is responsible for a particular case of ß-thalassemia, one can apply particular direct molecular tests. Some of these entail the approach of allele-specific oligonucleotides (ASOs) that enable one to detect specific single base-pair mutations. In addition, it may be desirable to actually clone the mutant ß-globin genes (or cDNAs) from the patient for comparison with a normal ß-globin gene. Cloning of individual mutant genes (or portions of genes) from a patient's material is facilitated by use of polymerase chain reaction (PCR) to specifically generate many million of copies of a particular gene fragment. Once the mutant gene is isolated, one can then analyze it at the finest level possible by determining the DNA sequence of base pairs in the mutant gene for comparison with the normal gene. In this way, the specific mutation responsible for the genetic disorder in the patient can be determined and used to develop direct screening tests for that mutation in the patient's family.
Several tests can be used initially to examine the gross integrity of the ß-globin gene itself and its mRNA. Is the gene present in the patient in both normal amount (i.e., two copies) and structure? Or is one or both copies of the gene deleted or structurally rearranged, as has been described in some cases of ß-thalassemia? If the gene is present, is it transcribed? The Southern blotting technique is now a standard method for using a cloned gene probe (in this case, for the ß-globin gene) to examine the integrity of a DNA sample. Southern blotting can address the question of whether it is grossly normal in structure. By this method one can detect large molecular defects that are well below the level of sensitivity of chromosome analysis. However, as currently used in diagnostic laboratories, it cannot reveal the presence of single mutations, such as base-pair changes or very small deletions of only a few base pairs. In order to examine whether mRNA is present, a technique called Northern blotting is used. This approach also enables one to detect major changes in mRNA levels or structure for a specific gene, but not to detect minor alterations.
Having asked whether there are gross changes in the gene or in its mRNA, one can proceed to a number of methods developed to examine gene structure and expressions at increasingly finer levels of analysis. In ß-thalassemia, as in many other genetic disorders, particular mutations responsible for the disease in other patients. To determine whether one of these already known mutations is responsible for a particular case of ß-thalassemia, one can apply particular direct molecular tests. Some of these entail the approach of allele-specific oligonucleotides (ASOs) that enable one to detect specific single base-pair mutations. In addition, it may be desirable to actually clone the mutant ß-globin genes (or cDNAs) from the patient for comparison with a normal ß-globin gene. Cloning of individual mutant genes (or portions of genes) from a patient's material is facilitated by use of polymerase chain reaction (PCR) to specifically generate many million of copies of a particular gene fragment. Once the mutant gene is isolated, one can then analyze it at the finest level possible by determining the DNA sequence of base pairs in the mutant gene for comparison with the normal gene. In this way, the specific mutation responsible for the genetic disorder in the patient can be determined and used to develop direct screening tests for that mutation in the patient's family.
Electrophoresis - Seperation and purification of DNA fragments
Electrophoresis refers to carrying something by applying electricity. It is an analytical device commonly used for separation and purification of DNA fragments. A gel is used in electrophoresis which is either polyacrylamide or agarose. The former is preferred for smaller DNA fragments and the latter for larger ones. Agarose is a purified powder isolated from agar, a gelatinous material of sea weeds. Agarose powder when dissolved in water and boiled results into gel form. The gel prepared in a mixture of salt and water becomes a good conductor of electricity. The gel forms small pores the size of which varies depending on its amount in a given water. These pores act as molecular sieve. These allow the larger molecules to move slowly than the smaller molecules.
An Electrophoresis apparatusThe electrophoresis box consists of a positive and a negative electrode, a shelf designed to held the gel, a comb used to form the wells within the gel, and a power supply. The DNA to be electrophoresed is digested with restriction enzymes which yields DNA fragments of unequal length. The fragments are mixed with sucrose and a dye (ethidium bromide or methylene blue) which altogether is known as loading dye. Sucrose increases the density of DNA preparation and dye increases the visibility of the preparation.
DNA bands on gelThe preparation is loaded into wells at one end of the gel. At least one well is filled with reference DNA (i.e. DNA fragments of known length) for comparison with those of unknown length. Electric current is applied at opposite ends of electrophoresis chamber. A current is generated between a negative electrode at the top of loading end of the gel and a positive electrode at the bottom of the end of gel resulting in movement of fragments through pores of the gel. DNA molecules have a negative electric charges due to PO4(4-) which alternate with sugar molecules. Opposite electric charges tend to attract one another. The small DNA molecules move at faster speed as compared to larger ones. All DNA molecules of a given length migrate nearly the same distance into the gel and form bands. Each band represents many copies of DNA fragments having about the same length. After completion of electrophoresis gel is removed from the chamber and stained to make bands easily seen either with ethidium bromide (EB) or methylene blue. When gel is illuminated with UV light, fluorescent orange bands appear due to EB; methylene blue results in blue bands under normal room temperature.
Embryology of the reproductive system
The embryology of the male and the female reproductive systems:
By the sixth week of development in both sexes, the primordial germ cell have migrated from their earlier extra embryonic location to the gonadal ridges, where they are surrounded by the sex cords to form a pair of primitive gonads. Up to this time, the developing gonad, whether chromosomally XX or XY, is bipotential.
The current concept is that development into an ovary or a testis is determined by the coordinated action of a sequence of genes that lead to ovarian development when no Y chromosome is present or to testicular development if a Y is present. The ovarian pathway is followed unless a gene on the short arm of the Y, designated TDF (testis-determined factor), acts as a switch, diverting development into the male pathway. The search for the major testis-determined gene is one of the leading current problems in medical genetics.
In the presence of Y Chromosome, the medullary tissue forms typical testes with seminiferous tubules and Leydig cells, which, under the stimululation of human chorionic gonadotropin from the placenta, become capable of androgen secretion. The spermatogonia, derived from the primordial germ cells by 200 or more successive mitosis, form the walls of the semiferous tubules together with supporting Sertoli cells.
If no Y chromosome is present, the gonad, by default, forms an ovary; the cortex develops, the medulla regresses, and oogonia begin to develop within follicles. The oogonia are derived from the primitive germ cells by a series of about 30 mitoses, for fewer than the number required for spermatogenesis. Beginning at about the end of the third month, the oogonia enter meiosis I, but this this process is arrested at a stage called dictyotene, in which the cell remains until ovulation occurs many years later. Many of the oogonia degenerate before birth, and only about 400 mature into ova during the 30 years or so of sexual maturity of the female.
While the primordial germ cells are migrating to the genital ridges, thickenings in the ridges indicate the developing genital ducts, the mesonephric (formerly called Wolffian) and paramesonephric (formely called Mullerian) ducts. In the male, the Leydig cells of the fetal testes produce androgen, which stimulates the mesonephric ducts to form the male genital ducts, and the Sertoli cells produce a hormone that suppresses formation of the paramesonephric ducts. In the female (or in an embryo with no gonads), the mesonephric ducts regress, and the paramesonephric ducts develop into the female duct system. In the early embryo, the external genitalia consist of a genital tubercle, paired labioscrotal swellings, and paired urethral folds. From this undifferentiated state, male external genitalia develop under the influence of androgens, or, in the absence of a testis, female external genitalia are formed regardless of whether an ovary is present.
By the sixth week of development in both sexes, the primordial germ cell have migrated from their earlier extra embryonic location to the gonadal ridges, where they are surrounded by the sex cords to form a pair of primitive gonads. Up to this time, the developing gonad, whether chromosomally XX or XY, is bipotential.
The current concept is that development into an ovary or a testis is determined by the coordinated action of a sequence of genes that lead to ovarian development when no Y chromosome is present or to testicular development if a Y is present. The ovarian pathway is followed unless a gene on the short arm of the Y, designated TDF (testis-determined factor), acts as a switch, diverting development into the male pathway. The search for the major testis-determined gene is one of the leading current problems in medical genetics.
In the presence of Y Chromosome, the medullary tissue forms typical testes with seminiferous tubules and Leydig cells, which, under the stimululation of human chorionic gonadotropin from the placenta, become capable of androgen secretion. The spermatogonia, derived from the primordial germ cells by 200 or more successive mitosis, form the walls of the semiferous tubules together with supporting Sertoli cells.
If no Y chromosome is present, the gonad, by default, forms an ovary; the cortex develops, the medulla regresses, and oogonia begin to develop within follicles. The oogonia are derived from the primitive germ cells by a series of about 30 mitoses, for fewer than the number required for spermatogenesis. Beginning at about the end of the third month, the oogonia enter meiosis I, but this this process is arrested at a stage called dictyotene, in which the cell remains until ovulation occurs many years later. Many of the oogonia degenerate before birth, and only about 400 mature into ova during the 30 years or so of sexual maturity of the female.
While the primordial germ cells are migrating to the genital ridges, thickenings in the ridges indicate the developing genital ducts, the mesonephric (formerly called Wolffian) and paramesonephric (formely called Mullerian) ducts. In the male, the Leydig cells of the fetal testes produce androgen, which stimulates the mesonephric ducts to form the male genital ducts, and the Sertoli cells produce a hormone that suppresses formation of the paramesonephric ducts. In the female (or in an embryo with no gonads), the mesonephric ducts regress, and the paramesonephric ducts develop into the female duct system. In the early embryo, the external genitalia consist of a genital tubercle, paired labioscrotal swellings, and paired urethral folds. From this undifferentiated state, male external genitalia develop under the influence of androgens, or, in the absence of a testis, female external genitalia are formed regardless of whether an ovary is present.
The chromosomal basis for sex determination
The X and Y chromosomes have long attracted attention and interest because they differ between the sexes, because they have their own specific patterns of inheritance, and because they are involved in primary sex determination, They are structurally quite distinct and are subject to different forms of genetic regulation, yet they pair in male meiosis. For all these reasons, they require special attention.
The chromosomal basis for sex determination:
It has been known for decades that human male and female cells have different sex chromosomes (Painter, 1921) and that the difference is visible in interphase as well as in mitosis (Barr and Bertram, 1949). Although Painter's discovery of the human sex chromosomes could not be exploited clinically at the time because cytogenetic techniques were inadequate, the discovery of sex chromatin masses (Barr Bodies) in interphase cells of females but not of males was soon followed by the development of a simple technique that allowed Barr bodies to be studied in buccal smears. As a result, it was quickly recognized that although most females were "chromatin positive" and most males were "chromatin negative", there were exceptions. It was especially noteworthy that many short, infertile females with a condition known as Turner syndrome had no Barr bodies, whereas tall, infertile males with a condition known as Klinefelter syndrome did have Barr bodies.
Soon after cytogenetic analysis became feasible, the chromosomal basis for these discrepancies became apparent. Because the anomalous sex chromotin findings had suggested that the Turner and Klinefelter syndromes were characterized by unusual sex chromosome constitutions, they were two of the first conditions for which chromosome studies were performed. Patients with Klinefelter syndrome were found to have 47 chromosomes with two X Chromosomes as well as a Y Chromosome (karyotype 47, XXX ; Jacobs and Strong, 1959), whereas most Turner syndrome patients were found to have only 45 chromosomes with a single X Chromosome (Karyotype 45, X; Ford et al.,1959). These findings promptly established the crucial role of the Y chromosome in normal male development.
The next step in the understanding of the human sex chromosomes was an explanation of sex chromatin in terms of X inactivation. As additional sex chromosome abnormalities were identified, the number of Barr bodies seen in interphase cells was observed to be always one less than the total number of X chromosomes per cell:
The theory of X inactivation (the Lyon hypothesis) is that in somatic cells in normal females (but not in normal males), one X chromosome is inactivated, thus equalizing the expression of X-linked genes in the two sexes. The Barr body represents the late-replicating, inactive X chromosome. The replication asynchrony between active (early-replicating) and inactive (late-replicating) X chromosomes can be recognized cytogenetically by specialized banding procedures called "replicating banding". In patients with extra X chromosomes, any X chromosome in excess of one is inactivated and forms a Barr body (see table above). Thus all diploid somatic cells in both males and females have a single active X chromosome, regardless of the total number of Xs or Ys present.
Even though many abnormalities of the sex chromosomes have been defined and their clinical consequences reported in detail, there still remain mysteries about the precise role of the sex chromosomes in sexual determination. There are exceptions, not yet fully understood, to the rule that females are always XX and males always XY. These exceptions, which include XX males, XY females, and XX true hermaphrodites, suggest that the entire Y chromosome is not the sole determinant of phenotyoic sex. Molecular analysis is currently being used to find an explanation for these unusual
The chromosomal basis for sex determination:
It has been known for decades that human male and female cells have different sex chromosomes (Painter, 1921) and that the difference is visible in interphase as well as in mitosis (Barr and Bertram, 1949). Although Painter's discovery of the human sex chromosomes could not be exploited clinically at the time because cytogenetic techniques were inadequate, the discovery of sex chromatin masses (Barr Bodies) in interphase cells of females but not of males was soon followed by the development of a simple technique that allowed Barr bodies to be studied in buccal smears. As a result, it was quickly recognized that although most females were "chromatin positive" and most males were "chromatin negative", there were exceptions. It was especially noteworthy that many short, infertile females with a condition known as Turner syndrome had no Barr bodies, whereas tall, infertile males with a condition known as Klinefelter syndrome did have Barr bodies.
Soon after cytogenetic analysis became feasible, the chromosomal basis for these discrepancies became apparent. Because the anomalous sex chromotin findings had suggested that the Turner and Klinefelter syndromes were characterized by unusual sex chromosome constitutions, they were two of the first conditions for which chromosome studies were performed. Patients with Klinefelter syndrome were found to have 47 chromosomes with two X Chromosomes as well as a Y Chromosome (karyotype 47, XXX ; Jacobs and Strong, 1959), whereas most Turner syndrome patients were found to have only 45 chromosomes with a single X Chromosome (Karyotype 45, X; Ford et al.,1959). These findings promptly established the crucial role of the Y chromosome in normal male development.
The next step in the understanding of the human sex chromosomes was an explanation of sex chromatin in terms of X inactivation. As additional sex chromosome abnormalities were identified, the number of Barr bodies seen in interphase cells was observed to be always one less than the total number of X chromosomes per cell:
| Sexual Phenotype | Karyotype | Barr Bodies |
|---|---|---|
| Male | 46,XY;47,XYY 47,XXY;48,XXYY 48,XXXY,49,XXXYY 49,XXXXY | 0 1 2 3 |
| Female | 45,X 46,XX 47,XXX 48,XXXX 49,XXXXX | 0 1 2 3 4 |
The theory of X inactivation (the Lyon hypothesis) is that in somatic cells in normal females (but not in normal males), one X chromosome is inactivated, thus equalizing the expression of X-linked genes in the two sexes. The Barr body represents the late-replicating, inactive X chromosome. The replication asynchrony between active (early-replicating) and inactive (late-replicating) X chromosomes can be recognized cytogenetically by specialized banding procedures called "replicating banding". In patients with extra X chromosomes, any X chromosome in excess of one is inactivated and forms a Barr body (see table above). Thus all diploid somatic cells in both males and females have a single active X chromosome, regardless of the total number of Xs or Ys present.
Even though many abnormalities of the sex chromosomes have been defined and their clinical consequences reported in detail, there still remain mysteries about the precise role of the sex chromosomes in sexual determination. There are exceptions, not yet fully understood, to the rule that females are always XX and males always XY. These exceptions, which include XX males, XY females, and XX true hermaphrodites, suggest that the entire Y chromosome is not the sole determinant of phenotyoic sex. Molecular analysis is currently being used to find an explanation for these unusual
Glucose-6-Phospate Dehydrogenase (G6PD) Deficiency
Deficiency of G6PD, a ubiquitous X-linked enzyme, is the most common disease-producing enzyme defect of humans, estimated to affect 400 million people worldwide; one allele, the A variant, is found in 1 in 20 Black males in the United States. With over 300 variants described, G6PD deficiency also appears to be the most genetically heterogeneous disorder yet recognized (Luzzatto and Mehta, 1989). The high gene frequency of G6PD variants in some populations appears to reflect the fact that G6PD deficiency, like sickle cell hemoglobin and thalassemia, confers some protection against malaria. This enzymopathy originally came to attention when the antimalarial drug primaquine was found to induce hemolytic anemia in Black males, who were subsequently found to have G6PD deficiency.
The mechanism of the drug-induced hemolysis is reasonably clear. One of the products of G6PD, nicotinamide-adenine dinucleotide phospate (NADPH), is the major source of reducing equivalents in the red blood cell. NADPH protects the cell against oxidative damage by regenerating reduced glutathione from the oxidized form. In G6PD deficiency, oxidant drugs such as primaquine deplete the cell of reduced glutathione, and the consequent oxidative damage leads to hemolysis. Additional offending compounds include sulfonamide antibiotics, sulfones such as dapsone (widely used in the treatment of leprosy and Pneumocystis carinii infection), naphthalene (moth balls), and a few others. The role of some drugs in producing hemolysis in G6PD deficiency is ambiguous because of the uncertain significance of other genetic factors (such as genetically determined ethnic and individual variation in pharmacokinetics) and nongenetic determinants (such as infection, which itself can induce hemolysis in severe variants of G6PD deficiency).
Favism, a sever hemolytic anemia that results from ingestion of the broad bean Vicia faba and that has been known since ancient times in parts of the Mediterranean, is due to extreme G6PD deficiency. The enzyme defect makes the cells vulnerable to oxidnants in fava beans (Pythagoras, the Greek mathematician, warned his followers of the danger of eating these beans). In areas where severe deficiency variants like the Mediterranean allele are prevalent, they are a major cause of both neonatal jaundice and congenital nonspherocytic hemolytic anemia.
The common deficiency alleles of American Blacks and of the Mediterranean region migrate electrophoretically at the same rate as A and B variants but have much lower activities, and so are called A -ve and B -ve variants, respectively. Although G6PD deficiency is far more common in males, an appreciable number (at least 1 in 400) of American Black females are genetically A-ve/A-ve, and are clinically susceptible to drug-induced hemolysis.
The A-ve Variant has decreased stability
In addition to reduced catalytic activity, instability of the A-ve variant is a major factor in the pathological response to drug ingestion. Synthesis of the A-ve protein is unaffected by the mutation, but because the molecule is relatively unstable, its abundance decreases more quickly than the normal as the red cell ages. (Remember that the mature cell is nucleate, and thus new protein synthesis is limited). After drug ingestion, patients with this allele have hemolysis only as long as it takes (usually about a week) to destroy the fraction of older red cells that have lost, through aging, a critical amount of G6PD activity. Even if the drug administration is continued, the hemolytic phase comes to an end because the young cells produced in response to hemolysis have sufficient newly synthesized G6PD A-ve prevent oxidative damage.
The mechanism of the drug-induced hemolysis is reasonably clear. One of the products of G6PD, nicotinamide-adenine dinucleotide phospate (NADPH), is the major source of reducing equivalents in the red blood cell. NADPH protects the cell against oxidative damage by regenerating reduced glutathione from the oxidized form. In G6PD deficiency, oxidant drugs such as primaquine deplete the cell of reduced glutathione, and the consequent oxidative damage leads to hemolysis. Additional offending compounds include sulfonamide antibiotics, sulfones such as dapsone (widely used in the treatment of leprosy and Pneumocystis carinii infection), naphthalene (moth balls), and a few others. The role of some drugs in producing hemolysis in G6PD deficiency is ambiguous because of the uncertain significance of other genetic factors (such as genetically determined ethnic and individual variation in pharmacokinetics) and nongenetic determinants (such as infection, which itself can induce hemolysis in severe variants of G6PD deficiency).
Favism, a sever hemolytic anemia that results from ingestion of the broad bean Vicia faba and that has been known since ancient times in parts of the Mediterranean, is due to extreme G6PD deficiency. The enzyme defect makes the cells vulnerable to oxidnants in fava beans (Pythagoras, the Greek mathematician, warned his followers of the danger of eating these beans). In areas where severe deficiency variants like the Mediterranean allele are prevalent, they are a major cause of both neonatal jaundice and congenital nonspherocytic hemolytic anemia.
The common deficiency alleles of American Blacks and of the Mediterranean region migrate electrophoretically at the same rate as A and B variants but have much lower activities, and so are called A -ve and B -ve variants, respectively. Although G6PD deficiency is far more common in males, an appreciable number (at least 1 in 400) of American Black females are genetically A-ve/A-ve, and are clinically susceptible to drug-induced hemolysis.
The A-ve Variant has decreased stability
In addition to reduced catalytic activity, instability of the A-ve variant is a major factor in the pathological response to drug ingestion. Synthesis of the A-ve protein is unaffected by the mutation, but because the molecule is relatively unstable, its abundance decreases more quickly than the normal as the red cell ages. (Remember that the mature cell is nucleate, and thus new protein synthesis is limited). After drug ingestion, patients with this allele have hemolysis only as long as it takes (usually about a week) to destroy the fraction of older red cells that have lost, through aging, a critical amount of G6PD activity. Even if the drug administration is continued, the hemolytic phase comes to an end because the young cells produced in response to hemolysis have sufficient newly synthesized G6PD A-ve prevent oxidative damage.
Pharmacogenetic Diseases
Pharmacogenetics is the special areas of biochemical genetics that deals with variation in drug response and the contribution of genetics to such variation. In broad terms, pharmacogenetics can be said to encompass any genetically determined variation in response to drugs: for instance, the effect of barbiturates in precipitating attacks of porphyria in people with the gene for acute intermittent porphyria or the effect of alcohol use by pregnant woman on the incidence of fetal alcohol syndrome. In a narrower sense, pharmacogenetics can be restricted to those genetic variations that are revealed only by response to drugs or other chemicals.
The origin of polymorphisms by which they are maintained pose a problem. They obviously have not developed in response to drugs, since they antedate the drugs concerned. The handling of and the response to drugs require many specific bio-chemical reactions, and the enzymes involved may participate in the metabolism of ordinary food substances.
Recognizing that there is normal variation in response to drugs, pharmacologists define the "potency" of a drug by the dose that produces a given effect in 50 percent of the population. For genetic traits, continuous variation is usually best explained on the basis of multi factorial inheritance or by a combination of genetic and environmental factors. But response to drugs can also show discontinuous variation, with sharp distinctions between different degrees of response. The finding of a bimodal or trimodal population distribution of activity of a drug-metabolizing enzyme may indicate that the enzyme is coded by genes at a polymorphic locus.
The origin of polymorphisms by which they are maintained pose a problem. They obviously have not developed in response to drugs, since they antedate the drugs concerned. The handling of and the response to drugs require many specific bio-chemical reactions, and the enzymes involved may participate in the metabolism of ordinary food substances.
Recognizing that there is normal variation in response to drugs, pharmacologists define the "potency" of a drug by the dose that produces a given effect in 50 percent of the population. For genetic traits, continuous variation is usually best explained on the basis of multi factorial inheritance or by a combination of genetic and environmental factors. But response to drugs can also show discontinuous variation, with sharp distinctions between different degrees of response. The finding of a bimodal or trimodal population distribution of activity of a drug-metabolizing enzyme may indicate that the enzyme is coded by genes at a polymorphic locus.
Cultivation of Dhingri (Pleurotus sajor-caju)
Pleurotus is one of the important edible mushrooms gaining popularity in recent years. It is found growing naturally on dead organic materials rich in cellulose. Its several species are edible such as P . sajor-caju, P. sapidus, P. flabellatus, P. ostriatus, P. Florida, etc. These species can be cultured successfully on various agricultural, domestic, industrial and forestry waste materials. It is very versatile in nature as far as substrate reference and growth are concerned. However, it can be grown on paddy straw, gunny bags, rice husk, copped Parthenium stem, etc. The steps for cultivation of dhingri start with preparation of substrate for growth.
Paddy is cut into 2.5 cm long pieces and soaked in hot water at 60 Degree Celsius for about 30 minutes. Excess water is drained off from straw. About 4 kg of wet straw is transferred into the large sized polythene bags. About 5 grams of Bengal-gram powder with half bottle of spawn of fungus are mixed with straw. This mixture is filled in large-sized polythene bags. Mouth of the bags is tied and kept on a raised platform in well ventilated cropping room or in open when properly protected for about 15 days. At this time when mycelia are visible inside the polythene bags over the surface of paddy straw, the polythene bags are cut and gently removed. Now paddy straw forms a compact mass and does not lose its make up. This composite mixture is watered daily just to maintain moisture. The temperature where the compost has been kept should be between 20 and 25 Degree Celsius with relative humidity of 75%. After 15 days first flask of dhingri becomes apparent. These are harvested when become young. A photograph of P. sajor-caju is shown below.
Paddy is cut into 2.5 cm long pieces and soaked in hot water at 60 Degree Celsius for about 30 minutes. Excess water is drained off from straw. About 4 kg of wet straw is transferred into the large sized polythene bags. About 5 grams of Bengal-gram powder with half bottle of spawn of fungus are mixed with straw. This mixture is filled in large-sized polythene bags. Mouth of the bags is tied and kept on a raised platform in well ventilated cropping room or in open when properly protected for about 15 days. At this time when mycelia are visible inside the polythene bags over the surface of paddy straw, the polythene bags are cut and gently removed. Now paddy straw forms a compact mass and does not lose its make up. This composite mixture is watered daily just to maintain moisture. The temperature where the compost has been kept should be between 20 and 25 Degree Celsius with relative humidity of 75%. After 15 days first flask of dhingri becomes apparent. These are harvested when become young. A photograph of P. sajor-caju is shown below.
Wastes as Renewable Source of Energy
Waste is the spoilage, loss or destruction of either matter or energy, which is unsuable to man. Gradually increasing civilization through industrialization and urbanization, has led to increase in generation of wastes into environment from various sources. Waste generation is, therefore, a necessary outcome of consumption, and also because of insufficient process, general ignorance, wasteful habits and social attitudes (Ray, 1979).
Ray (1979) classified the wastes into energy wastes and material wastes. The main source of energy in the developed and developing countries is petroleum oil, followed by coal. In India, about 50 per cent oil is imported each year. Coal mines are concentrated only in a few regions. Coal is used in generation of electricity, steam engines and fire. Most potential energy of coal is wasted during electric generation in thermal power plants. Thermal loss in India is about 20-30 per cent because of lack of suitable technologies. Based on the chemical nature, material wastes are of various types;
(i) Inorganic wastes (those generated by metallurgical and chemical industries, coal mines, etc.),
(ii) Organic wastes (agricultural products, dairy and milk products, slaughter houses, sewage, forestry, etc.), and
(iii) Mixed wastes (those discharged from industries dealing with textiles, dyes, cake and gas, plastic, wool, leather, petroleum, etc.). The inorganic wastes may be recovered by chemical/ mechanical treatment, whereas organic and mixed wastes require biological as well chemical treatments.
Moreover, the wastes occur in three states, the solid, liquid and gaseous ones. The solid wastes can be burnt, thermally decomposed, anaerobically digested to get methane and other combustible gases or biologically converted to a variety of products. Liquid wastes are most troublesome, because of the presence of non-retractable chemicals, and their further return to environment through surface waters. Gaseous wastes include the toxic gases such as NO2, NO2, NH3, CO2, CO2, SO2, etc. When concentration of these gases increases in the atmosphere they cause gaseous pollution, which has its bad impact on plant and animal lives. The organic wastes and residues become a source of renewable energy in multifarious ways.
Ray (1979) classified the wastes into energy wastes and material wastes. The main source of energy in the developed and developing countries is petroleum oil, followed by coal. In India, about 50 per cent oil is imported each year. Coal mines are concentrated only in a few regions. Coal is used in generation of electricity, steam engines and fire. Most potential energy of coal is wasted during electric generation in thermal power plants. Thermal loss in India is about 20-30 per cent because of lack of suitable technologies. Based on the chemical nature, material wastes are of various types;
(i) Inorganic wastes (those generated by metallurgical and chemical industries, coal mines, etc.),
(ii) Organic wastes (agricultural products, dairy and milk products, slaughter houses, sewage, forestry, etc.), and
(iii) Mixed wastes (those discharged from industries dealing with textiles, dyes, cake and gas, plastic, wool, leather, petroleum, etc.). The inorganic wastes may be recovered by chemical/ mechanical treatment, whereas organic and mixed wastes require biological as well chemical treatments.
Moreover, the wastes occur in three states, the solid, liquid and gaseous ones. The solid wastes can be burnt, thermally decomposed, anaerobically digested to get methane and other combustible gases or biologically converted to a variety of products. Liquid wastes are most troublesome, because of the presence of non-retractable chemicals, and their further return to environment through surface waters. Gaseous wastes include the toxic gases such as NO2, NO2, NH3, CO2, CO2, SO2, etc. When concentration of these gases increases in the atmosphere they cause gaseous pollution, which has its bad impact on plant and animal lives. The organic wastes and residues become a source of renewable energy in multifarious ways.
Blood Test For Brain Injuries Gains Momentum
A blood test that can help predict the seriousness of a head injury and detect the status of the blood-brain barrier is a step closer to reality, according to two recently published studies involving University of Rochester Medical Center researchers.
News stories about tragic head injuries - from the death of actress Natasha Richardson to brain-injured Iraq war soldiers and young athletes - certainly underscore the need for a simpler, faster, accurate screening tool, said brain injury expert Jeffrey Bazarian, M.D., M.P.H., associate professor of Emergency Medicine, Neurology and Neurosurgery at URMC, and a co-author on both studies.
The S-100B blood test recently cleared a significant hurdle when a panel of national experts, including Bazarian, agreed for the first time that it could be a useful tool for patients with a mild injury, allowing them to safely avoid a CT scan.
Previous studies have shown the S-100B serum protein biomarker to increase rapidly after an injury. If measured within four hours of the injury, the S-100B test accurately predicts which head injury patients will have a traumatic abnormality such as hemorrhage or skull fracture on a head CT scan. It takes about 20 minutes to get results and could spare many patients unnecessary radiation exposure.
Physicians at six Emergency Departments in upstate New York, including the ED at Strong Memorial Hospital in Rochester, this year will continue to study the accuracy of the test among 1,500 patients. Scientists plan to use the data to apply for U.S. Food and Drug Administration approval.
"The S-100B blood test is an important part of the tool set we need to improve our treatment of patients with brain injuries," Bazarian said. "It's not the ultimate diagnostic test, but it may make things easier for patients, and it will help doctors sort through difficult clinical decisions."
The test is used routinely in 16 European countries as a screening device. If a person falls and gets a head injury in Munich, Germany, during Oktoberfest, for example, a neurosurgeon is on duty within 500 meters of the beer tent, ready to administer the blood test, Bazarian said.
But in the United States, the current, accepted standard screening tool for head injuries is still the CT scan, which shows bleeding in the brain but does not detect more subtle injury to the brain's neurons, which can result in lasting neurological defects. In fact, 95 percent of CT scans look normal for patients with a relatively mild but potentially life-altering injury, Bazarian said.
There are more than 1 million emergency visits annually for traumatic brain injury (TBI) in the U.S. The majority of these visits are for mild injuries, primarily the result of falls and motor vehicle crashes. The challenge for doctors is to identify which of these patients has an acute, traumatic intracranial injury, something that is not always evident, and which patients can be observed and sent home.
Widespread use of the blood test could result in a 30 percent reduction of CT scans, according to the report by the national panel of brain experts, which published updated clinical guidelines in the December 2008 Annals of Emergency Medicine, and the April 2009 Journal of Emergency Nursing.
Bazarian and colleague Brian J. Blyth, M.D., assistant professor of Emergency Medicine at URMC, additionally found that the S-100B test can relay critical information about how the blood-brain barrier (BBB) is functioning after a head injury. Blyth was the first author on this study, reported electronically March 3, 2009, in the Journal of Neurotrauma.
In the context of head injuries, the BBB acts like a gate between the brain tissue and peripheral circulation. The gate often opens after injury, but not always. Knowing the status of the BBB helps doctors to decide if medications given to repair damage will actually reach the brain. The time between injury and irreversible brain swelling is short - and many drug studies have failed to find a therapy that leverages this time frame and works as designed.
Before the S-100B blood test, the best way to know if the BBB was open was to perform an invasive procedure called a ventriculostomy. (Doctors insert a catheter through the skull and into the brain, withdrawal fluid, and compare the concentration of albumin protein in the cerebrospinal fluid to the concentration in the blood.)
In a pilot study of 20 patients, however, Blyth found that serum S-100B concentrations could accurately predict the function of the blood-brain barrier 12 hours after injury, eliminating the need for the invasive procedure.
The study compared levels of S-100B proteins to the CSF-serum albumin quotient (Qa), the gold standard measurement signaling a brain injury. Researchers compared nine people with a known severe head injury, to 11 people who suffered from non-traumatic headaches.
Blyth and Bazarian believe the research may impact future drug studies. "The disability and death rates from brain injuries have not improved much in the past 20 years," Blyth said. "Many clinical trials for new medications have failed, probably because it was difficult to know if the blood-brain barrier was open and the drugs were reaching its target. Our study shows that any diagnostic test for brain injury should incorporate a way to measure the status of the blood-brain barrier into its design."
News stories about tragic head injuries - from the death of actress Natasha Richardson to brain-injured Iraq war soldiers and young athletes - certainly underscore the need for a simpler, faster, accurate screening tool, said brain injury expert Jeffrey Bazarian, M.D., M.P.H., associate professor of Emergency Medicine, Neurology and Neurosurgery at URMC, and a co-author on both studies.
The S-100B blood test recently cleared a significant hurdle when a panel of national experts, including Bazarian, agreed for the first time that it could be a useful tool for patients with a mild injury, allowing them to safely avoid a CT scan.
Previous studies have shown the S-100B serum protein biomarker to increase rapidly after an injury. If measured within four hours of the injury, the S-100B test accurately predicts which head injury patients will have a traumatic abnormality such as hemorrhage or skull fracture on a head CT scan. It takes about 20 minutes to get results and could spare many patients unnecessary radiation exposure.
Physicians at six Emergency Departments in upstate New York, including the ED at Strong Memorial Hospital in Rochester, this year will continue to study the accuracy of the test among 1,500 patients. Scientists plan to use the data to apply for U.S. Food and Drug Administration approval.
"The S-100B blood test is an important part of the tool set we need to improve our treatment of patients with brain injuries," Bazarian said. "It's not the ultimate diagnostic test, but it may make things easier for patients, and it will help doctors sort through difficult clinical decisions."
The test is used routinely in 16 European countries as a screening device. If a person falls and gets a head injury in Munich, Germany, during Oktoberfest, for example, a neurosurgeon is on duty within 500 meters of the beer tent, ready to administer the blood test, Bazarian said.
But in the United States, the current, accepted standard screening tool for head injuries is still the CT scan, which shows bleeding in the brain but does not detect more subtle injury to the brain's neurons, which can result in lasting neurological defects. In fact, 95 percent of CT scans look normal for patients with a relatively mild but potentially life-altering injury, Bazarian said.
There are more than 1 million emergency visits annually for traumatic brain injury (TBI) in the U.S. The majority of these visits are for mild injuries, primarily the result of falls and motor vehicle crashes. The challenge for doctors is to identify which of these patients has an acute, traumatic intracranial injury, something that is not always evident, and which patients can be observed and sent home.
Widespread use of the blood test could result in a 30 percent reduction of CT scans, according to the report by the national panel of brain experts, which published updated clinical guidelines in the December 2008 Annals of Emergency Medicine, and the April 2009 Journal of Emergency Nursing.
Bazarian and colleague Brian J. Blyth, M.D., assistant professor of Emergency Medicine at URMC, additionally found that the S-100B test can relay critical information about how the blood-brain barrier (BBB) is functioning after a head injury. Blyth was the first author on this study, reported electronically March 3, 2009, in the Journal of Neurotrauma.
In the context of head injuries, the BBB acts like a gate between the brain tissue and peripheral circulation. The gate often opens after injury, but not always. Knowing the status of the BBB helps doctors to decide if medications given to repair damage will actually reach the brain. The time between injury and irreversible brain swelling is short - and many drug studies have failed to find a therapy that leverages this time frame and works as designed.
Before the S-100B blood test, the best way to know if the BBB was open was to perform an invasive procedure called a ventriculostomy. (Doctors insert a catheter through the skull and into the brain, withdrawal fluid, and compare the concentration of albumin protein in the cerebrospinal fluid to the concentration in the blood.)
In a pilot study of 20 patients, however, Blyth found that serum S-100B concentrations could accurately predict the function of the blood-brain barrier 12 hours after injury, eliminating the need for the invasive procedure.
The study compared levels of S-100B proteins to the CSF-serum albumin quotient (Qa), the gold standard measurement signaling a brain injury. Researchers compared nine people with a known severe head injury, to 11 people who suffered from non-traumatic headaches.
Blyth and Bazarian believe the research may impact future drug studies. "The disability and death rates from brain injuries have not improved much in the past 20 years," Blyth said. "Many clinical trials for new medications have failed, probably because it was difficult to know if the blood-brain barrier was open and the drugs were reaching its target. Our study shows that any diagnostic test for brain injury should incorporate a way to measure the status of the blood-brain barrier into its design."
How Does Microglia Examine Damaged Synapses?
Microglia, immune cells in the brain, is suggested to be involved in the repair of damaged brain, like a medical doctor. However, it is completely unknown how microglia diagnoses damaged circuits in an in vivo brain. Japanese group led by Professor Junichi Nabekura and Dr Hiroaki Wake of National Institute for Physiological Sciences, NIPS, Japan, successfully took a live image how microglia surveys the synapses in the intact and ischemic brains of mice by using two-photon microscopic technology. They report their finding in Journal of Neuroscience on April 1, 2009.
They took an intense tune-up of their two-photon microscopy and achieved to visualize the fine structures of neurons and glias of mice in the range of 0 to 1 mm from the brain surface (world-leading deep imaging technology).
Surprisingly even in the normal (intact brain), microglias actively reached out their processes selectively for neuronal synapses at an interval of one hour with a contact duration of 5 minutes. More frequently microglias contacted on more active synapses. Once the brain received the damage such as ischemic infarction, microglial surveillance of synapses was much prolonged in duration, up to 2 hours. Frequently after the prolonged survey by microglia, damaged synapses were eliminated. This is the first report to show how microglia actively surveys the synapses in vivo and determines the fate of synapses, remained or eliminated
"Dynamic change of microglial surveillance of neuronal circuits in damaged brain, observed here, could contribute to establish the therapeutic approach targeted to damaged circuits", said Professor Nabekura.
Manipulation of Reproduction and Transgenic Animals – Ovulation control & Sperm sexing
Ovulation control:
In many animals it is difficult to find out oestrous (sexual heat) in animals because it persists only for a few hours and occurs mostly at night. After ovulation (which is indicated by oestrous) females are inseminated. But in herd it would be economical, easy and simplified management if females are inseminated at a times. However, it is possible only when all the female ovulate at a time; in practice it is not possible to get synchronisation of oestrous. Moreover, it is possible to bring about ovulation in about 80 per cent of females by using hormones, for example progesterone and/or prostaglandin. These hormones regulate ovulation cycle of female and result in total synchrony of oestrous.
Sperm sexing:
Sperms are produced in the testes of males and ova in female’s ovaries. Sperms and ova contain half of chromosomes as compared to somatic cells. An ovum possesses autosomes and one X chromosome. Similarly, a sperm contains autosomes and one Y chromosome. In animals, sex is determined genetically i.e. by sex chromosomes. X chromosome determines femaleness and Y maleness. All the ova contain X chromosome, whereas a sperm consists of either X or Y chromosomes. One sperm ejaculate contains half X and half Y chromosomes.
In dairy industry demand of females is more than the males. Secondly, females have more desirable characteristics. The livestock industry prefers animals of one sex. Therefore, through artificial insemination technology X and Y chromosomes can be detected and sex of progenies determined accordingly.
There is fluorescence dye (Hoechst 33342) that stains X and Y chromosomes with different intensities. Thus, these two chromosomes possibly can be separated by using an instrument, fluorescent activated cell sorter (FACS). Sperms are present in the form of suspension. The FACS converts a suspension of sperms into micro droplets. Each droplet consists of a single sperm cell. Individual micro droplet passes through a laser beam. Micro droplets of different intensities are deflected into separate collection tubes as the fluorescence of dye is measured electronically. The sperms separated by using FACS have recently been used and pre-sexed calves have been produced through in vitro fertilization technique. Moreover, FACS is very expensive and slow. It takes about 24 hrs to process one semen ejaculate, whereas the sperm cannot remain viable for a long time. Therefore, more refinement in technique is required for its use on a large scale (Read and Smith, 1996)
In many animals it is difficult to find out oestrous (sexual heat) in animals because it persists only for a few hours and occurs mostly at night. After ovulation (which is indicated by oestrous) females are inseminated. But in herd it would be economical, easy and simplified management if females are inseminated at a times. However, it is possible only when all the female ovulate at a time; in practice it is not possible to get synchronisation of oestrous. Moreover, it is possible to bring about ovulation in about 80 per cent of females by using hormones, for example progesterone and/or prostaglandin. These hormones regulate ovulation cycle of female and result in total synchrony of oestrous.
Sperm sexing:
Sperms are produced in the testes of males and ova in female’s ovaries. Sperms and ova contain half of chromosomes as compared to somatic cells. An ovum possesses autosomes and one X chromosome. Similarly, a sperm contains autosomes and one Y chromosome. In animals, sex is determined genetically i.e. by sex chromosomes. X chromosome determines femaleness and Y maleness. All the ova contain X chromosome, whereas a sperm consists of either X or Y chromosomes. One sperm ejaculate contains half X and half Y chromosomes.
In dairy industry demand of females is more than the males. Secondly, females have more desirable characteristics. The livestock industry prefers animals of one sex. Therefore, through artificial insemination technology X and Y chromosomes can be detected and sex of progenies determined accordingly.
There is fluorescence dye (Hoechst 33342) that stains X and Y chromosomes with different intensities. Thus, these two chromosomes possibly can be separated by using an instrument, fluorescent activated cell sorter (FACS). Sperms are present in the form of suspension. The FACS converts a suspension of sperms into micro droplets. Each droplet consists of a single sperm cell. Individual micro droplet passes through a laser beam. Micro droplets of different intensities are deflected into separate collection tubes as the fluorescence of dye is measured electronically. The sperms separated by using FACS have recently been used and pre-sexed calves have been produced through in vitro fertilization technique. Moreover, FACS is very expensive and slow. It takes about 24 hrs to process one semen ejaculate, whereas the sperm cannot remain viable for a long time. Therefore, more refinement in technique is required for its use on a large scale (Read and Smith, 1996)
Manipulation of reproduction in animals - Artificial Insemination / Semen and its storage
Manipulation of reproduction in animals:
One of the natural laws of life is to be many from one which is possible through reproduction. In animals, sexual reproduction occurs through fertilization of female gametes (ova) by male gametes (sperms) followed by fusion of two nuclei coming from two gametes. Thus sexual reproduction maintains the genetic traits of organism. But offspring develop and born in natural way. To meet the demand of animal products due to gradual increasing propulsion there is need of increase in number of animals too which is possible bio technologically because natural process of increase in number is slow.
In nature female mammals produce only one egg in a month except pigs. Secondly, ruminant females can have only one offspring in a year. Therefore, biotechnology can help to meet increasing demand and need of quality products of animals. In this regard principles of artificial breeding system has been discussed that may be useful further in the area of animal biotechnology.
Artificial Insemination:
A male animal produces millions of sperms daily. Theoretically, it can inseminate females regularity and produce several offspring. This excess capacity of male has been utilized through developing new technologies for artificial insemination which can be said as the first animal biotechnology.
The most effective factor that has increased the productivity of cattle is the artificial insemination. However, the breeder must replace the nature through artificial insemination if he ensures about the ovulation of female in herd together at a time. In contrast, if a breeder awaits for female to ovulate and then inseminate separately, the importance and economic significance of artificial insemination get reduced. Therefore, through artificial insemination technology increased number of females can be inseminated by a male.
Semen and its storage:
In addition methods have been developed to produce semen from male by ejaculation. Semen ejaculate is collected and diluted (extended). Sperm motility and there number per millilitre are examined under the microscope. About 0.2 ml bull semen contains about 10 million motile sperms. The diluted sperms may be used fresh within a few days or cryopreserved at -196 Degree Celsius by using liquid nitrogen. The cryopreserved semen can be stored for a long time and easily transported across states or countries. Thus cryopreserved semen of a single male is capable of inseminating thousands of females of a country or other countries. For example, one ejaculate semen of a bull is sufficient to inseminate about 500 cows.
One of the natural laws of life is to be many from one which is possible through reproduction. In animals, sexual reproduction occurs through fertilization of female gametes (ova) by male gametes (sperms) followed by fusion of two nuclei coming from two gametes. Thus sexual reproduction maintains the genetic traits of organism. But offspring develop and born in natural way. To meet the demand of animal products due to gradual increasing propulsion there is need of increase in number of animals too which is possible bio technologically because natural process of increase in number is slow.
In nature female mammals produce only one egg in a month except pigs. Secondly, ruminant females can have only one offspring in a year. Therefore, biotechnology can help to meet increasing demand and need of quality products of animals. In this regard principles of artificial breeding system has been discussed that may be useful further in the area of animal biotechnology.
Artificial Insemination:
A male animal produces millions of sperms daily. Theoretically, it can inseminate females regularity and produce several offspring. This excess capacity of male has been utilized through developing new technologies for artificial insemination which can be said as the first animal biotechnology.
The most effective factor that has increased the productivity of cattle is the artificial insemination. However, the breeder must replace the nature through artificial insemination if he ensures about the ovulation of female in herd together at a time. In contrast, if a breeder awaits for female to ovulate and then inseminate separately, the importance and economic significance of artificial insemination get reduced. Therefore, through artificial insemination technology increased number of females can be inseminated by a male.
Semen and its storage:
In addition methods have been developed to produce semen from male by ejaculation. Semen ejaculate is collected and diluted (extended). Sperm motility and there number per millilitre are examined under the microscope. About 0.2 ml bull semen contains about 10 million motile sperms. The diluted sperms may be used fresh within a few days or cryopreserved at -196 Degree Celsius by using liquid nitrogen. The cryopreserved semen can be stored for a long time and easily transported across states or countries. Thus cryopreserved semen of a single male is capable of inseminating thousands of females of a country or other countries. For example, one ejaculate semen of a bull is sufficient to inseminate about 500 cows.
RNA genes
RNA genes (sometimes referred to as non-coding RNA or small RNA) are genes that encode RNA that is not translated into a protein. The most prominent examples of RNA genes are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation. However, since the late 1990s, many new RNA genes have been found, and thus RNA genes may play a much more significant role than previously thought. In the late 1990s and early 2000, there has been persistent evidence of more complex transcription occurring in mammalian cells (and possibly others). This could point towards a more widespread use of RNA in biology, particularly in gene regulation. A particular class of non-coding RNA, micro RNA, has been found in many metazoans (from Caenorhabditis elegans to Homo sapiens) and clearly plays an important role in regulating other genes. First proposed in 2004 by Rassoulzadegan and published in Nature 2006.
RNA is implicated as being part of the germline. If confirmed, this result would significantly alter the present understanding of genetics and lead to many question on DNA-RNA roles and interactions.RNA Deatiles,ScienceRibonucleic acid (RNA) is a nucleic acid polymer consisting of nucleotide monomers, that acts as a messenger between DNA and ribosomes, and that is also responsible for making proteins out of amino acids. RNA polynucleotides contain ribose sugars and predominantly uracil unlike deoxyribonucleic acid (DNA), which contains deoxyribose and predominantly thymine. It is transcribed (synthesized) from DNA by enzymes called RNA polymerases and further processed by other enzymes.
RNA serves as the template for translation of genes into proteins, transferring amino acids to the ribosome to form proteins, and also translating the transcript into proteins. Nucleic acids were discovered in 1868 (some sources indicate 1869) by Johann Friedrich Miescher (1844-1895), who called the material 'nuclein' since it was found in the nucleus. It was later discovered that prokaryotic cells, which do not have a nucleus, also contain nucleic acids. The role of RNA in protein synthesis had been suspected since 1939, based on experiments carried out by Torbjörn Caspersson, Jean Brachet and Jack Schultz. Hubert Chantrenne elucidated the messenger role played by RNA in the synthesis of proteins in ribosome.
The sequence of the 77 nucleotides of a yeast RNA was found by Robert W. Holley in 1964, winning Holley the 1968 Nobel Prize for Medicine. In 1976, Walter Fiers and his team at the University of Ghent determined the complete nucleotide sequenceDNA Bases Bio TechnologyDeoxyribonucleic acid, or DNA is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all living organisms. The main role of DNA is the long-term storage of information and it is often compared to a set of blueprints, since DNA contains the instructions needed to construct other components of cells, such as proteins and RNA molecules.
The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. Chemically, DNA is a long polymer of simple units called nucleotides, which are held together by a backbone made of alternating sugars and phosphate groups. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription. Most of these RNA molecules are used to synthesize proteins, but others are used directly in structures such as ribosomes and spliceosomes.
Within cells, DNA is organized into structures called chromosomes and the set of chromosomes within a cell make up a genome. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms such as animals, plants, and fungi store their DNA inside the cell nucleus, while in prokaryotes such as bacteria it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA, which helps control its interactions with other proteins and thereby control which genes are transcribed Biotechnology Indroduction. The convention recognized for the first time in international law that the conservation of biological diversity is "a common concern of humankind" and is an integral part of the development process.
The agreement covers all ecosystems, species, and genetic resources. It links traditional conservation efforts to the economic goal of using biological resources sustainably. It sets principles for the fair and equitable sharing of the benefits arising from the use of genetic resources, notably those destined for commercial use. It also covers the rapidly expanding field of biotechnology through its Cartagena Protocol on Biosafety, addressing technology development and transfer, benefit-sharing and biosafety issues. Importantly, the Convention is legally binding; countries that join it('Parties') are obliged to implement its provisions .
Apply Bio Technology Science The convention reminds decision-makers that natural resources are not infinite and sets out a philosophy of sustainable use. While past conservation efforts were aimed at protecting particular species and habitats, the Convention recognizes that ecosystems, species and genes must be used for the benefit of humans. However, this should be done in a way and at a rate that does not lead to the long-term decline of biological diversity The convention also offers decision-makers guidance based on the precautionary principle that where there is a threat of significant reduction or loss of biological diversity, lack of full scientific certainty should not be used as a reason for postponing measures to avoid or minimize such a threat. The Convention acknowledges that substantial investments are required to conserve biological diversity. It argues, however, that conservation will bring us significant environmental, economic and social benefits in return.In this situation, your range of choices is very broad and many packages will meet these limited.
RNA is implicated as being part of the germline. If confirmed, this result would significantly alter the present understanding of genetics and lead to many question on DNA-RNA roles and interactions.RNA Deatiles,ScienceRibonucleic acid (RNA) is a nucleic acid polymer consisting of nucleotide monomers, that acts as a messenger between DNA and ribosomes, and that is also responsible for making proteins out of amino acids. RNA polynucleotides contain ribose sugars and predominantly uracil unlike deoxyribonucleic acid (DNA), which contains deoxyribose and predominantly thymine. It is transcribed (synthesized) from DNA by enzymes called RNA polymerases and further processed by other enzymes.
RNA serves as the template for translation of genes into proteins, transferring amino acids to the ribosome to form proteins, and also translating the transcript into proteins. Nucleic acids were discovered in 1868 (some sources indicate 1869) by Johann Friedrich Miescher (1844-1895), who called the material 'nuclein' since it was found in the nucleus. It was later discovered that prokaryotic cells, which do not have a nucleus, also contain nucleic acids. The role of RNA in protein synthesis had been suspected since 1939, based on experiments carried out by Torbjörn Caspersson, Jean Brachet and Jack Schultz. Hubert Chantrenne elucidated the messenger role played by RNA in the synthesis of proteins in ribosome.
The sequence of the 77 nucleotides of a yeast RNA was found by Robert W. Holley in 1964, winning Holley the 1968 Nobel Prize for Medicine. In 1976, Walter Fiers and his team at the University of Ghent determined the complete nucleotide sequenceDNA Bases Bio TechnologyDeoxyribonucleic acid, or DNA is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all living organisms. The main role of DNA is the long-term storage of information and it is often compared to a set of blueprints, since DNA contains the instructions needed to construct other components of cells, such as proteins and RNA molecules.
The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. Chemically, DNA is a long polymer of simple units called nucleotides, which are held together by a backbone made of alternating sugars and phosphate groups. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription. Most of these RNA molecules are used to synthesize proteins, but others are used directly in structures such as ribosomes and spliceosomes.
Within cells, DNA is organized into structures called chromosomes and the set of chromosomes within a cell make up a genome. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms such as animals, plants, and fungi store their DNA inside the cell nucleus, while in prokaryotes such as bacteria it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA, which helps control its interactions with other proteins and thereby control which genes are transcribed Biotechnology Indroduction. The convention recognized for the first time in international law that the conservation of biological diversity is "a common concern of humankind" and is an integral part of the development process.
The agreement covers all ecosystems, species, and genetic resources. It links traditional conservation efforts to the economic goal of using biological resources sustainably. It sets principles for the fair and equitable sharing of the benefits arising from the use of genetic resources, notably those destined for commercial use. It also covers the rapidly expanding field of biotechnology through its Cartagena Protocol on Biosafety, addressing technology development and transfer, benefit-sharing and biosafety issues. Importantly, the Convention is legally binding; countries that join it('Parties') are obliged to implement its provisions .
Apply Bio Technology Science The convention reminds decision-makers that natural resources are not infinite and sets out a philosophy of sustainable use. While past conservation efforts were aimed at protecting particular species and habitats, the Convention recognizes that ecosystems, species and genes must be used for the benefit of humans. However, this should be done in a way and at a rate that does not lead to the long-term decline of biological diversity The convention also offers decision-makers guidance based on the precautionary principle that where there is a threat of significant reduction or loss of biological diversity, lack of full scientific certainty should not be used as a reason for postponing measures to avoid or minimize such a threat. The Convention acknowledges that substantial investments are required to conserve biological diversity. It argues, however, that conservation will bring us significant environmental, economic and social benefits in return.In this situation, your range of choices is very broad and many packages will meet these limited.
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