BIOTECH 4
There are certain environmental factors that affect the bioremediation processes such as temperature, pressure, oxygen level, salinity, pH, turbulence, background concentration of inorganic nutrients and the type of contaminants. As expressed in the Congress Report most sea water is between -2 and 35¡C. Biodegradation has been recorded to be faster at the higher end of this scale. As the temperature decreases the rate decreases and sometimes decreases dramatically. Pressure seems to have an inverse effect. As pressure increases biodegradation rates have decreased. This apparently causes problems in deep oceans. Oxygen is one of the most important factors in the biodegradation process since most of the degradation is done by means of aerobic respiration. Usually it is not a limiting factor on or near surface of the ocean; however, as one goes deeper rates decrease. Factors such as pH, salinity and turbulence are known not to have major influences on the process. The presence of nutrients is one of the major factors affecting degradation. Since this has been noticed, scientists have been working on methods to solve this issue more than anything else. As a result there has been vast experimentation on fertilization techniques, as explained before. Another important factor would be related to the contaminant- its susceptibility and the complicating factors that accompany. Certain complicating factors as the National Research Council has put it, are unavailability of contaminants to the organisms, toxicity of contaminants to the organisms, presence of multiple contaminants and natural organic chemicals, incomplete degradation of contaminants and inability to remove contaminants to low concentrations. The susceptibility of the contaminant has been, however, of major concern. After long series of experimentation Hinchee and his colleagues have come to the following conclusion: within families of compounds, the more substituted the compound, the slower the rate at which the constituent was lost by degradation. Generally, these results are not inconsistent with the widely held view that the order of decreasing susceptibility to biodegradation of petroleum constituents has been n-alkanes> branched alkanes > low-molecular weight aromatics> cyclic alkanes> high-molecular weight alkanes> polar compounds. The order of biodegradation of petroleum products by Prince William Sound, Alaska microorganisms was hexadecane=napthalene> pristane> phytane> fluorenes> dibenzothiophene=phenanthrene> chrysene. It is important to note that the results have proven the general notion that phytane and pristane are difficult to degrade, incorrect.
One must view the advantages and the disadvantages of bioremediation before evaluating its efficiency. As is summarized in the Congress Report, bioremediation usually involves minimal physical disruption, has no significant adverse effects when used correctly, is helpful in removing certain toxic components of oil, offers a simpler and more thorough solution than mechanical technologies and is probably less costly than other approaches. However, it does not remain without disadvantages. It is of undetermined effectiveness for many types of spills, may not be appropriate at open sea but rather the coastlines, takes time to work, approach must be specifically tailored for each polluted site and optimization requires substantial information about spill site and oil characteristics.
What is Biotechnology?
Imagine a microorganism that will clean up ocean oil spills, a new antibiotic that can be quickly altered to combat drug-resistant strains of bacteria, or a cancer vaccine that will help prolong human life. This is biotechnology.
Biotechnology uses biological systems, such as bacteria, to create goods and services. And while it's been around for thousands of years - the use of yeast in the making of cheese, bread and beer is biotechnology - our growing understanding of DNA, the basic genetic material of living organisms, has ushered this technology into an era of new discovery.
Canada is a world leader in biotech products. From Frederick Banting and Charles Bests' discovery of life-saving insulin in 1921, to the development of 3TC, an anti-viral drug effectively used to prolong the life of people infected with the AIDS virus, in 1997, Canadians have made headlines with their biotech discoveries.
The use of a biological science to create a new product was revolutionized in the 1940s and '50s with the discovery of the role and structure of the DNA molecule. Today, the technology breaks down into six areas:
Genetic Engineering - Selective breeding and the manipulation of genes to change the characteristics of an organism. Cell Culture Technology - Growing animal and plant cells in a lab, using cultured cells as production systems. Cell Fusion Technology - Fusing cells using chemicals or electric shock to create hybrids, and using artificial cells as delivery mechanisms for new drugs and other therapies. Enzyme Technology - The use of enzymes, (enzyme proteins control all of the chemical reactions that make up life) to bio-convert, degrade or synthesize materials. Fermentation Technology - The ability to grow cells in very large quantities. Immobilization Technology - The ability to attach cells, enzymes and DNA to solid, inorganic surfaces.
According to the Biotechnology Human Resource Council, the term biotechnology was first used in Canada in 1983 to describe the "manipulation of hereditary traits manually by transferring genes from one organism to another."
Modern biotechnology refers to the tools used to identify, understand and take advantage of genetic variations in animals, plants or humans. Researchers study genes and DNA (deoxyribonucleic acid) to understand complicated biological processes that will hopefully uncover the causes of certain human diseases.
The knowledge gained from researching human genes is used to develop new and innovative ways to help and heal people suffering from a variety of diseases and to improve our quality of life.
Biotechnology has the potential of great benefits to humankind through innovations in food production, pharmaceuticals and general genetic research. Many of these areas are controversial due to a variety of deeply held beliefs, and legislation to control types of research and public information have become hot political issues throughout the world.
Biotechnology can be described in many different ways, but a straightforward definition is :
Biotechnology is the use of biological organisms or enzymes in the synthesis, breakdown or transformation of materials in the service of people.
The polymerase chain reaction has enabled scientists to make large quantities of DNA from tiny samples. This in turn has made much of the most recent and exciting DNA technology possible. The ability to sequence DNA, identifying genes and what they do, the Human Genome Project, and DNA fingerprinting – all depend for their success on this ingenious technique.
The Human Genome Project, which has sequenced the whole of the human DNA, has been a massive international effort of biotechnology. The results are being used to help design very specifically targeted pharmaceutical molecules. These will provide better treatments which can be given at lower doses. What is more, these new medicines should have relatively few side effects because they will work with our individual genetic makeup rather than against it.
The new DNA technologies are helping scientists develop many new medicines which should enable us to treat many diseases more effectively. Knowledge of the human genome is also making it easier to test for genetic diseases. Gene probes have been developed to test for known genetic disorders. What’s more, in the future it seems likely that doctors will be able to find out our genetic tendency to develop diseases like cancer and heart disease. This in turn will help us to make lifestyle plans to help us remain healthy – choosing our diets, our exercise levels and our jobs to make sure we avoid situations our genes are not well-equipped to cope with.
Biotechnology is the use of living things, or life processes to make something useful. The term was coined in 1915, and understood to include such ancient practices as brewing and baking, cheesemaking, selective breeding of plants and livestock, and later, vaccine and pharmaceutical development. In recent years (since the late 70's) it has come to indicate the application of a much more sophisticated set of techniques and tools, as our understanding of how biological things work has rapidly accelerated (especially at the level of DNA). b, a, j, j, h. These tools and techniques, taken from biochemistry, immunology, microbiology, cell biology and chemistry, are used to address a variety of problems. Public institutions and private companies are engaged in research, development and manufacturing of products and procedures in medicine (diagnostics and therapeutics), agriculture, and environmental science.
One area of biotechnology which nearly everyone has heard of is genetic engineering. We now have genetically-modified organisms ranging from bacteria to cows and sheep which produce life-saving medicines including vaccines, insulin and blood-clotting factors. Genetically-modified bacteria can even make the lung surfactant needed to save the life of a premature baby. Hundreds of thousands of people benefit from the chemicals these very special organisms produce.
Gene therapy is another area of medical biotechnology which is still in the early stages of development. The hope is that gene technology will help scientists develop ways to correct mistakes in the DNA code which lead to genetic diseases such as SCID (severe combined immunodeficiency).
Medicine also benefits from many sensitive tests which indicate the presence or absence of substances in body fluids. Biotechnological advances in the use of immobilised enzymes and monoclonal antibodies mean these tests have become increasingly rapid and accurate in recent years. A common example is the pregnancy test in the picture (left). This used to take weeks – now it can be done at home on the first day of a missed period and the results are ready in minutes!
Cloning – making genetically identical copies of an organism – is not new biotechnology. Gardeners and farmers have being cloning plants for centuries. The new developments which have caused much controversy involve the cloning of mammals, from sheep to pigs to humans. The technology holds out many exciting possibilities for producing herds of genetically modified animals making useful medicines in their milk. There is also the very real possibility of producing new tissues for transplantation which would not cause any rejection problems – because they would be cloned from the patient themselves. But there are some real concerns about the ethics of this work and society is still deciding what is the right way to go.
More controversial developments in biotechnology involve research into the use of stem cells. These cells, taken from the hollow ball of cells which make up the early human embryo, have the potential to grow and develop into new tissues or organs to replace others which are worn out or diseased. This area of biotechnology is still at a very early stage but the potential for medical advances is enormous. The use of human embryos means that it is also, for some people, very controversial.
Most of the developments in biotechnology have taken place in a very short space of time, beginning with the revelation of the structure of the DNA molecule 50 years ago. The biotechnology timeline shows just how rapidly DNA technology has developed.
What is biotechnology?
Biotechnology is a set of scientific tools which uses living things to solve problems and make products.
The use of yeasts and bacteria to make bread, beer, wine and cheese are techniques that have been used for centuries. Traditional plant and animal breeding techniques are of more recent origin. Such methods can be seen as 'biotechnology' even though the term biotechnology was not coined until 1917. So-called traditional biotechnology is also used to extract and purify active components from plants and animals to produce cosmetics, drugs and health foods.
Modern biotechnology uses new techniques which provide much more understanding of, and control over, living processes. These new approaches are producing applications such as genetically engineered crops, DNA fingerprinting and cloning.
An even newer development is the integration of biotechnology with other disciplines such as information and computer technology and materials science. This approach is producing novel products like biosensors, which provide physical objects with the sensory capabilities of living things, and gene chips in which many thousands of DNA fragments are attached to a chip for rapid screening of genetic characteristics.
The following pages in this booklet outline some of the technologies and applications that fit within the scope of biotechnology. Rather than covering the full breadth of biotechnology, this booklet focuses on the newer biotechnologies about which there may be significant social, ethical or safety issues. Many of these relate to gene technologies, such as genetic engineering.
Biotechnology is “any technique that uses living organisms or substances from those organisms, to make or modify a product, to improve plants or animals, or to develop microorganisms for specific purposes” (Office of Technology Assessment, United States Congress).
Although the term sounds contemporary, biotechnology is not new. Over 9,000 years ago, people discovered that microorganisms could be used to make bread, brew alcohol, and produce cheese. Although this process of fermentation was not thoroughly understood at the time, its use still constitutes a traditional application of biotechnology.
What is new, however, is the extent of applications and sophistication of biotechnology techniques currently employed. Researchers can manipulate living organisms and transfer genetic material between organisms. Genetic engineering, the specific modification or transfer of genetic material, underlies modern biotechnological innovation
These current applications of biotechnology are predominantly practiced in the fields of agriculture and medicine. Modern techniques allow for the production of new and improved foods. Virus resistant crop plants and animals have been developed and advances in insect resistance have been made. Biotechnology applications in the field of medicine have resulted in new antibiotics, vaccines for malaria, and improved ways of producing insulin. Diagnostic tests for detecting serious diseases such as hereditary cancers and Huntington’s chorea have been developed as well as ways of detecting and treating AIDS.
Biotechnology is also being applied in the areas of pollution control, mining and energy production. Genetically engineered microorganisms and plants are used to clean up toxic wastes from industrial production and oil spills. Biotechnology applications have also been introduced into the forestry and aquaculture industries. These strategies offer hope for conservation biologists. Genetic methods can be used to identify particular populations of endangered species. Thanks to biotechnology, minute traces of animal or plant remains can be used to track and convict poachers. Genetic analysis can help botanical gardens, zoos, and game farms improve their breeding programs by determining the genetic diversity of various plant and animal populations.
Overall, biotechnology has significantly impacted and improved the quality of life for people on this planet. And it doesn’t end there. Complementing the creative endeavors of researchers and engineers are the efforts to commercialize biotechnology products with the input of business management and marketing personnel. The expertise of intellectual property and patent lawyers are also a necessary component in the process. New career opportunities in the area of bioinformatics are on the increase.
An important part of biotechnology is food biotechnology. This involves steps from the food production process and delivery to consumption. Cheaper raw materials such as food enzymes, engineered products such as delayed-ripening tomatoes and low-saturated-fat soybean oil ensure that we are given fresher, tastier, and healthier food products at lower cost. Future developments in Biotechnology Gene therapy is a promising and active area of biotechnology. It refers to the application of genetic engineering techniques in curing diseases, especially hereditary diseases. g, g, l, i, k. Basically, a sick person may have genes from his father or mother that are defective, causing the disease. Genetic engineers can find ways to add a functional gene to the individual's cells so that he may be able to produce the needed substances that will stop the disease.
Biotechnology can be defined as the controlled and deliberate manipulation of biological systems (whether living cells or cell components) for the efficient manufacture or processing of useful products. The fact that living organisms have evolved such an enormous spectrum of biological capabilities means that by choosing appropriate organisms it is possible to obtain a wide variety of substances, many of which are useful to man as food, fuel and medicines. Over the past 30 years, biologists have increasingly applied the methods of physics, chemistry and mathematics in order to gain precise knowledge, at the molecular level, of how living cells make these substances. By combining this newly-gained knowledge with the methods of engineering and science, what has emerged is the concept of biotechnology which embraces all of the above-mentioned disciplines.
"Biotechnology is the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services" (Organization for Economic Co-operation and Development).(Takeoka GR, et al., eds. 1996)
This broad definition of biotechnology encompasses the use of traditional techniques such as the use of yeasts and bacteria for leavening, brewing, fermentation and culturing yogurt. Selective breeding of plants and animals to create more productive breeds is a form of biotechnology.
The term "biotechnology" for many people refers specifically to the genetic engineering techniques that have been developed in the past two to three decades. These techniques have beneficial applications in medicine, agriculture, food processing, food safety, waste management, and crime detection.
Biotechnology is a key technology for many industrial sectors including pharmaceuticals, diagnostics, agriculture and waste treatment. Through the application of biotechnology there have already been major developments in healthcare, including the production of new medicines, new methods for the large-scale production of existing medicines and new, more accurate diagnostic tests for diseases such as HIV. In other areas biotechnology is focusing on developing replacement processes that are less polluting and energy consuming, and provides a basis for sustainability, the concept by which Nature’s resources are recycled, thus protecting the environment.
Although the science of biotechnology has been used for some time, there is still some public confusion over the nature of the technology. In general terms, biotechnology is the use of biological processes to make useful products (including modified organisms, substances and devices). In healthcare alone, biotechnology products include those naturally produced by the body to fight disease, as well as products which are manufactured by chemical synthesis but have used biological processes and screening methods for their initial discovery.
In many ways, biotechnology is an old science. Without understanding the principles of fermentation or genetics, mankind has used some biotechnology processes since antiquity, for example, in the production of cheese, bread, alcohol, penicillin or the selective breeding of animals and plants.
It is also a new science in that, only since the 1970s, have advances in molecular biology and other sophisticated techniques enabled scientists to have a better knowledge of how individual cells and their components work in the body. This has enabled scientists to develop new methods for isolating genes and instructing cells outside the body to make large quantities of human proteins.
One of the results is medicines that are based on - or even replicate - the body’s own disease-fighting mechanisms: medicines that work with the body rather than against it. Biotech medicines are already being used in the treatment of many problems, including cancers, anaemia, heart attacks, rheumatoid arthritis, viral infections and multiple sclerosis.
The techniques of biotechnology are also being used to produce, in large quantities, substances such as Factor VIII for the treatment of haemophilia, or human growth hormone to help overcome pituitary dwarfism. Diabetes can also be treated with a biotechnology-produced version of human insulin to overcome the deficiency in sufferers. Previously, these materials had to be extracted from human and animal tissue with some difficulty and with potential risks for the patient. Biotechnology has provided a better alternative. One of the first great successes of biotechnology in healthcare has been to create a safer and more effective vaccine against Hepatitis B. New types of therapeutic vaccines are being developed to treat, rather than prevent disease, including HIV, herpes and various cancer-causing infections. Biotechnology is also leading to the development of new medicines for diseases, which, until now, have been otherwise untreatable. Beta-interferon, for example, is giving new hope to multiple sclerosis patients, where before there has been no hope of treatment.
Biotechnology is likely to come up with new therapies for many more unconquered diseases. The technology will help in the earlier diagnosis of disease and will help to identify more accurately gene abnormalities that may lead to the treatment of certain hereditary diseases.
In agriculture, biotechnology has the promise to produce foods with enhanced nutrition and flavour, and crops with increased yield. Most of today’s hard cheese products are made with an enzyme chymosin, which is produced by biotechnology. In the UK the first recombinant tomato paste was launched in supermarkets earlier this year. Biotechnology has the potential to make crops more nutritious, to taste better, last longer and naturally resist insects, viruses and herbicides. This can reduce our dependence on chemical pesticides. Therefore, Biotechnology can contribute to solving some of the World’s food problems.
What Is Biotechnology And Why Do We Need It?
Biotechnology refers to the techniques that allow scientists to modify DNA, the genetic material of living organisms, to enhance their tolerance to pests and diseases, increase yield and improve quality and nutritional value. Biotechnology can bring many benefits to medicine, the environment and industry. It also has a wide range of possible applications in food and agriculture. The benefits to agriculture include:
Improving Crop Yield
Genetically improved plants (GIPs) have been developed to be more tolerant to disease, weeds insects, and drought and be able to grow in difficult environmental conditions. Considering the devastating impact of pests, weeds and disease on yields, the agronomic and economic benefit when plants have built in tolerance is enormous.
Increasing a crop's yield enables us to use less land to produce the same amount or more food. This also allows us to preserve other lands such as native forests and delicate ecosystems for the benefit of the environment and wildlife. Without increasing crop yields more and more non-arable land will be brought into production to feed the growing population. In many cases, this land is unsuitable for long term cultivation and its use will result in environmental degradation, or the destruction of the few remaining wild lands and native forests.
Less Chemical Usage
Biotechnology can also help farmers reduce the amount of pesticides they need to use on crops. For instance, by making crops tolerant to a specific herbicide, weeds can be killed without damaging the crop. The amount of herbicide used per acre of crop can also be reduced relative to regular practices. Insect-tolerant crops not only reduce the volume of insecticide sprayed but also encourage natural and biological control by not affecting the beneficial insects. Insect-tolerant crops can form the basis of extremely effective Integrated Pest Management practices.
Improved Food Quality
Biotechnology can give our food improved quality characteristics. Scientists have the ability to improve the taste, appearance and the nutritional quality of fruits and vegetables.
Another advantage created by biotechnology is genetically improving the gene responsible for ripening. For example, delayed-ripening tomatoes reduce the waste that occurs during transport. In some countries, up to 20% of fresh produce is destroyed on the way to market due to unsuitability of these crops for transport. In India, where the food transportation sector is not completely developed and huge losses occur, delayed-ripening fruits and vegetables would benefit consumers living far from agricultural areas.
Environment Friendly
Among the environmental benefits of biotechnology is a reduction in the use of pesticides and herbicides, the prospect of more food production from the same unit of land and more nutritious food produce. In addition, scientists have developed methods utilizing biotechnology to clean up pollution (bioremediation), caused by, for instance, oil spills.
Biotechnology can be defined as making use of the natural processes or products of living things. This covers most of what we think of as biotechnology:
medical biotechnology, which uses microorganisms (such as bacteria or fungi) to make antibiotics or vaccines
industrial biotechnology, which uses microorganisms to make enzymes (e.g. to add to biological washing powders), or to produce beer, cheese or bread
environmental biotechnology, which uses microorganisms or plants to clean up land or water that is polluted with sewage or industrial waste and
agricultural biotechnology which aims to produce better crops, ‘natural’ fertilizers, or feed additive New technologies, discoveries and a better understanding of the natural processes of living organisms are extending this list almost daily to give opportunities for developing new medicines and improved products for agriculture and industry.
Defining biotechnology in agriculture and food production When we consider agriculture and food production, ‘biotechnology’ is more difficult to define. How is making milk from cows, or flour from wheat different from making antibiotics from bacteria? All these products are normally produced in Nature, but we have harnessed the natural ability of organisms that produce them for our own use. By breeding plants and animals, or choosing the best antibiotic-producing strains of bacteria or fungi, we have selected organisms with the genetic make-up that gives them the ‘improved’ characteristics we desire.
‘Traditional’ biotechnology
‘Traditional’ biotechnology is nearly as old as humankind: brewing, breadmaking and cheesemaking are all ancient ways of using natural fermentation processes of microorganisms to either preserve food produced through agriculture, or create new tastes and textures – indeed, entirely new foods. These same processes of microorganisms are used today for a wider range of applications, to produce valuable products such as antibiotics or enzymes for medicine or industry, too.
‘Modern’ biotechnology
‘Modern’ biotechnology attempts to achieve the same goals – new or better products – but more efficiently. Because we can now identify which genes code for particular characteristics, and can move the genes from one organism to another, we can achieve the most useful combination of genes in plants more quickly by genetic modification than by breeding or selection. Sometimes, genes are moved between closely related organisms – for example, from a plant that makes a particularly valuable product but only in small amounts, to a plant that has the machinery to make vast amounts of product. Alternatively, genes can be moved between very different organisms: genes for human insulin moved into bacteria provide a cheap, safe and plentiful supply of insulin to treat diabetics.
Literally speaking, biotechnology refers to the application of our knowledge of biological phenomena to serve our economic and social needs. However, this extremely broad definition is not very useful, as it embraces almost all aspects of our society from agriculture to beer and wine fermentations, to medicine, cheese and bread making, plant and animal breeding, cloning, embryo transfer, and even mining of certain metals with bacteria.
A more modern and focused definition which is more consistent with current usage would limit the term to the application of modern techniques of molecular biology and bioinformatics to solve industrial and scientific problems. However, this definition is still quite broad as it includes such applications as transgenic animals and plants, marine natural products, recombinant bacteria and fungi for the production of proteins, comparative genomics for research into evolutionary relationships and the genetic basis of disease, gene arrays for the study of gene expression and developmental biology, and the myriad applications of cell and tissue culture in research and industry.
If you were to ask 20 people this question, you would probably receive 20 differing responses. That's because biotechnology is a collection of technologies that are being deployed across a whole host of industries including: agriculture, environment, food, forestry, health, and mining.
This section is designed to provide a background to what biotechnology entails and why there is so much excitement about the biotechnology industry in general. Considering all that has been written about the industry this introduction is not meant to be all embracing. Instead, we have provided useful links in order for you to delve further into both the science and the industry itself.
Biotechnology deals with the manipulation of the DNA molecule in very precise and directed ways, generally in the context of industrial or commercial applications. The common element to the technology is the use of cells, cellular processes, and the manipulation of molecular events, in ways that achieve predictable and reproducible results. Biotechnology has many goals, including:
J Appl Microbiol, 1997 Apr, 82(4), 441 - 7 Biodegradation of aniline, anthracene, chlornitrophen, fenitrothion and linear alkylbenzene sulphonate in pond water; Nishihara T et al.; Biodegradation of five chemicals (aniline, anthracene, chlornitrophen (CNP), fenitrothion (FNT) and linear alkylbenzene sulphonate (LAS)) by aquatic bacteria in three different types of ponds was determined according to the cultivation method developed by this group . The degradability toward these chemicals was varied among the ponds, except for LAS which was decomposed well in all samples . Higher degradability towards the two agrochemicals, CNT and FNT, was found in the pond surrounded by paddy fields, whereas aniline and anthracene were decomposed more rapidly in the pond located in the industrial area . Water from the pond in the botanical garden, with the least exposure to any chemicals, exhibited the lowest degradation toward all chemicals tested . There was no significant seasonal variation in the biodegradation of chemicals in these ponds . It was deduced that biodegradability toward certain chemicals could be a result of acclimatization of the microbial community by chemical contamination present and past, suggesting the possible use of biodegradation profiles as an indicator for chemical pollution in the aquatic environment.
Protein Expr Purif, 1997 Apr, 9(3), 346 - 54 Expression, purification, and characterization of a catalytically active human cytochrome P450 1A2:rat NADPH-cytochrome P450 reductase fusion protein; Parikh A et al.; An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression in Escherichia coli . A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5' end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3' end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser-Thr . This fusion plasmid was expressed in E . coli and the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP-agarose, and hydroxylapatite chromatographies . The purified protein has the spectral characteristics of human P450 1A2 and cytochrome c reduction activity comparable to rabbit NADPH-P450 reductase . The fusion protein catalyzed 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation to appreciable levels in the presence of NADPH and phospholipid . While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components . Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase in E . coli enhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.
Chemosphere, 1997 Apr, 34(8), 1813 - 22 The ISO Headspace CO2 Biodegradation Test; Battersby NS; Two published methods for evaluating the aerobic biodegradability of organic compounds by measuring inorganic carbon (CO2) production in sealed vessels, under a headspace of air were combined into one protocol . This was ring tested in 1995 by the International Organization for Standardization (ISO) using aniline and 1-octanol as the test substances . This paper describes the ISO method and discusses the results of the inter-laboratory calibration exercise in terms of the method's precision and performance.
Biomaterials, 1997 Apr, 18(7), 567 - 75 In vitro and in vivo degradation of films of chitin and its deacetylated derivatives; Tomihata K et al.; Chitin was deacetylated to various extents with NaOH to obtain partially and thoroughly deacetylated chitins . The specimens used in this study were deacetylated by 0 (chitin), 68.8, 73.3, 84.0, 90.1 and 100 mol% (chitosan) . Films with a thickness of 150 microns were prepared from these specimens by the solution casting method . The equilibrated water contents of the films were 52.4 (chitin), 73.8 (68.8 mol%), 64.2 (73.3 mol%), 61.8 (84.0 mol%), 57.8 (90.1 mol%) and 49.7 wt% (chitosan), while the tensile strengths of the water-swollen films were 244 (chitin), 197 (68.8 mol%), 232 (73.3 mol%), 320 (84.0 mol%), 293 (90.1 mol%) and 433 g mm-2 (chitosan) . The maximum water content and the minimum tensile strength observed for a specimen deacetylated between 0 and 68.8 mol% may be ascribed to the lowered crystallinity by deacetylation of chitin, since both chitin and chitosan are crystalline polymers . Unlike their physical properties, in vitro and in vivo degradations of these films occurred less rapidly without passing a maximum or minimum, as their degree of deacetylation became higher . The in vitro degradation was carried out by immersing the films in buffered aqueous solution of pH 7 containing lysozyme at 37 degrees C, while the in vivo degradation was studied by subcutaneously implanting the films in the back of rats . It was found that the rate of in vivo biodegradation was very high for chitin and 68.8 mol% deacetylated chitin, compared with that for the 73.3 mol% deacetylated chitin . The films which were more than 73.3 mol% deacetylated showed slower biodegradation . Interestingly, the tissue reaction towards highly deacetylated derivatives including chitosan was very mild, although they had cationic primary amines in the molecule.
Artif Organs, 1997 Apr, 21(4), 287 - 92 Myristoyl gelatin as a sealant for Dacron vascular prostheses; Sasajima T et al.; Myristoyl gelatin (MG) retains its gel structure at temperatures above body temperature without any crosslinking . As a coating material, MG adheres well to polyester fibers, and the outermost layers of the sealant that are in contact with blood or surrounding tissue become hydrophilic . We produced MG-impregnated knitted Dacron vascular prostheses (MG graft {MGG}) and investigated the usefulness of MG as a sealant by replacing the thoracic aorta of dogs . MGGs (5 cm long with an inner diameter of 10 mm) were implanted in 5 mongrel dogs (10-20 kg), and the grafts were retrieved at intervals of 4 h and 2, 4, 8, and 15 weeks after grafting . There was no thrombus formation on the flow surface of the MGGs, indicating adequate antithrombogenic properties . No resorption of MG occurred until after 2 weeks, and neither immune reaction nor excessive foreign body reaction was noted . Fragmentation of the sealant induced by cell infiltration began to occur at 4 weeks, yet the sealing effect persisted . The organization of MGG was almost complete at 8 weeks . Because of its pliability and effective adhesion to polyester fibers, its antithrombogenicity, and the persistent sealing effect due to delayed biodegradation and resorption, we conclude that MG is an extremely useful sealant for polyester vascular prostheses.
J Biomed Mater Res, 1997 Mar 15, 34(4), 519 - 30 Role of oxygen in biodegradation of poly(etherurethane urea) elastomers; Schubert MA et al.; It is generally accepted that biodegradation of poly(etheruethane urea) (PEUU) involves oxidation of the polyether segments on the surface where leukocytes are adhered . The influence of dissolved oxygen, which is known to control oxidation of polymers in more traditional environments, was explored in this study . Specimens treated in vitro with hydrogen peroxide-cobalt chloride for 12 days exhibited a brittle, degraded surface layer about 10 microm thick . Attenuated total reflectance-Fourier transform infrared spectroscopy of the surface revealed that the ether absorbance at 1110 cm(-1) gradually decreased with in vitro treatment time to 30% of its initial value after 12 days . In contrast, 6 days in vitro followed by 6 days in air produced a decrease to 12% of the initial volume . Therefore, removing a specimen from the in vitro solution after 6 days and exposing it to air for the remainder of the 12 days actually resulted in more oxidation than leaving it in the in vitro solution for the entire 12 days . These results suggest that PEUU degrades by an autooxidation mechanism sustained by oxygen . By successfully modeling the depth of the surface degraded layer with a diffusion-reaction model, it was demonstrated that PEUU biodegradation is controlled by diffusion of oxygen into the polymer.
J Biomed Mater Res, 1997 Mar 15, 34(4), 493 - 505 Comparison of two antioxidants for poly(etherurethane urea) in an accelerated in vitro biodegradation system; Schubert MA et al.; Vitamin E (+/-alpha-tocopherol) was recently investigated as an antioxidant for implanted poly(etherurethane urea) (PEUU) elastomers . In that work, vitamin E prevented chemical degradation of biaxially strained PEUU up to 5 weeks implantation, and prevented pitting and cracking of the PEUU surface for the duration of the 10-week cage implant study . The promising results of the in vivo studies motivated a detailed comparison of vitamin E with Santowhite, the standard antioxidant used in PEUU elastomers . To evaluate vitamin E and Santowhite as antioxidants in PEUU, an accelerated in vitro treatment system was used that mimics the in vivo degradation of PEUUs . Vitamin E was even more effective than Santowhite in preventing pitting and cracking to the biaxially strained PEUU elastomers . The inhibition of ether oxidation was greater with vitamin E than with Santowhite when compared by equivalent concentrations and molar concentrations, respectively . It is hypothesized that the increased effectiveness of vitamin E in this system, compared to Santowhite, is due to differences in antioxidant mechanism(s) . Vitamin E is more efficient in preventing PEUU oxidation than Santowhite because its phenoxy radical is more stable and it can terminate more than one chain per vitamin E molecule.
Chem Biol, 1997 Mar, 4(3), 215 - 21 Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase; Yeung BK et al.; BACKGROUND: Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase . The Mn-binding site in MnP is of particular interest . Oxidation of Mn(II) to Mn(III) is a key step in the biodegradation of lignin, a complex phenylpropanoid polymer, as well as many aromatic pollutants . Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese . Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure and function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds . RESULTS: Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (Gly41-->Glu, Val45-->Glu, His181-->Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity . Titrating MnSO4 into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP . The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CCP were also similar to MnP . Titration with MnSO4 resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP . None of the spectral features were observed in wild-type CcP . In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP . CONCLUSIONS: A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP . The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.
Chem Biol, 1997 Mar, 4(3), 169 - 74 Diverse mechanistic approaches to difficult chemical transformations: microbial dehalogenation of chlorinated aromatic compounds; Copley SD; Chlorinated aromatic compounds represent an important class of environmental pollutants . Microbial dehalogenases play a crucial role in the biodegradation of these compounds . The three major classes of aromatic dehalogenases are discussed in this minireview.
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