Occupational health problems in Silk production: A Review

Rakesh.K.Pandey

Regional Sericultural Research Station, Miransahib, Jammu, 181101, India

Abstract

 Even though, the Silkworm life cycle is eco friendly, silk industry involves certain health risks such as carbon monoxide poisoning in temperate area like Kashmir due to the use of burnt coal to raise room temperature, handling of diseased worms and excreta with bare hands, use of formaline and bleaching powder for disinfection, use of bed disinfectants made from paraformaldehyde, use of organophosphates pesticides to control mealy bug, leaf roller and white fly, use of chlorpyrifos to control termites. Beside, Grainage workers suffer from moth scales, which trigger asthma and conjunctivitis. While cocoons are put in hot water to loosen silk fibres for unwinding, the workers also put their bare hands in hot water, resulting in blisters in their hands leading to secondary infection, such as dermatitis. About 70 Benzidine based silk colorants such as azo dyes, release carcinogenic aromatic amines. Similarly, heavy metal complex dyes, are known to damage mental and central nervous function, lower energy levels and damage blood composition, lungs, kidneys and liver. Trivalent chromium used to fix silk dyes undergoes oxidation into hexavalent chromium, which leads to skin irritation, ulcers, sensitization and allergic contact dermatitis. Lead acetate used in dyeing silk cloth is a neurotoxin. It affects the human brain as well as reproductive system. Lead also affects reading and reasoning abilities in children. Dye factories across the world are dumping millions of tons of dye effluent into rivers without any effluent treatment. Pentachlorophenol, which is used in spray starch before ironing silk garment to protect from mould attack also, pose severe health problems. Formaldehyde resins routinely applied on silk to reduce shrinkage and wrinkling, cause eczematous rashes. Contact with silk cloth with a pH outside the accepted range (5.5), turns the skin flora out of balance and causes irritation. Dermatitis, narcosis, dizziness, fatigue, nausea, headache, eye irritation, adverse reproductive hazards including increased risk of miscarriage and serious neurological problems can all result from the processes of screen printing, where toluene, xylene and methyl ethyl ketone are used as solvents of the inks, thinners and clean up materials. Several health hazards are also associated with weaving and related activities, which cause stress and strain to weavers. Need of including eco parameters testing in issuing silk mark certificate is discussed

Sericulture industry is labour oriented agro industry employing 70 lakh people (5 lakh are sericulture farmers and the rest are stakeholders such as reelers, twisters, weavers, printers etc.) in India. About 23060 tonnes of silk is produced in India annually, which generates a turnover of Rs.25000 crore of which Rs.2500 crore is exported.

Silk production today is a blend of ancient techniques and modern innovations. The first stage of silk production is hatching the silkworm eggs, which have been previously examined and shown to be free from disease. Larvae are then fed cut-up mulberry leaves and after the fourth molt climb a twig placed near them and spin their silken cocoons. The silk is a continuous-filament fiber consisting of fibroin protein secreted from two salivary glands in the head of each larva, and a gum called sericin, which cements the two filaments together. Pupae within cocoons are killed by steam or fumigation to prevent adult emergence, which would cut and tangle the silk filaments. Cocoons are later softened in hot water to remove the sericin, thus freeing silk filaments for reeling. Single filaments are drawn from cocoons in water bowls and combined to form yarn. This yarn is drawn under tension through several guides and eventually wound onto reels. The yarn is dried, packed according to quality, and is now raw silk ready for marketing. Indian silk industry produces basically four types of silks namely Mulberry, Muga, Tasar, and Eri silk. Karnataka, Andhra Pradesh, Tamil Nadu, Jharkhand, Chhattisgarh, Orissa, Jammu & Kashmir and West Bengal are the major hubs of Indian silk industry. The traditional silk sarees like the Kanjeevaram sarees, Banarasi sarees, Konrad sarees, Mysore silk sarees, Pochampally Ikat sarees, Chanderi sarees, Paithani sarees, Patola sarees, Baluchari sarees, Bomkai sarees, Tasar sarees etc., are the exclusive creations of the artisans who use silk as the base material for these sarees. Among non-mulberry silks, Tasar is mostly produced by tribal people settled in different parts and regions of India.

Workers in sericulture industry are exposed to a number of health hazards. Various reproductive and menstrual risk factors for endometrial cancer have been identified, whereas few occupational or environmental risk factors have been explored. Several studies have investigated exposure to insecticides, specifically Organochlorine compounds, but no consistent associations have been reported. Although solvents are considered endocrine disruptors, the only study to date to specifically address solvent exposure and endometrial cancer detected no association (Wernli et al.2008). An association between occupational exposures and health nested within a large cohort of silk workers in India is reviewed here.

Health risk factors during silkworm rearing 

Wani and Jaiswal (2011) reported that majority of the rearers in Kashmir are suffering from health problems like, eye irritation, injuries, back pain, allergies, respiratory problems and headache.

Carbon monoxide

Coal Sigri used in silkworm rearing in temperate Kashmir

Carbon monoxide is known as a silent killer because it has no smell, colour or taste and can be produced by a faulty or poorly ventilated fuel-burning appliance such as partially burnt coal sigri used in Kashmir and other temperate areas to raise room temperature in cold condition. Symptoms of carbon monoxide toxicity consisting of headache, vertigo, nausea and vomiting. We lost a worker at Patnitop (J&K) in rearing room due to CO beyond tolerance level (100ppm). Cherry red lividity is seen in human body due to CO poisoning

Illness caused by unhygienic conditions of rearing

Unhygienic conditions due to accumulation of unutilized leaves and silkworm excreta pose health risk. Any negligence of hygiene leads to silkworm mortality due to two main diseases Grasserie & Flacherie. The dead silkworms, if not removed immediately, putrify and cause illness among rearers. 

Health Risk from Formalin used in the disinfection to maintain hygiene

Formaline spray with body mask

HCHO has strong irritant effect on the eyes and nasal mucous, when present in the air at levels exceeding 0.1ppm. HCHO is a   carcinogen. 2% solution of formaline is employed for spray in the rearing room and on rearing appliances such as trays in a closed room a day before the onset of rearing work.

Health Risk from Bleaching powder

Contact of bleaching powder with skin and eyes may cause severe injury, burns or death. Use of Only 2% solution is recommended for washing trays and rearing rooms

 Health Risk from Bed disinfectant (Paraformaldehyde) 

Bed disinfectants employed during silkworm rearing mainly consist of slaked lime and paraformaldehyde. Human Skin contact with bed disinfectants result in sensitization, inflammation of the eye (redness, watering and itching)

Health risk factors in mulberry plantation

Risk from 2,4-D Amine used for broad leaf Dicot weeds

Over the past 40 years, dozens of studies have shown the connection between 2,4-D and cancers of the blood

Parthenium: a problematic weed

 Health Risk from glyphosate for monocot weeds

Glyphosate is employed to control monocotyledon weeds such as Liver seed grass, Urochloa panicoides and Kyllinga brevifolia etc.Carcinogenic potential of Glyphosate , at extremely low concentration, is confirmed. 

Health Risk from chlorpyrifos for control of termites

Chlorpyrifos is used to control termites’ world over. Poisoning from chlorpyrifos affect the central nervous system, the cardio vascular, and the respiratory system in human being. It is also a skin and eye irritant

Health Risk from-hazardous organophosphate

Dichlorvos (DDVP) is employed to control pest attack on mulberry, such as tukra, leaf roller and white fly. Dichlorvos is known to induce neurophysiological and behavioural changes in human being

Tukra, Leaf roller, white fly in mulberry

 Health Risk from moth scales in eggs production (Grainage)

In the process of silkworm eggs production, male and female moths emerge from the cocoons, copulate and female moths lay eggs. The wings of moth are minutely scaled. The scale allergens trigger asthma, and Conjunctivitis. Around 9% grainage workers suffer from these problems.

Health Risk of Dermatitis in Reeling

While Cocoons are immersed in hot water to loosen fibres, workers tend to put their bare hands in hot water to see if the fibres are loosened to pick silk thread for unwinding. Hot water gives blisters in fingers leading to secondary infection like dermatitis

Dermatitis symptoms, reeling process

Health Risk from azo dyes

Government of India has notified Indian Eco Standards (Table-I) and prohibited manufacture and use of Benzidine based 70 dyes, which are known to break down into aryl amines.

The enzyme, azo-reductase, breaks down azo dye molecules into aromatic amines which are then absorbed in the intestine. These are especially hazardous to the workforce. The exporter of silk, have to make sure that their products do not carry any of the azo dyes that are prohibited in the western countries (EU).

Release of carcinogenic Benzidine from azo dyestuff

 Typical eco-parameters under the Eco-labels for finished textiles

 Health risk from Heavy metal complex dyes

Metal Complex Dyes are known for its fastness properties and hence find application in dyeing of silk.  Antimony, arsenic, bismuth, cadmium, cerium, chromium, cobalt, copper, gallium, gold, iron, lead, manganese, mercury, nickel, platinum, silver, tellurium,t hallium, tin, uranium,vanadium,and zinc comes under heavy metals category. Heavy metal toxicity can result in damaged or reduced mental and central nervous function, lower energy levels, and damage to blood composition, lungs, kidneys,and liver.

Health Risk from Chromium in metal complex dyes

Trivalent Chromium is used to fix silk dyes. It undergoes oxidation into Hexavalent Chromium. Exposure to Hexavalent chromium is hazardous to human health and increase the risk of developing lung cancer. Dermal exposure to Cr (VI) leads to skin irritation, ulcers, sensitization, and allergic contact dermatitis.

Effect of Hexavalent Chromium

Health risk from Lead absorption 

Lead is used in Lead acetate for dyeing of silk cloth, Lead molybdate in pigments used in dyestuffs and Lead nitrate in mordant in dyeing and oxidizer in dyeing .Lead is a neurotoxin – it affects the human brain and cognitive development, as well as the reproductive system. Some of the kinds of neurological damage caused by lead are not reversible. Specifically, it affects reading and reasoning abilities in children, and is also linked to hearing loss, speech delay, balance difficulties and violent tendencies.

Effect of different Lead levels (microgram/decilitre) in blood

 From Dyeing effluents

Dye factories across the world are dumping millions of tons of dye effluent into rivers. The dyeing effluents contain azo dyes, chromium, heavy metals etc.

In accordance with a Madras High court order, around 400 dyeing units were shut down by 2012 and their operating licences were cancelled and the power connections snapped. Only the factories that installed reverse osmosis equipment were allowed to operate.

Coloured Dye effluent into Ganga river at Varansi

 Health Risk from Pentachlorophenol (PCP) Organochlorine compound such as PCP are used in spray starch before ironing silk saree/garment to protect from mould attack. In humans, its bioaccumulation takes place and poses severe health hazard. Pentachlorophenol is a common ingredient in spray starch that often irritates the lungs. 

Mould attack on silk

Pentachlorophenol formula

 Health Risk from Formaldehyde resins

Use of formaldehyde in clothing is extremely widespread. There have even been lawsuits alleging high levels of it in Victoria's Secret bras. Formaldehyde is linked to a 30% increase in lung cancer, plus skin/lung irritation and contact dermatitis. It is found in fabrics claiming to be:

1. Anti-cling, anti-static, anti-shrink
2. Waterproof
3. Perspiration-proof
4. Moth-proof and mildew resistant
5. Chorine resistant

It is also used in dyes and printing to fix the design and prevent "running". Eczematous scaly rash occurred after the patient wore a cap containing formaldehyde resins in the silk inner lining.

Eczematous rash on forehead due to cap

Health Risk from pH value

Human skin has a pH of about 5.5, acting as a barrier to bacteria, viruses and contaminations. Contact with materials with a pH outside the accepted range turns the skin flora out of balance and causes irritations. Eco mark (India) restricts pH of aqueous extract of the fibres between 4.0 and 7.5.

Health risk from screen printing solvents

The major hazard comes from exposure to the solvents, Toluene,Xylene,methyl ethyl ketone  in the inks, thinners, clean-up materials, (permitted<1 .0="" b="" ppm="">

Dermatitis, narcosis, dizziness, fatigue, nausea, headache, eye irritation, adverse reproductive hazards including increased  risk of miscarriage, and serious neurological problems can all result from the processes of screen printing

Health Risk of Handloom Silk weavers

Several health hazards are associated with weaving and related activities which may cause stress and strain to weavers and pose several health related risk factors to them

Ailments of silk weavers

Conclusion

Various types of occupational disorders are associated with silk industry such as, respiratory disorders, injuries, eyesight problems, nerve disorders, and carcinogenic skin problems. Most of these health risk factors can be avoided by proper precautions. Awareness programme and local group discussions are essential for improving the health status of these workers. There must be some provision of protecting equipments such as face masks, first aid facility, gloves and proper uniform. Silk mark protects the interests of consumers, who are being cheated by traders by selling spurious products in the name of silk. So far silk mark authorities test cocoons and raw silk only.  In order to safe guard health of the consumers, the silk mark should be clubbed with silk testing with reference to eco parameters of Textile testing laboratories as per the norms of Bureau of Indian Standards.

References

Nadiger G.S.(2001)Azo ban,econorms and testing.

Indian J. Fibre Text.Res.Mar-Jun, 55-60.

Wani, K.A. and Y.K.Jaiswal (2011_a) Health hazards of rearing silkworms and environmental impact assessment of rearing households of Kashmir, India.

Nature Environment and Pollution Technology, 10, (1), 85-90.

Wani, K.A. and Y.K.Jaiswal (2011_b) Occupational health risk factors in carpet industry:

A review. Asian J.Exp.Biol.Sci.2 (1), 135-138.

Wernli, K.J.,R.M.Ray, D.LiGao, E.D.,Fitzgibbons,J.E.,Camp,,G.Astrakianakis,   W.Li, A.J.DeRoos, Z. Feng, D. B Thomas, and H. Checkoway (2008). Occupational Risk Factors for Endometrial Cancer among Textile Workers in Shanghai, China.

Am J Ind Med. Sep 2008; 51(9): 673–679.

Yousuf, T., I Khan, T. Yousuf and T.Raja (2013) Socio economic profile of silk weavers: A micro level study of Srinagar city. European Acad.Res. 1, (3), June, 319-331.

Key words: Cancer, occupation, silk, textile

‘Bivoltine is not for small farmer’- TH. Somasekhar

Interview by GK. Rajesh

Dr. TH. Somasekhar is India’s leading silk expert. Recipient of the Louis Pasteur Award (2005) of

Dr. TH. Somasekhar

International Sericulture Commission, he led the country’s silk technology research & development for fifteen years as Director of Central Silk Technological Research Institute (CSTRI). Dr. Somasekhar is a PhD in Fibre Science from University of Strathclyde, Glasgow. He obtained B Tech and M. Tech from Bangalore University and Indian Institute of Technology, New Delhi respectively. In an illustrious career spanning more than three decades he has held many positions such as Head of Textile Technology Division, Silk & Art Silk Mills Research Association, Mumbai, Senior Scientific Officer of Ahmedabad Textile Industry’s Research Association, Chief Technical Advisor of M/s. Swan Silk, Ltd., Bangalore etc. Dr. Somashekar is an internationally renowned expert on textiles especially silk. He is widely travelled and associated with all silk producing countries. In this interview, recorded at his residence at Bangalore, India, Dr. Somasekhar spoke at length about India’s sericulture and silk industry, demystifying popular perceptions on the future of silk as an apparel fibre.

Dr. Somasekhar can be contacted at: thsomashekar@gmail.com       

GK: Sericulture is an important agro-industry in many parts of India, especially the South. A large number of stake holders such as farmers, silk reelers, weavers, traders and other manual labourers are involved. Silk has been considered a very important commodity by its Royal patrons and during British Rule. The fact that the first commodity board in independent India was constituted for silk is indicative of the importance attached to it. But the industry has been showing a declining trend over past few years. What do you think are the reasons?

Dr. Somasekhar: For last few years particularly for a decade sericulture has been declining and the decline is due to various reasons. For example in Karnataka, a large track of land which was under sericulture in the past has been lost. The main reason around Bangalore Rural district has been urbanisation. Now the new Bangalore international air port has come up. As a result of that development the lands around it have gone for various purposes other than sericulture. Another reason is perhaps sericulture all along has been a labour intensive activity and we have learnt from experience of other countries that sericulture has thrived and survived as long as the contribution of family labour was in bulk. As it went towards engaging labour from outside the economics has not been working out and then also we have known that as the standards of living among the farming community has gone beyond a certain level sericulture has been lost. Another aspect is that there are many other alternatives for the farming community where, with less effort they are able to earn more. See for example in Karnataka there are more profitable alternatives like horticulture, floriculture etc with ready market and less risk. And then we have not been able to provide a stable market for the producer. The cocoon and raw silk prices fluctuate; something that they never wanted. They have always asked, ‘give us a stable price’.

Another thing that has happened in recent years is imports. Large scale imports have affected the local industry and that has been a major factor. As a nation we may have various compulsions for allowing imports- particularly after globalisation of the markets we have to participate survive and fight it out in the open market and that is something which has not been conducive for the progress of sericulture. So as a result we have seen all these factors put together in various dosages contributed to the decline of sericulture.

Globalisation, in fact could have been an opportunity rather than a threat for development. Many domestic industries utilised it either as a means to get cheap imports of essential raw material or as an avenue for large scale export of domestic goods. But silk industry seems to have failed miserably in utilising this opportunity. What is your observation?

We have seen that while imports have increased the exports have not been increasing. Exports in quantity terms have come down. The case of raw material exports is different. For example silk waste export doesn’t help the local spun silk industry. As such the number of spun silk mills in the country has come down. We had a string of spun silk mills numbering almost a dozen in the private sector. They have all closed down now. The reason is they are not getting raw material at a price which is economical to them. We have all the technologies imported. We have Japanese technology, Italian technology for de gumming and spinning and considerable amount of money have been invested in getting the suitable machinery, the line of machines, required for spun silk manufacture.

Then we also see in the recent years that the import duties on raw silk have been brought down to almost 5% and this is going to have a major impact, a perceivable effect on the growth of the industry. The reason is it is not that we will be getting imported silk at a cheaper rate like in the past. Gone are the days when we could import low quality silk when a lot of silk was made available to weavers at import prices as low as $12-16 per kg. Today the prices have gone up beyond $37-38. So what is happening? We are having bulk imports at high prices. So this is something which is going to create problems. Our own production is not much and at least the farmer and silk reeler would have commanded a better price if the imports were not there. The import price is also closely linked to the availability of local silk. When the local silk availability is low, prices automatically go up and import prices also will be at a higher side. These are the changes that have taken place and sericulture is taking a beating from all angles.

India obtained a couple of anti-dumping sanctions against China in case of cheap imports of silk yarn. Do you think China has been dumping silk in India?

You can’t say dumping. A few years ago cheap silk was coming. But today silk is not cheap. The silk we are importing is expensive. Why that is happening is because we have a lot of demand. There is a huge gap between demand and supply. Our own raw silk production is not meeting the requirement. 7 to 8000 tonnes of raw silk is being legally imported. Our production may not be more than ten thousand tonnes, though we claim that we have 17 to 18,000 tonnes of raw silk. On observing the availability in the field that is the farmer activity, cocoon market activity reeler and weaver activity, I get a feeling that we don’t have the kind of raw silk production that we clam to have. That is the reason we are forced to import so much of raw silk. Our requirement has always been estimated around 24 to 25,000 tonnes. If we had 17 to 18,000 tonnes local production 7 to 8,000 tonnes should have been imported. My own understanding of the weaver’s situation is that they are all not doing silk alone. This is because of the increased prices and as ordinary people can’t afford pure silk. So there is a lot of imitation material and combinations. Cotton and silk, Polyester and silk, polyester alone . . . but you have the same designs. Thus the prices have decreased the demand for pure silks. Therefore I feel that this 7,000 to 8,000 tonnes added to our own requirement may not be 25,000 tonnes. It could be far less. Our own production should be around 10 to 12,000 tonnes and our total requirement should be around 18 to 20,000 tonnes.

As I understood, in the past, there were lot of loop holes in imports and large quantities of raw silk were getting into the country through unofficial channels. But all those have been plugged now and the situation also has changed. There is no incentive for people to import silk when the price is high. Even in China, they are not giving you silk less than $ 37 to 38. The overall market situation is such that we have a gap and because of our own production being low, this gap is being filled by imports.

I was in China a few years ago and I had an opportunity to discuss with the companies which manufacture and sell silk. They have lot of buyers in India. I asked them why have they increased the prices, and we can’t afford silk at $ 37 to 38 and even more. The answer I got was: “after all our interest is to sell more. But we will have to see at what prices we are producing raw silk”. The MD of the company told me that for past few years they have been forced to give double the salaries being paid until then. That has (almost) doubled the cost of production. The standards of living in China have increased and the situation that existed in the past is no more relevant. Given this kind of a situation silk is becoming an expensive material and I don’t think it will be cheap in the future and the kind of prices that we have seen in the past may not come again. In such situation countries like India may see further drop in production. We may feel that China has a monopoly. But in the years to come it is quite possible that their interest may also go down and the total silk availability come down.

Is silk going to stay for ever?

I foresee a situation where we will definitely have less and less silk in the coming years and we have to live with it and kind of decide where we need silk. This is going to be a big question. Do we need silk for apparels? In the recent years we see a whole lot of uses of silk other than apparels. With the total availability coming down we should also look at other possibilities which could open opportunities for expensive silk in much better and more important uses. In bio-medical engineering silk is seen as a scaffolding material. It helps growth of cells in the body. Can we look at these possibilities and expand into these areas? We need not worry oh! We are losing silk. In the years to come we should also plan out sericulture from these angles. When we look at silk for apparel purposes the quantity requirements are too huge and we try to increase productivity, leading to use of large quantities of inorganic fertilizers. That is how the quality of land deteriorates. Diverting silk to non apparel purposes will reduce the quantity demand pressure. This would be far more scientific. When we use silk for bio-medical purposes there shouldn’t be even traces of inorganic material in it. This reduces pressure on environment. Perhaps more such uses may arise.

India implemented at least two ambitious externally aided projects: the Karnataka Sericulture Project (KSP) and National Sericulture Project (NSP) both were declared failure by those who did final assessment. Where did we go wrong? What are the lessons?

KSP & NSP were in many ways projects for the sericulturist, for upgrading himself, to adopt new practices and to accept new technologies. The model of sericulture that we had is something very important. In fact I had projected three models of sericulture existing in different parts of the world.

1. The Indian model which consist of small producer and small converter

2. The Chinese model which has small producer and large converters

3. The Japanese model (that has been practiced by Brazil) where you have large producers and large converters

The inputs that we gave in KSP & NSP were mainly to the Indian model. That is small producer – small converter. The support that was given to the industry was not much. The planners at that time thought that we have to support the supply side and that is how all the support was given to the farmer. That was to strengthen the base. What has happened is there for you to see. Now, while looking back one gets the feeling; should we have supported the demand side, for that would have created a demand pull and perhaps resulted in better sericulture?  All said and done, there is an absolute need that we change the Indian model of small producer & small converter. May not be totally towards the Chinese or Japanese side; but somewhere in between. For this I had proposed certain changes. There was a committee which had looked into these things and given a request to the Research Coordination Committee (RCC) of central Silk Board which was chaired by Prof. Chopra. He said that the suggestions of the committee should be put into practice at least by the leading states (Karnataka, Andhra Pradesh and Tamil Nadu). But I don’t think that has happened. We haven’t made course corrections. We tend to stay where we are. That is not good. We should adopt ourselves to the changes that are taking place. Everything is market driven today. We can’t say ‘I have a poor farmer, please buy’. I can’t go and ask people ‘buy Indian silk’. If the demand is met, if the quality is met, any silk is acceptable to a customer. Market doesn’t wait for you.

The focus of both NSP and KSP was popularisation of Bivoltine (BV) sericulture. India received a lot of technical support from abroad, especially from Japan and until recently Japanese stayed in India to ‘bivoltinise’ Indian sericulture. In this quest we have grossly over looked the strengths of local varieties of silk worm: the multivoltines and cross breeds etc. Given that the current percentage of diffusion of Bivoltine hybrid in the country is as low as around 10%, what is your assessment on India’s strategy on BV popularisation? How important is BV sericulture to India?
I don’t say, looking at the Indian sericulture, the situation is very conducive for bivoltine. Even in Karnataka we can practice only two crops of bivoltine out of 5 crops per year. Thus bivoltine is not favoured so much, climate wise.

We put in efforts since 1975 for developing Bivoltine sericulture in India. Beginner’s luck, you can say, we had very good results with bivoltine. But we have not been able to sustain it. As a person with long term association with silk industry, I would say that bivoltine is not for small scale farmer. That means bivoltine is not for our model of sericulture. Our farmers are poor. They don’t have the strength to absorb the technological advancements. They don’t have the capacity to invest. Under the project we supported them technically, financially and by way of extension. All these helped them to get the result which was very good. But the moment all these supports were withdrawn after the project it went back to square one.

We were quite successful in developing bivoltine for tropical conditions. The races we developed in Mysore were very good. There is no doubt about it. But the performance of Cross Breed (CB) was exceptionally good. Even today it is close to bivoltine and without much effort. Now the farmer realise why should he take all the trouble for bivoltine when there is no market for it. When he goes to the market, he s a loner, for very small quantities of bivoltine comes there. As a result there is not a demand for his product. Instead of that CB has give good results. Now the question is ‘was bivoltine developed for the improvement of CB?’ Honestly I feel: YES. Looking from both farmer’s and reeler’s side, yes it was. From our old multi-bivoltines like PM X NB4D2 etc., PM X CSR2 is a big jump. As that has happened I am quite happy even if bivoltine is not there. At least there has been a significant improvement in CB production which has helped the economics of the farmer. If cross breed is the answer, we can use BV for improvement of CB. So I still feel it is a big improvement and we should recognise that fact and then we should focus more on the growth of the industry in these lines rather than beating only BV

But the now celebrated Cross Breed (Kolar gold) has not been a research breakthrough. This has been a contribution from the graineurs.

Yes. This is the point. In research we had aimed at something and the fall out has been something else. We have succeeded in research. It is only the extension and transfer and sustenance of this knowledge at the farmer level (we have failed). I am not talking about a small number of farmers who are always under scrutiny and support. I am talking about farmers at large. But the entry of PM X CSR 2 and PM X CSR 4 has changed the whole situation.

Sericulture research has been a prerogative of Central Silk Board, as the government money for research goes to CSB. Has CSB been monopolising sericulture research in the country?

The fact is that apart from CSB research institutes other institutes were not developed to carry out bivoltine research. They didn’t have that kind of background. Where ever you talked about Bivoltine sericulture a scientist found elsewhere was from CSB or formerly from CSB. Two state institutes came up; one in Karnataka and the other in AP. Basically they were all meant for carrying out applied research. Sericulture research is basically applied. During the NSP period CSB invited proposals from umpteen numbers of universities. Some 80- 100 projects were approved and funds were made available. But their interest dried up after the project. They didn’t pursue it and try to get funding from elsewhere. Even today CSB supports research from universities but not to the extent one has been during NSP period. The bottom line there is the interest shown by people.

CSB dint monopolise any research. There was no need. Whatever facilities (equipments and man power) created in institutes and universities during NSP were all limited to the project period. Afterwards they haven’t really done much. The universities also don’t have any long term research plans. Research in our country has been influenced by the demands that have come up. There was this need to expend. That is we needed to produce more, reduce imports. This is the kind of situation that influenced our research. So we addressed productivity and quality. I agree that productivity and quality are not achieved at the final stage. One has to work from the beginning. But that kind of effort has been less. Again the interest of individuals also matter. Large number of scientists would like to delve more on applied areas rather than basic areas because they all feel that the results are quicker and they would be able to show something to the people. The number of people who have shown interest in basic research is less. It is not that we don’t have facilities for basic research in CSB. We do. Basic research is time consuming. You don’t get results over night. So we need people interested in these areas. The pressure of the market shouldn’t be coming to these people. Let the applied people take care of the market situation. I think the CSB would do well to create in each of its research institutes in Berhampore (West Bengal), Mysore (Karnataka) and Pampore (J&K); certain number of people and groups to go into basic research.

It was also thought during the NSP period (as I remember) to entrust universities with basic research. Universities were not expected to do applied research. But that thought didn’t materialise. There were wrong decisions made too. The Karnataka Sericulture Development Institute (KSSDI) later on became KSSRDI (Karnataka Sericulture Research and Development Institute). The research part was not there as it was conceived during the KSP period. The KSSDI was supposed to take out the output of Central Institute and ‘package’ for field requirements. But that responsibility was not taken by KSSDI. It became KSSRDI and became a competitor to Central Institute. That wasn’t required. Today there are problems. They are not able to get funds for salaries. A re-look is desirable and possible even today. KSSRDI could drop the ‘R’ and become a sequel to the research that is done at Mysore institute. Same holds good for APSSRDI of Andhra Pradesh. Their job is very important.

Indian silk reeling industry is visibly in peril. The farm sector of sericulture seems to have stolen the entire attention of policy makers, leaving silk reelers in hardship. Apparently the obsolete technologies prevailing in the reeling sector has made it incapable of absorbing whatever quantities of high quality bivoltine cocoons produced in the country. What has been happening to the reeling sector?   

For the reeling industry to do well, quality of cocoons is primary. In raw silk production, 90-92% of cost of production is cocoon price (during normal times). Sometimes it can be even more than 100% where the reeler looses. He can only be compensated by selling by-products. Unless you have good quality cocoons it becomes impossible for the reeler to adopt new technologies.  Central Silk Technological Research Institute (CSTRI) Bangalore developed multi-end reeling package and as many as 300 or more small units (which was the economic size at that point of time) were manufactured.   They were all planted in a number of reeling areas. The quality of yarn came out of them was good. Only thing was the way the BV has not been able to influence the (cocoon) market, multi-end raw silk could not influence the raw silk market. The ‘influence factor’ comes with quantities in the market. Quantity influences the market. If BV has to influence, it has to be made available in very large quantities. Similarly multi-end silk has to be made available in very large quantities.

For the reeler, when he goes to the market, his buying ability restricted by the amount of money he has. None of these small units enjoy the kind of working capital support the bigger industries enjoy. We have worked on this also. There was a scheme to support these people. We went to banks, talked to them and some kind of support was made available. But still the reeler lacks the much needed large working capital base. Today it is once in 2-3 days the reeler has to go to the cocoon market. Buy cocoons, convert to silk, and sell silk and again go back to buy cocoon in 2-3 days. Whereas in the organised sector; it is at least a couple of months. A small reeler who is producing 10 kg silk per day would require at least 70 kg cocoons and if he has to survive well he needs to have a stock for 20-25 days. That means 1.5to 2 tonnes of good quality cocoons, which would cost at least Rupees 35 to 45 lakhs. Is this kind of money available to our reeler? That is why he is finding it difficult. Our own cocoons, for the quality they have, are the highest priced of the whole world. Nowhere in the world cocoons are as expensive as in India.  That’s the reason why the reeler has not been able to change and he continues to work in a dirty situation with smoke, smell and all this.

This is where we have to look at the models of sericulture. There is a large number of people working in this sector. We can’t go on like this, talking about taking care of these many people. See, in the years to come frankly speaking, if you want to take care of these people as a state, you have to give them alternate areas to work. Take them out of this misery and keep only the required number of people in this activity to make it economical. And if something is not economical, I don’t think there is a need to practice it.  If sericulture is not good for certain state why should they do it? Just because a sericulture department has been created, you don’t have to waste money. Sericulture is a culture. If it is a culture for a farmer in Kolar, because he has been doing it for generations and he knows what it is. You can’t take this culture and ask a person somewhere in another state who has not even seen a silkworm, into sericulture. That’s why we have failed. There are certain practices, very native. If I have to take it to Kerala, I have to see if it is acceptable to Keralites. The family and the neighbourhood should accept it. It is a culture by itself.  

Is the silk weaving sector any better off? How well equipped is our weaving sector in catering to the needs (of high quality silk fabric) of the international silk market?

Indian textile industry is quite modern now, thanks to the Government of India interventions. More than 25,000 crores of Rupees has been infused into the industry and lot of modernisation has taken place. They are able to produce high quality yarn, a huge quantity of which is being exported. Formation of producer consortia has helped the situation by enabling the weavers to place bulk orders for raw material at competent prices. But the case has not been the same with silk weaving sector. It is too small a sector for taking to such changes. We have three sub sectors in silk weaving: handlooms, power looms and the highly organised shuttle-less loom sector. 95% of the silk fabrics produced is from the handlooms and power looms the bulk of which is coming from the handlooms. The conditions of the handloom weaver are not very good. He has no capacity to buy his raw material, which is silk yarn. He takes the yarn from a master weaver and converts it to fabric on wages. Thus he is only a converter or labourer, not an entrepreneur. The silk reeler is at least an entrepreneur. He is investing a few thousands of rupees every day. But a weaver doesn’t. So the handloom weaver is affected by the market conditions. And his situation is pretty bad. There is not enough demand for the handloom product. The reason is that he is not weaving fabric as per the requirements of the market. He knows to weave something which he continues to weave whether it sells or not. The handloom weaving industry is not reacting to market requirements.

Finally, what has been the contribution of Central Silk Technological Research Institute, to Indian silk Industry?

CSTRI has been a partner to bivoltine development, all along. Then we became the prime mover to improve quality of silk reeling. That’s where the multi end technology came into being. May be we dint succeed much in the reeling sector, because of the reasons I have already explained. But we succeeded in other areas like silk yarn dying. We successfully moved the dyers to new technology which has also influenced the market. Today the yarn dying industry has come of age. We developed a handloom and an electronic jacquard, which have diffused well in the industry. Another important development was computer aided designing coupled with a card cutting machine and automatic lacing of card. You see a large number of people using these technologies now. In Dharmavaram area itself as many as 150 computer aided design units are working. In the non-mulberry sector, the reeling and spinning machines we developed has gone to the filed in thousands, through supporting schemes. CSTRI is now recognised as a National Research Institute. It is also recognised as one of the Textile Research Associations by Ministry of Textiles, Government of India. We take up a lot of consultancy services for companies like Hindustan Lever, developing chemicals and evaluating their soaps and detergents. We have helped IFB, the washing machine people in developing a wash program for silks. We dint advocate continuous revolving of the drum. For silks, we said it should rock!

RESEARCH UPDATES 02

9 papers on silkworm, 4 papers on silk proteins and 5 papers on medicinal applications of sericulture

A. SILKWORM

1. Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori.
Uno T, Nakada T, Okamaoto S, Nakamura M, Matsubara M, Imaishi H, Yamagata H, Kanamaru K, Takagi M.
Arch Insect Biochem Physiol. 2007 Oct;66(2):89-97.
The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser-132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in-gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17879235&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

2. The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori.
Shao YM, Dong K, Zhang CX.
BMC Genomics 2007, 8:324doi:10.1186/1471-216

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based on their genome sequences. The silkworm Bombyx mori is a model insect of Lepidoptera, among which are many agricultural pests. Identification and characterization of B. mori nAChR genes could provide valuable basic information for this important family of receptor genes and for the study of the molecular mechanisms of neonicotinoid action and resistance. RESULTS: We searched the genome sequence database of B. mori with the fruit fly and honeybee nAChRs by tBlastn and cloned all putative silkworm nAChR cDNAs by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. B. mori appears to have the largest known insect nAChR gene family to date, including nine alpha-type subunits and three beta-type subunits. The silkworm possesses three genes having low identity with others, including one alpha and two beta subunits, alpha9, beta2 and beta3. Like the fruit fly and honeybee counterparts, silkworm nAChR gene alpha6 has RNA-editing sites, and alpha4, alpha6 and alpha8 undergo alternative splicing. In particular, alternative exon 7 of Bma8 may have arisen from a recent duplication event. Truncated transcripts were found for Bma4 and Bma5. CONCLUSIONS: B. mori possesses a largest known insect nAChR gene family characterized to date, including nine alpha-type subunits and three beta-type subunits. RNA-editing, alternative splicing and truncated transcripts were found in several subunit genes, which might enhance the diversity of the gene family.

FullText:http://www.biomedcentral.com/1471-2164/8/324 and http://www.ncbi.nlm.nih.gov/entrez/utils/fref.fcgi?PrId=3196&itool=AbstractPlus-def&uid=17868469&db=pubmed&url=http://www.biomedcentral.com/1471-2164/8/324

3. A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.
Tomita M, Hino R, Ogawa S, Iizuka M, Adachi T, Shimizu K, Sotoshiro H, Yoshizato K. (Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima, 739-0046, Japan.)
Transgenic Res. 2007 Aug;16(4):449-65. Epub 2007 Apr 6.
A silk thread of the silkworm, Bombyx mori, is composed of the insoluble inner fibroin and the hydrophilic outer sericin layer, which are synthesized in the posterior and middle silk gland (MSG), respectively. This study aimed to develop a novel sericin 1 gene (ser1) promoter-driven recombinant expression system using transgenic silkworms, in which recombinant proteins are synthesized in MSG and secreted into the sericin layer. To obtain a high level of gene expression, we tested whether a baculovirus-derived enhancer, hr3, and a trans-regulator, IE1, are capable of stimulating the transcriptional activity of the ser1 promoter, using a transient gene expression system. The results showed that hr3 and IE1 cooperatively increased the ser1 promoter activity more than 30-fold. Then, transgenic silkworms were generated which expressed the EGFP with the signal peptide in MSG under the control of the hr3-linked ser1 promoter and IE1 gene. The silkworms exclusively secreted the EGFP into the sericin layer of cocoons as predicted. The expressed EGFP was extractable from cocoons through a simple procedure with neutral pH buffer solution. The expression system developed in this study enables us to produce recombinant proteins in bulk that can be easily extracted and purified.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17415674&ordinalpos=20&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

4. Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin
Ogawa S, Tomita M, Shimizu K, Yoshizato K.(Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan.)
J Biotechnol. 2007 Feb 20;128(3):531-44. Epub 2006 Nov 17.
In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days later, the transplants were immunohistochemically analyzed, which showed that middle silk gland (MSG) cells expressed rHSA and secreted it into the MSG lumen. Then, silkworm eggs were injected with the vector and developed to larvae. The obtained transgenic silkworms spun silk threads whose sericin layers contained rHSA at 3.0microg/mg of cocoons. Most (83%) of the rHSA in cocoons was extracted with phosphate buffered saline, which was then subjected to ammonium sulfate precipitation and affinity chromatography. Finally, we obtained 2.8mg of 99%-pure rHSA from 2g of cocoons. Measurements of circular dichroism spectra of rHSA, and equilibrium dissociation constants of rHSA to warfarin and naproxen indicated that rHSA was conformationally and functionally identical to natural plasma HSA. Germline transgenic silkworms will be useful for producing various recombinant proteins in the sericin layer of cocoons.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17166611&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

5. Silk gland specific secretory expression of egfp gene in silkworm Bombyx mori with rAcMNPV system.
Guo XY, Guo TQ, Wang SP, Wang JY, Lu CD. (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, PR China.)
Arch Virol. 2005 Jun;150(6):1151-60. Epub 2005 Feb 10.
To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californica nucleopolyhedrovirus (AcserpegfpDeltaEGT) carrying the reporter gene egfp downstream of silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus AcserpegfpDeltaEGT was injected into the haemocoel of newly ecdysed 5thHendekl) instar silkworm larvae at the amount of 10(6) pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15703851&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

6. Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori.
Liu Y, Yu L, Guo X, Guo T, Wang S, Lu C. (College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027, China.)
Biochem Biophys Res Commun. 2006 Mar 31;342(1):273-9. Epub 2006 Feb 6
The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209bp region by overlapping deletion studies showed that a 25bp region (-500 to -476) suppresses
the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25bp fragment. These results suggest that this 25bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16480950&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

7. Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.
Li BC, Zhang SQ, Dan WB, Chen YQ, Cao P. (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Jiangsu, Nanjing, PR China.)
Biotechnol Lett. 2007 Jul;29(7):1031-6. Epub 2007 Mar 21.
The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17375264&ordinalpos=21&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

8. Expression of the antibacterial peptide CM4-like gene of Chinese silkworm Bombyx mori in Escherichia coli and its antibacterial activity analysis
Li BC, Chen YQ, Liu P, Zhang SQ. (The Life Science School of Nanjing Normal University, Nanjing 210097. jslbc@126.com)
Fen Zi Xi Bao Sheng Wu Xue Bao. 2007 Apr;40(2):98-102
[Article in Chinese] According to the amino acid sequence of CM4 and the bias for preferred condons of E. coli, the CM4-like gene was obtained by a recursive PCR (rPCR) strategy using two lapping oligonucleotides. The synthesized gene was coloned into the expression vector pET32(a) and transformed into E. coli BL21 (DE3). Recombinant CM4-like gene expression was driven by the T7 promoter on the vector upon addition of IPTG and high level of expression was achieved. The solube protein was purified by Ni-chelating agarose and treated with formic acid. After cleavege, the recombinant peptide was purified by another Ni(2+)-NTA-Agarose affinity chromatography and cation-exchange chromatography. Results of agarose diffuse assay and liquid turbidity analysis indicated that the recombinant peptide exhibited the antibacterial activity.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17580662&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

9. Structural Basis of Ligand Binding and Release in Insect Pheromone-binding Proteins: NMR Structure of Antheraea polyphemus PBP1 at pH 4.5.
Damberger FF, Ishida Y, Leal WS, Wuthrich K.
J Mol Biol. 2007 Aug 17; [Epub ahead of print]
The NMR structure of the Antheraea polyphemus pheromone-binding protein 1 at pH 4.5, ApolPBP1(A), was determined at 20 degrees C. The structure consists of six alpha-helices, which are arranged in a globular fold that encapsulates a central helix alpha7 formed by the C-terminal polypeptide segment 131-142. The 3D arrangement of these helices is anchored by the three disulfide bonds 19-54, 50-108 and 97-117, which were identified by NMR. Superposition of the ApolPBP1(A) structure with the structure of the homologous pheromone-binding protein of Bombyx mori at pH 4.5, BmorPBP(A), yielded an rmsd of 1.7 A calculated for the backbone heavy-atoms N, C(alpha) and C' of residues 10-142. In contrast, the present ApolPBP1(A) structure is different from a recently proposed molecular model for a low-pH form of ApolPBP1 that does not contain the central helix alpha7. ApolPBP1 exhibits a pH-dependent transition between two different globular conformations in slow exchange on the NMR chemical shift timescale similar to BmorPBP, suggesting that the two proteins use the same mechanism of ligand binding and ejection. The extensive sequence homology observed for pheromone-binding proteins from moth species further implies that the previously proposed mechanism of ligand ejection involving the insertion of a C-terminal helix into the pheromone-binding site is a general feature of pheromone signaling in moths.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17884092&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

B. SILK PROTEINS

1. Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution
Tao W, Li M, Zhao C.(School of Material Engineering, Stem Cell Research Laboratory of Jiangsu Province, Suzhou University, Campus Box 64, No. 178 East Gan-Jiang Road, Suzhou 215021, China.)
Int J Biol Macromol. 2007 Apr 10;40(5):472-8. Epub 2006 Nov 24.
Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, (13)C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol-gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were alpha-helix and random coil in solution. There also existed a little beta-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no alpha-helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol-gel transformation. The velocity increased obviously when the temperature was above 30 degrees C. During the sol-gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to beta-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173967&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

2. Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: grafting of chitosan under heterogeneous reaction conditions.
Freddi G, Anghileri A, Sampaio S, Buchert J, Monti P, Taddei P. (Stazione Sperimentale per la Seta, via Giuseppe Colombo 83, Milano, Italy. freddi@ssiseta.it)
J Biotechnol. 2006 Sep 1;125(2):281-94. Epub 2006 Apr 18
The capability of mushroom tyrosinase to catalyze the oxidation of tyrosine residues of Bombyx mori silk fibroin was studied under heterogeneous reaction conditions, by using a series of silk substrates differing in surface and bulk morphology and structure, i.e. hydrated and insoluble gels, mechanically generated powder and fibre. Tyrosinase was able to oxidize 10-11% of the tyrosine residues of silk gels. The yield of the reaction was very low for the powder and undetectable for fibres. FT-Raman spectroscopy gave evidence of the oxidation reaction. New bands attributable to vibrations of oxidized tyrosine species (o-quinone) appeared, and the value of the I853/I829 intensity ratio of the tyrosine doublet changed following oxidation of tyrosine. The thermal behaviour of SF substrates was not affected by enzymatic oxidation. o-Quinones formed by tyrosinase onto gels and powder were able to undergo non-enzymatic coupling with chitosan. FT-IR and FT-Raman spectroscopy provided clear evidence of the formation of silk-chitosan bioconjugates under heterogeneous reaction conditions. Chitosan grafting caused a beta-sheet --> random coil conformational transition of silk fibroin and significant changes in the thermal behaviour. Chitosan grafting did not occur, or occurred at an undetectable level on silk fibres. The results reported in this study show the potential of the enzymatically initiated protein-polysaccharide grafting for the production of a new range of bio-based, environmentally friendly polymers.

3. Chemical and physical properties of sulfated silk fabrics.
Taddei P, Arosio C, Monti P, Tsukada M, Arai T, Freddi G.(Centro di Studio sulla Spettroscopia Raman, Dipartimento di Biochimica G. Moruzzi, Università di Bologna, via Belmeloro 8/2, Bologna 40126, Italy.)
Biomacromolecules. 2007 Apr;8(4):1200-8. Epub 2007 Mar 6.
Silk fabrics were treated with chlorosulphonic acid in pyridine for different times. The amount of sulfur bound to silk increased during the first 2 h of reaction and then reached a plateau. The amino acidic pattern of sulfated silk remained essentially unchanged for short reaction times (< or ="2"> or =2 h). Spectroscopic analyses performed by FT-IR and FT-Raman showed the appearance of new bands attributable to various vibrations of sulfated groups. The IR bands at 1049 and 1014 cm-1, due to organic sulfate salts, were particularly intense. Bands assigned to alkyl sulfates and sulfonamides appeared in the 1300-1180 cm-1 range. Organic covalent sulfates displayed a weak but distinct IR band at 1385 cm-1. Both IR and Raman spectra revealed that silk fibroin mainly bound sulfates through the hydroxyl groups of Ser and Tyr, while involvement of amines could not be proved. Changes observed in the amide I and II range indicated an increase of the degree of molecular disorder of sulfated silk. Accordingly, the I850/I830 intensity ratio between the two Tyr bands at 850-830 cm-1 increased from 1.41 to 1.52, indicating a more exposed state of Tyr residues in sulfated silk. TGA, DSC, and TG analyses showed that sulfated silk attained a higher thermal stability. A thermal transition attributable to sulfated silk fibroin fractions appeared at about 260 degrees C in the DSCthermograms.

http://www.ncbi.nlm.nih.gov/sites/entrez?=pubmed&Cmd=ShowDetailView&TermToSearch=17338562&ordinalpos=22&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

4. A study on the flow stability of regenerated silk fibroin aqueous solution.
Wang H, Zhang Y, Shao H, Hu X.(State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, Shanghai 200051, PR China).
Int J Biol Macromol. 2005 Jul;36(1-2):66-70
The flow stability of silk fibroin (SF) aqueous solutions with different concentrations under different temperatures was investigated. It was found that the flow stability decreased quickly with the increase of solution concentration and temperature. X-ray diffraction, Fourier transform infrared (FTIR) and Raman spectroscopy analysis showed that silk fibroin in aqueous solution was mainly in random coil and alpha-helix conformation. However, it turned into alpha-helix and beta-sheet conformation after gelation, and both silk I and silk II crystalline structures appeared accordingly. The investigation implies that the original dilute regenerated SF aqueous solution should be stored under low temperature and concentrated just before spinning.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15916801&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

C. MEDICINAL APPLICATIONS
1. Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration.
Yang Y, Ding F, Wu J, Hu W, Liu W, Liu J, Gu X.
Biomaterials. 2007 Sep 18; [Epub ahead of print]
Slk fibroin (SF), derived from natural silk long used as a textile material, has recently become an important biomaterial for tissue engineering applications. We have previously reported on good in vitro biocompatibility of SF fibers with peripheral nerve tissues and cells. In the present study, we developed a novel biomimetic design of the SF-based nerve graft (SF graft) which was composed of a SF-nerve guidance conduit (NGC) inserted with oriented SF filaments. The SF-NGC prepared via well-established procedures exhibits an eggshell-like microstructure that is responsible for its superior mechanical and permeable properties beneficial to nerve regeneration. The SF graft was used for bridge implantation across a 10-mm long sciatic nerve defect in rats, and the outcome of peripheral nerve repair at 6 months post-implantation was evaluated by a combination of electrophysiological assessment, FluoroGold retrograde tracing and histological investigation. The examined functional and morphological parameters show that SF grafts could promote peripheral nerve regeneration with effects approaching those elicited by nerve autografts which are generally considered as the gold standard for treating large peripheral nerve defects, thus raising a potential possibility of using these newly developed nerve grafts as a promising alternative to nerve autografts.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4PPFTBC-4&_user=989289&_coverDate=09%2F19%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000049901&_version=1&_urlVersion=0&_userid=989289&md5=2be7c99e0c8bc73d65966ad569342c95

2. Silk-fibroin-coated liposomes for long-term and targeted drug delivery.
Gobin AS, Rhea R, Newman RA, Mathur AB. (University of Texas MD Anderson Cancer Center, Laboratory of Reparative Biology and Bioengineering, Plastic Surgery, Houston, TX 72230, USA.)
Int J Nanomedicine. 2006;1(1):81-7
Many barriers to drug delivery into a tumor site require careful consideration when designing a new drug. In this study, the adhesive targeting and drug specificity of modified liposomal vesicles on human-scar-producing cells, keloid fibroblasts, were investigated. Keloids express abundant levels of mucopolysaccharides and receptor tyrosine kinase (RTK). In this report, the structural properties, drug release kinetics, and therapeutic availability of silk-fibroin-coated, emodin-loaded liposomes (SF-ELP), compared with uncoated, emodin-loaded liposomes (ELP), were investigated. SF-ELP had a highly organized lamellae structure, which contributed to 55% of the liposomal diameter. This modified liposomal structure decreased emodin release rates by changing the release kinetics from a swelling and diffusional process to a purely diffusional process, probably due to steric hindrance. SF-ELP also increased adhesion targeting to keloid fibroblasts. Increased retention of SF-ELP is most likely due to the interaction of the fibrous protein coating around the ELP with the pericellular molecules around the cell. SF-ELP also decreased survival rate of keloids that expressed high levels of RTK. These results demonstrated that SF-ELP enhanced emodin delivery by improved diffusion kinetics and specific cell targeting.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17722265&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

3. Nanolayer biomaterial coatings of silk fibroin for controlled release
Wang X, Hu X, Daley A, Rabotyagova O, Cebe P, Kaplan DL. (Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, USA.)
J Control Release. 2007 Aug 28;121(3):190-9. Epub 2007 Jun 14
An all-aqueous, stepwise deposition process with silk fibroin protein for the assembly of nanoscale layered controlled release coatings was exploited. Model compounds, Rhodamine B, Even Blue and Azoalbumin, representing small molecule drugs and therapeutically relevant proteins were incorporated in the nanocoating process and their loading and release behavior was quantified. In addition, the structure and morphology of the coatings were characterized. Release studies in vitro showed that control of beta-sheet crystal content and the multilayer structure of the silk coatings correlated with the release properties of the incorporated compounds. In particular, higher crystallinity and a thicker silk capping layer suppressed the initial burst of release and prolonged the duration of release. These novel coatings and deposition approach provide a unique option to regulate structure and morphology, and thus release kinetics. The results also suggest these systems as a promising framework for surface engineering of biomaterials and medical devices to regulate the release of drugs, when considered with the all-aqueous process involved, the conformal nature of the coatings, the robust material properties of silk fibroin, and the degradability and biocompatibility of this family of protein.

FullText:http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17628161&ordinalpos=14&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

4. Silk fibroin mediated delivery of liposomal emodin to breast cancer cells.
Cheema SK, Gobin AS, Rhea R, Lopez-Berestein G, Newman RA, Mathur AB. (University of Texas M.D. Anderson Cancer Center, Departments of Biomedical Engineering and Plastic Surgery, Unit 602, P.O. Box 301402, Houston, TX 72230-1402, United States.)
Int J Pharm. 2007 Aug 16;341(1-2):221-9. Epub 2007 Apr 3.
The efficacy of a drug is dependent on its mode of delivery and its potency at the tumor site. In this study, the drug delivery and efficacy of silk fibroin coated liposomes (SF-ELP), encapsulating a receptor tyrosine kinase inhibitor, emodin, on Her2/neu over-expressing breast cancer cells, was investigated. This study demonstrates that SF-ELP was more efficacious in suppressing the growth of Her2/neu over-expressing breast cancer cells MDA-MB-453 and BT-474 as compared to uncoated emodin loaded liposomes (ELP). Reduced levels of phosphorylated Her2/neu correlated with growth inhibition observed in the MDA-MB-453 cells, treated with both ELP and SF-ELP. ELP treatment of MDA-MB-453 breast cancer cells resulted in inhibition of the PI3K pathway whereas SF-ELP treatment inhibited both the PI3K and MAPK pathways, which contributed to the enhanced growth inhibitory effects of Her2/neu over-expressing breast cancer cells. Coating of ELP with silk fibroin did not alter the target specificity of emodin, on the other hand the emodin efficacy was enhanced. Higher uptake of emodin delivered by SF-ELP lead to increased cell death as compared to emodin delivery via ELP. Silk fibroin coating around the liposome imparts an extra layer that emodin has to extravasate in order to release from the encapsulating liposome. This increases retention of the drug in the cell for a longer time and protects emodin from quick release and metabolism. Longer intracellular retention may lead to the longer availability of emodin for down-modulation of various Her2/neu pathways. This study demonstrates that silk fibroin coating enhanced emodin delivery in Her2/neu over-expressing breast cancer cells thereby increasing the overall efficacy of the drug.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17499461&ordinalpos=19&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

5. Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans
Kimura T, Nakagawa K, Kubota H, Kojima Y, Goto Y, Yamagishi K, Oita S, Oikawa S, Miyazawa T. (Food & Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. kmr@affrc.go.jp)
J. Agric. Food Chem., 55 (14), 5869 -5874, 2007. 10.1021

Mulberry 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor, has been hypothesized to be beneficial for the suppression of abnormally high blood glucose levels and thereby prevention of diabetes mellitus. However, DNJ contents in commercial mulberry products were as low as about 0.1% (100 mg/100 g of dry product), implying that the bioavailability of DNJ might not be expected. We carried out studies in two directions: (1) production of food-grade mulberry powder containing a maximally high DNJ content; (2) determination of the optimal dose of the DNJ-enriched powder for the suppression of the postprandial blood glucose through clinical trials. The following method was used: (1) DNJ concentrations in mulberry leaves from different cultivars, harvest seasons, and leaf locations were determined using hydrophilic interaction chromatography with evaporative light scattering detection. (2) Healthy volunteers received 0, 0.4, 0.8, and 1.2 g of DNJ-enriched powder (corresponding to 0, 6, 12, and 18 mg of DNJ, respectively), followed by 50 g of sucrose. Before and 30-180 min after the DNJ/sucrose administration, plasma glucose and insulin were determined. The following results were obtained: (1) Young mulberry leaves taken from the top part of the branches in summer contained the highest amount of DNJ. After optimization of the harvesting and drying processes for young mulberry leaves (Morus alba L. var. Shin ichinose), DNJ-enriched powder (1.5%) was produced. (2) A human study indicated that the single oral administration of 0.8 and 1.2 g of DNJ-enriched powder significantly suppressed the elevation of postprandial blood glucose and secretion of insulin, revealing the physiological impact of mulberry DNJ (effective dose and efficacy in humans). This study suggests that the newly developed DNJ-enriched powder can be used as a dietary supplement for preventing diabetes mellitus.

Full text: http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2007/55/i14/abs/jf062680g.html