Friday, February 29, 2008

Human Coagulation factor VII in Fish

A human blood-clotting factor used to treat some people with haemophilia and accident victims suffering serious bleeding has been produced using genetically modified fish.
There is still a long way to go before any product reaches the market, but if the fish project is a commercial success many other proteins might be made in this way.
"We have a list of 20 other human therapeutic proteins that could be produced via fish to treat lung disease, liver problems, even tumours," says Norman Maclean of the University of Southampton in the UK.
Maclean has been working on producing human coagulation factor VII in fish together with AquaGene of Alachua, Florida. Factor VII can be purified directly from human blood, but there is a risk of diseases being transmitted this way.
The only alternative, called NovoSeven, is produced using genetically modified hamster cells. But growing mammalian cells is very expensive, and the cost of a single injection can be as high as $10,000.
Gunshot wounds
Factor VII is used to treat people with a rare form of haemophilia that means they cannot make the protein themselves, and it is often needed to treat other forms of the disease as well.
Many doctors, including US army medical staff in Iraq, are now also using it to stem internal bleeding caused by accidents or gunshot wounds, even though NovoSeven is not approved for this purpose.
AquaGene is hoping to produce a much cheaper rival product using tilapia, a fast-growing freshwater fish widely farmed for food. Maclean has now managed to produce several lines of transgenic tilapia that produce human factor VII.
His team added a genetic switch from the tilapia to the human gene. This ensures that the gene is switched on in the liver of modified fish, and the protein secreted into the blood.
"Each millilitre of human blood has about 500 nanograms of the protein. We were able to match that yield in the blood of our fish," says Maclean. He hopes to produce tilapia that will make 10 times that level within a year.
Silkworm larvae
The next step will be to convince regulators that the fish-derived protein is the same as the human form, and that it is safe. The researchers have already tested it on samples of blood taken from patients with haemophilia, but many more studies will have to be done.
Other groups are exploring rival ways of producing proteins, from plants and chicken eggs to silkworm larvae and cattle, but Maclean thinks fish are a serious contender.
There is no evidence that any disease can be transmitted from fish to humans, for starters. Transgenic fish are also relatively cheap and easy to make, whereas it can cost millions to produce transgenic cattle.
Because tilapia breed so quickly, production could easily be adjusted to meet demand. "But escape is a concern," says John Matheson of the US Food and Drug Administration. For commercial production, transgenic tilapia could be grown in contained facilities.

Blood-staunching bandages

Conventional gauze bandages do not work well enough because, although they absorb blood, they do not prevent its flow. Thomas Fischer and colleagues at the Francis Owen Blood Research Laboratory at the University of North Carolina at Chapel Hill think they may have the solution.
His team has discovered that bandages made from about 65% glass fibre and 35% bamboo fibre not only absorb blood but also stimulate the body's ability to staunch the flow by triggering the release of blood-clotting factors such as thrombin or fibrinogen. They say the bandages work even better if they are themselves impregnated with blood-clotting factors.
Given the number of military casualties in Afghanistan and Iraq this is an idea that could well be fast-tracked. The idea is part-owned by a company called Entegrion, which was co-founded by Fischer. So all the pieces are in place for the bandages to be commercialised soon.
Read the full patent application for blood-staunching bandages.

Shrimp-shell wound healant to get space test

A biopolymer produced from shrimp shells that has proved invaluable in treating wounded soldiers will be put to a new test in August – aboard the space shuttle Endeavour.
A commercial experiment will assess how the material, called chitosan, affects human immune cells in space, where they are less responsive than on the ground.
The US Army equips its troops in Iraq with chitosan-laden bandages both to speed blood clotting in fresh wounds, and to stop bacterial infections. NASA does not expect astronauts to fight battles in space, but has to plan for accidents, and worries that slow healing or infection of wounds could imperil long-duration missions to Mars or other distant targets.
The test also could pay dividends on the ground. They are being paid for by Hawaii Chitopure, which makes the highly purified chitosan used in military bandages and hopes to show that chitosan can reduce inflammation, as well as kill bacteria. That could reduce scarring and other harmful byproducts of immune response, says Shenda Baker, a chemist at Harvey Mudd College in California, US, and president of BioSTAR west, the company which will carry out the experiments.
"While mammalian cells don't like microgravity, bacteria grow very well," Baker told New Scientist. The best-known example was the thriving colony of terrestrial bacteria that contaminated the Russian Mir space station so badly that cosmonauts became sick.
Biofilms have also been found on the space shuttle. Without help from materials like chitosan, bacteria could overwhelm mammalian immune systems during long space missions.
Charged exoskeleton
Chitosan is a water-soluble form of chitin, an abundant long-chain natural biopolymer that is a key component of the semi-transparent exoskeletons of arthropods from insects to lobsters, and in the cell walls of fungi.
Some researchers believe natural chitin helps protect arthropods from bacterial infection, important because they lack a conventional immune system. The soluble chitosan carries a positive charge that attracts the negatively charged membranes of bacteria, stopping them from multiplying and in some cases killing them. The charge also initiates clotting of red blood cells.
Baker's experiment will monitor the behavior of human monocytes, a type of white blood cell that rapidly responds to wounds and infections.
Three sets of monocyte samples will fly on the shuttle – one mixed with fragments of bacterial cell walls known to trigger immune reactions, one mixed with chitosan alone, and a third mixed with both chitosan and bacterial fragments. Microarrays will monitor activity of the monocytes, including the genes activated, proteins produced, and short chains of RNA that mediate gene activity.
By comparing the space cultures with identical samples from the ground, Baker and her colleagues at California-based BioSTAR West hope to learn how the immune system turns on and off in both environments.
Their key goal is to see if chitosan can stop the bacterial threat well enough to block the immune system's normal inflammatory response, which causes scarring and other harmful effects.

Life after death for empty shells: Crustacean fisheries create a mountain of waste shells, made of a strong natural polymer, chitin. Now chemists are helping to put this waste to some surprising uses

Chitin is the main structural component of the shells of crustaceans, molluscs and insects. It also makes up parts of the jaws and body spines of certain worms, and is found in the cell walls of fungi and in some algae. Henri Bracannot was the first to describe chitin - he called it fungine - as long ago as 1821. We now know that it is a natural polymer that strongly resembles cellulose, the main component of plant cell walls. Chitin is almost as common as cellulose - an estimated billion tonnes are synthesised every year - and this ubiquity holds a clue to some of its potential uses.
At first sight, however, chitin does not look at all promising. Chemically, it is a fairly dull molecule. Like cellulose, it can be broken down by enzymes, but only slowly, and it will not dissolve in most ordinary solvents like water or alcohol. It is usually bound to porteins to form large, complex molecules and its purity varies enormously. Even chitin from the same animal varies in the length of its molecular chain, its cyrstalinity, and in the number of acetyl (CH3C)O groups hanging off the chain.
But in 1959, a chemist called Rouget found that heating chitin with a very concentrated sodium hydroxide converts it to a related and much more useful chemical, called chitosan. This reaction removes some of the acetyl groups from the molecular chain, leaving behind complete amino (NH2) groups (see Figure). Increasing the temperature or the strength of the sodium hydroxide solution removes more acetyl groups. In this way chemists can produce a range of chitosan molecules with different properties and applications. Unlike chitin, chitosan dissolves easily in acidic solvents like acetic acid.
Chitosan's versatility depends almost entirely on its amino (NH2) groups. When dissolved in acids, these groups add proton, becoming (NH3)+ and giving chitosan a positive electrical charge overall. This makes the molecule extremely effective for removing negatively charged particles that are dissolved or suspended in water, such as lignosulphates and natural tannins. Chitsan form ionic, or sometimes hydrogen bonds with these molecules, desotabilising the suspension so that they precipitate out as insoluable solids.
One of the earliest uses of chitin was to purify waste water from the processing of shellfish. Processing plants produce contaminated water as well as solid waste, such as shells and viscera. Crustacean fisheries are very wasteful - up to 85 per cent by weight of each animal is thrown away, which amounts to over 3 million tonnes of solid waste every year. Some fisheries already use chitin derived from the solid waste to purify their own waste water. A study of one crawfish processing plant in Louisiana in 1989 showed that chitosan derived from the waste could be used to remove 97 per cent of the solids suspended in waste water this way.
Now some companies are promoting chitin for the clarification and purification of other types of contaimined water. Chitin and chitosan are also good chelators. This means they can bind at several points, rather like the grip of a claw, to metal atoms in solution, especially heavy metals such as mercury, lead and uranium, although no one knows quite how. This useful property could be exploited as the basis of a method for treating waste water that is toxic or radioactive. Japanese firms such a Kurita Industries sell chitosan as a flocculant. So does the Norwegian company Protan, which recommends it for the clarification of swimming pools and spas as its flocculates microbes and removes metals.
Waste-water treatment is only one of many suggested and proven uses for chitin and chitosan. Of these, cosmestics is one of the longest established. Chitin was first used in cosmetics in 1969; more recently, Japanese and German companies have been developing chitosan salts - soluble in water, and formed simply by treating chitosan with acid - for use is cosmetics for skin and hair. The German cosmetics giant Wella has been researching chitosan as hair treatment for 10 years. It has experimented with the film-forming properties of chitosan in hair sprays and nail varnishes and uses its thickening effects in creams and conditioners. In Japan, at least five companies manufacture chitin and chitosan, mainly from crab shells.
Chito-Bios of Ancona in Italy sell N-carboxybutyl chitosan, under the trade name EvalsanR, for shampoos, bath foams, liquid soaps, toothpaste, personal-hygiene detergent and face creams. The company uses this derivative of chitin as a replacement for hyaluronic acid, a common component of creams and lotions. It emphasises that chitosan is 'more than a comestic ingredient' and could be useful for dressing wounds, for surgery and dentistry.
But perhaps the greatest potential application is paper manufacture. Adding only 1 per cent by weight of chitin to pulp increases the strength of the paper, speeds up the rate at which water drains from the pulp and increases the quantity of fibres retained when making sheets of paper. So manufacturers can use cheaper, weaker fibres, without reducing quality, while saving up to 90 per cent of the energy they use to beat the pulp. Chitin also makes the paper easier to print on.
Paper that incorporates chitin has greatly improved wet strength - an advantage for diposable nappies, shopping bags and paper towels. But these benefits must be offset against the problems of supply. The current world production of chitin from all sources would be overwhelmed by an industry which in 1986 produced 172 million tonnes of newsprint. Any move towards the general use of chitin in paper manufacture would require a huge increase in chitin production. Where would it come from?
Maintaining fisheries of shellfish or molluscs just to harvest their chitin is unlikely to be economic, as they only contain around 1 per cent chitin by weight. This leaves two possible sources of chitin: shellfish waste and fungal fermentation. The pharmaceutical industries of most countries already exploit molecules, including vitamin C and penicillin. The process also produces large quantities of chitinous waste - estimates are difficult to find, but in 1977, one researcher gave a figure of 790,000 tonnes. Unlike shellfish waste, this source of chitin is predictable - a set input will produce a set output - and its quality can be controlled.
Several countries, including the US, Japan, Norway, Italy and India, already have chitin/chitosan plants based on shellfish waste as their source. The little they produce is used by the pharmaceutical industry and in the treatment of waste waters. There are no reliable data for how much chitin should in theory be available from crustacean fisheries, but according to the FAO's latest figures, in 1987 the world crustacean harvest was 3.69 million tonnes. Assuming chitin forms 1 per cent of the wet weight of a crustacean, on average, we are squandering about 36,700 tonnes of chitin each year as waste from the processing of shrimps, prawns, lobsters and crabs. The main problem is that it would be unecomomic to collect the waste from many small processing plants, so this source can only be tapped where large quantities of crustaceans are being handled.
The largest potential source of animal chitin is the zooplankton that inhabit the upper layers of the sea. But only one crustacean which could be loosely considered a member of the zooplankton is currently being harvested to any significant degree. This is the Antarctic krill. In 1989/90, fishing fleets caught 375,000 tonnes, making it the largest crustacean fishery in the world ('Who's counting on krill?', New Scientist, 11 November 1989). The krill fishery is only marginally economic. Most of the catch is either processed for its tail meat, which is destined for human consumption, or is used whole for aquaculture.
Peeling krill is no easy task and leaves 85 per cent by weight as waste. Of this waste, 85 per cent is recoverable protein. Almost a quarter of the deproteinised waste is chitin, compared wiht 3.2 per cent in the whole animal. About 90 per cent of this chitin can be recovered by conventional extraction techniques. This is half as efficient again as from crab chitin, although the figure would not be so high on board a trawler.
The fishery will probably expand from its current levels and the total krill stock in the southern ocean is now thought to be between about 100 and 400 million tonnes. Several millions tonnes of this could be harvested annually. Such a catch would dominate the total world curstacean catch of about 4 million tonnes and would be a major potential source of chitin. Other sources include squid, whose pens are 40 per cent chitin and largely free of minerals and bivalve molluscs, whose shells oftain contain a high proportion of minerals. (Minerals add to the weight of material and therefore the cost of processing.) Insects have chitin but it is quinone tanned, which makes it difficult to extract, and there is no consistent source.
So it seems that the major source of chitin in the future will probably be biotechnology rather than seafood waste. The infant chitin/chitosan industry will probably develop by using cheap supplies of waste materials, but if demand increased sufficiently, manufacturers could develop genetically engineered microorganisms to produce these useful molecules. Cultured strains of microorganisms will be able to produce chitin with desired properties under controlled conditions and in fixed quantities. This would sever the link between chitin production and the widely fluctuating market for protein. Chitin is easily extracted from fungal hyphae and some species even produce up to 14 per cent by weight of chitosan. Culturing chitosan-producing strains would eliminate the deacetylation step that converts chitin to chitosan. Although this step is fairly simple, it makes chitosan nearly twice as expensive to product as chitin.
Certain algae produce pure chitin in the form of extracellular fibres which can be between 10 and 15 per cent of the dry weight of the cells and can be readily separated from the non-chitinous structures with a yield of 80 per cent. But these algae grow only slowly under normal conditions. Researchers hope that advances in biotechnology will give them fast-growing strains that retain large amounts of chitin.
Chitin and its derivatives are shaping up to be as versatile as plastics. Unfortunately, although chitin and its derivatives can do many things well, there are few functions that they alone can carry out. Chitin-based products usually have to compete with those produced by established biochemical technologies. On the other hand, a 'natural' material that uses up waste, is biodegradable and does not damage the environment may have a bright future.
* * *
Industry shells out for chitin
Many chemical, medical and pharmaceutical companies are now researching and in some cases developing and patenting chitin-based products. Protan, a Norwegian company, has been producing and selling chitin and chitosan from shellfish waste since 1984. It lists 13 broad areas for its products, from 'personal care' to detoxification of industrial waste.
Other applications include treatment of sewage, dairy waste, paper mill effluent, food-factory waste, liquid radioactive waste and purfication of drinking water. In Japan about 500 tonnes of chitin are used every year as a water purifier, and the US Environmental Protection Agency rates chitosan as acceptable for the purfication of drinking water.
Using chitosan to remove suspended solids from food-processing wastes, such as cheese whey, has an additional benefit. As well as purified effluent, the method yields coagulated by-products rich in proteins which can be added to feed for domestic animals. This seems to make the feed more digestible.
Chitin and its derivatives also have some very useful properties in the medical field. Between 1968 and 1975 researchers working for the American pharmaceuticals company Lescarden of Goshen, New York, filed five patents for the use of chitin and chitosan to accelerate wound healing. They found the chitin mats, fibres, sponges, sutures and films were much better than standard cartilage-based ones. The pharmaceuticals company Katakurachikkarin based in Hokkaido makes an artifical skin - a chitosan-collagen composite - that appears to enhance recovery from surgical wounds or burns. In 1983, doctors working for the Veterans Administration Medical Center in Omaha discovered that chitosan could also speed up blood clotting and used it to reduce the loss of blood following blood vessel grafts.
Chitosan can be produced in numerous forms - powder, paste, solution, film, fibre or spray - giving manufacturers huge scope for incorporating it into bandages, dressings, salves, sutures or disposable contact lenses. The body does not seem to reject these and they break down slowly to harmless carbohydrates, carbon dioxide and water. Because chitosan is absorbed completely in the body, it is an ideal carrier for drugs that must be released slowly. After tests on rats in 1978, some Japanese researchers claimed that chitosan reduces serum cholestrol. In Japan you can now buy biscuits and noodles sold for the alleged benefits of the chitin they contain.
The food industry is developing ways of exploiting the emulsifying properties of chitosan to make mayonnaise and peanut butter. Chitosan could eventually find its way into an area where non-toxic, high strength films are required, form sausage casings to oven wraps and food packaging.
Some researchers even think these chemicals will be the basis of a biodegradable plastic. Technics, the hi-fi manufacturer, of Schizuoka in Japan has even made the vibrators of flat-panel speakers from chitosan, an idea which is supposedly based on the acoustic properties of crickets' wings.