Structural studies of Bombyx mori silk fibroin during regeneration from solutions and wet fiber spinning

Regenerated silk fibroin materials show properties dependent on the methods used to process them. The molecular structures of B. mori silk fibroin both in solution and in solid states were studied and compared using X-ray diffraction, FTIR, and (13)C NMR spectroscopy. Some portion of fibroin protein molecules dissolved in formic acid already have a beta-sheet structure, whereas those dissolved in TFA have some helical conformation. Moreover, fibroin molecules were spontaneously assembled into an ordered structure as the acidic solvents were removed from the fibroin-acidic solvent systems. This may be responsible for the improved physical properties of regenerated fibroin materials from acidic solvents. Regenerated fibroin materials have shown poor mechanical properties and brittleness compared to their original form. These problems were technically solved by improving the fiber forming process according to a method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the native silk fiber and their physical and chemical properties were characterized by X-ray diffraction, solid state (13)C NMR spectroscopy, SinTech tensile testing, and SEM.     

From EST sequence to spider silk spinning: identification and molecular characterisation of Nephila senegalensis major ampullate gland peroxidase NsPox

Spider dragline silk is renowned as one of the toughest materials of its kind. In nature, spider silks are spun out of aqueous solutions under environmental conditions. This is in contrast to production of most synthetic fibres, where hazardous solvents, high temperatures and pressure are used. In order to identify some of the chemical processes involved in spider silk spinning, we have produced a collection of cDNA sequences from specific regions of Nephila senegalensis major ampullate gland. We examined in detail the sequence and expression of a putative Nephila senegalensis peroxidase gene (NsPox) from our EST collection. NsPox encodes a protein with similarity to Drosophila melanogaster and Aedes aegypti peroxidases. Northern analysis and in situ localisation experiments revealed that NsPox is expressed in major and minor ampullate glands of the spider where the main components of the dragline silk are produced. We suggest that NsPox plays a role in dragline silk fibre formation and/or processing.     

Solvent and solid-phase extraction of natural and synthetic anabolic steroids in human urine

Liquid–liquid (using dichloromethane) and liquid–solid extraction processes (using disposable C₁₈ cartridges) were applied to human urine samples spiked with 15 androgenic anabolic steroids (natural and synthetic). The extraction recoveries were assessed from different HPLC separations of anabolic steroids using water–acetonitrile mobile phase, and using calibration graphs obtained by injection into HPLC of standard samples of these compounds before and after extraction. The procedures, including sample preconcentration, showed extraction efficiencies over 90% which were independent on a wide range of concentrations tested. Solid phase extraction yielded poor results for oximetolone, danazol and dehydroepiandrosterone. For real urine samples, hydrolysis using β-glucuronidase and washing using sodium hydroxide before and after solvent extraction, respectively, is recommended.     

Hollow fiber supported gas membrane for in situ removal of ammonium during an antibiotic fermentation

To study the influence of ammonium on an antibiotic cultivation, mass transfer measurements of ammonium through microporous hydrophobic membranes using different stripping methods were carried out and compared. The higher overall mass transfer coefficients for ammonium were obtained with an acid stripping solution compared to water, vacuum, or sweeping air. A hollow fiber module for in situ removal of ammonium during cultivation was designed and operated in an external bypass to a 10-L fermentor. Compared to a control fermentation, the cell dry mass could be increased 2.6 times and the antibiotic concentration 8 times, if the in situ ammonium removal was in operation.     

Differentiation of fiber types in wing muscles during embryonic development: effect of neural tube removal

The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a sample muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.     

Spider silks: recombinant synthesis,assembly, spinning,and engineering of synthetic proteins

Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins.     

Solvent extraction method used in separating the protected product of synthetic oligodeoxyribonucleotides

A solvent extraction method for separating synthetic protected oligodeoxyribonucleotides was used in our laboratory based on the lipophilic property of the protecting group of 5'-OH of the oligomers. The extraction of synthetic products protected with MMTr is complete by ether or ether-chloroform (6:1 V/V) for mononucleosides, by chloroform for dinucleoside monophosphates, by dichloromethane:n-butanol (4:1 V/V) for trimer or tetramer, and is nearly complete by dichloromethane:n-butanol (2:1 V/V) for hexamer. The 5'-end phosphorylated nucleotides, oligonucleotides and their symmetrical pyrophosphates remain in water phase. The following synthetic products of protected oligodeoxyribonucleotides have been isolated with this method, all above 85% in purity: (Formula: see text).     

Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber.

Spider dragline silk is a unique protein fiber possessing both high tensile strength and high elasticity. A partial cDNA clone for one dragline silk protein (Spidroin 1) was previously isolated. However, the predicted amino acid sequence could not account for the amino acid composition of dragline silk. We have isolated a partial cDNA clone for another dragline silk protein (Spidroin 2), demonstrating that dragline silk is composed of multiple proteins. The amino acid sequence exhibits an entirely different repetitive motif than Spidroin 1. Spidroin 2 is predicted to consist of linked beta-turns in proline-rich regions which alternate with beta-sheet regions composed of polyalanine segments. This structure for Spidroin 2 provides a model for dragline silk structure and function.     

废水中的脱氮除硫研究

随着氮、硫污染物对江河湖泊污染的扩大,同步脱氮除硫已成为净化水体的一个重要课题。试验中采用UASB反应器,接种含有厌氧氨氧化菌的污泥处理含有氮硫的废水。通过硫酸盐取代亚硝酸作为电子受体,驯化同步脱氮除硫。进水浓氨氮和硫酸盐浓度分别控制在50～60mg·L^-1和210～240mg·L^-1的情况下,60天后得到一定的稳定处理效果,出水氨氮和硫酸盐浓度为30mg·L和160mg·L^-1。这种新型的脱氮除硫现象有助于为氮硫循环开辟新的途径。     

Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1

. Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater. The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration. The presence of viable, intact cells is strictly necessary for this removal to take place. Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks. Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company. Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant. Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress.     

Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1

Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater. The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration. The presence of viable, intact cells is strictly necessary for this removal to take place. Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks. Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company. Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant. Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress.     

Gender differences in the sweat response during spinning exercise

The purpose of this field study was to examine gender differences in the sweat response reported in the literature in trained men and women during indoor cycling. In the present study, 14 men and 12 women took part in a 90-minute spinning class in preparation for a 108-km road race. Delta body mass, corrected for the volume of water consumed, was used to estimate sweat loss during the exercise period. Men had a significantly higher sweat rate (1.12 L.h(-1)) compared to women (0.57 L.h(-1)), despite the fact that there were no significant gender differences in ad libitum fluid intake. Future research should focus on determining whether women may be more efficient in sweat production and evaporation and whether men may have a greater reserve capacity for increased sweating.     

Structure of model peptides based on Nephila clavipes dragline silk spidroin (MaSp1) studied by 13C cross polarization/magic angle spinning NMR

To obtain detailed structural information for spider dragline spidroin (MaSp1), we prepared three versions of the consensus peptide GGLGGQGAGAAAAAAGGAGQGGYGGLGSQGAGR labeled with 13C at six different sites. The 13C CP/MAS NMR spectra were observed after treating the peptides with different reagents known to alter silk protein conformations. The conformation-dependent 13C NMR chemical shifts and peak deconvolution were used to determine the local structure and the fractional compositions of the conformations, respectively. After trifluoroacetic acid (solvent)/diethyl ether (coagulant) treatment, the N-terminal region of poly-Ala (PLA) sequence, Ala8 and Ala10, adopted predominantly the alpha-helix with a substantial amount of beta-sheet. The central region, Ala15, Ala18, and Leu26, and C-terminal region, Ala31, of the peptide were dominated by either 3(1)-helix or alpha-helix. There was no indication of beta-sheet, although peak broadening indicates that the torsion angle distribution is relatively large. After 9 M LiBr/dialysis treatment, three kinds of conformation, beta-sheet, random coil, and 3(1)-helix, appeared, in almost equal amounts of beta-sheet and random coil conformations for Ala8 and Ala10 residues and distorted 3(1)-helix at the central region of the peptide. In contrast, after formic acid/methanol and 8 M urea/acetonitrile treatments, all of the local structure tends to beta-sheet, although small amounts of random coil are also observed. The peak pattern of the Ala Cbeta carbon after 8 M urea/acetonitrile treatment is similar to the corresponding patterns of silk fiber from Bombyx mori and Samia cynthia ricini. We also synthesized a longer 13C-labeled peptide containing two PLA blocks and three Gly-rich blocks. After 8 M urea/acetonitrile treatment, the conformation pattern was closely similar to that of the shorter peptide.     

Endotoxin removal using a synthetic adsorbent of crystalline calcium silicate hydrate

A synthetic adsorbent of crystalline calcium silicate hydrate, the product LRA by Advanced Minerals Corp., has been studied for endotoxin removal from aqueous solutions. This adsorbent removes endotoxin effectively, and the removal is greatly enhanced by the presence of an electrolyte such as NaCl, Tris-HCl, or Na2HPO4. It has an endotoxin removal capacity as high as 6 million endotoxin units (EU) per gram. Its endotoxin removal kinetics is fast, and for instance, over 99.9% endotoxin in a 5000 EU/mL solution was removed by mixing for 2 min at an adsorbent usage of 10 g/L. Using the chromatographic column method to treat a 5000 EU/mL solution, an endotoxin log-reduction factor of 6.2 was achieved with a single pass. This adsorbent also demonstrated significantly better performance when compared to many commonly used endotoxin removal agents, such as ActiClean Etox Endotoxin Removal Resin, Affi-Prep Polymyxin Support, Detroxi-Gel Endotoxin Removing Gel, Q Sepharose Fast Flow Media, and Sigma Endotoxin Removal Solution. Furthermore, it demonstrated a high selective removal of endotoxin from a solution of lambda DNA. This adsorbent provides opportunities for developing disposable, scaleable, and cost-effective methods for endotoxin reduction in many biotechnological and pharmaceutical processes.     

Ethene removal from a synthetic waste gas using a dry biobed

A packed granular activated carbon (GAC) biobed, inoculated with the ethane-degrading strain Mycobacterium E3, was used to study ethene removal from a synthetic waste gas. Ethene, for which the dimensionless partition coefficient for an air-water system at 20 degrees C is about 7.6, was used as a model compound for poorly water soluble gaseous pollutants. In a first mode or operation, the GAC biobed was sprinkled intermittently and the waste gas influent was continuously pre-humidified, establishing relatively moist conditions (water content >40% to 45%). A volumetric ethene removal rate of 0.382 kg COD . m(-3) . d(-1) (0.112 kg ethene . m(-3) . d(-1)) was obtained for an influent concentration of 125 ppm, a superficial waste gas velocity of 3.6E-3 m . s(-1) and a pseudo residence time of 45 s. However, in the second mode of operation, omitting the pre-humidification of the waste gas influent and establishing a "dry" biobed (water content <40% to 45%), and thus obtaining better mass transfer to the biofilm, the ethene removal could be doubled for otherwise comparable operating parameters. Furthermore, under decreased wetting and for the given experimental conditions (influent concentration 125 to 816 ppm, waste gas superficial velocity 3.0E-3 m .s(-1), pseudo waste gas residence time 43 s), the ethene removal was not limited by mass transfer of ethene through the water layer covering the biofilm. (c) 1994 John Wiley & Sons, Inc.     

Propene removal from synthetic waste gas using a hollow-fibre membrane bioreactor

 Hollow-fibre modules containing microporous membrane material were evaluated as bioreactors for waste gas treatment. The reactors were inoculated with the propene-utilizing strain Xanthobacter Py2, which formed a biofilm on the inner side of the fibres. The removal of the poorly soluble volatile propene from synthetic waste gas was monitored for up to 170 days. The maximum removal rates were 70–110 g propene per m³ reactor per hour. A gas residence time of 80 s was required to remove 95% of an initial propene concentration of 0.84 g/m³. The presence of ammonium in the liquid medium resulted in the development of an additional population of nitrifying organisms. Therefore, nitrate was used as the source of nitrogen in later experiments. During long-term operation, the propene removal rates gradually decreased. At low liquid velocities (1–5 cm/s) clogging of individual fibres with excess biomass was observed. Elevation of the liquid velocity in the fibres to 90 cm/s resulted in the formation of a dense biofilm and prevented clogging of the fibres. However, also at this high liquid velocity a gradual decrease in propene removal rate was observed. These results suggest that aging of biofilms is a very important factor in long-term operation of hollow-fibre bioreactors. Received: 24 November 1995 / Received revision: 14 February 1996 / Accepted: 20 February 1996     

棒络新妇卵袋结构与组成

采用SEM和氨基酸自动分析仪对棒络新妇 Nephila clavata 卵袋进行了结构观察和氨基酸组成分析.结果 表明棒络新妇的卵袋呈开放的网筛椭球状,结构简单,分为框架(大壶状腺丝)、金黄色或白色外层(主要含大壶状腺丝)和白色内层(主要含柱状腺丝)三部分,内外层都是由大(approx. 6～13 μm)、中(1.25～3.0 μm)、小(approx. 0.7～0.8 μm)直径的丝纤维构成.卵袋框丝和外层丝的氨基酸组成相似,甘氨酸(分别占22.9%和32.5%)和丙氨酸(占24%左右)的含量最丰富,其次是谷氨酸(占16.5%左右)和丝氨酸(分别占8.3%和3%),苏氨酸含量低(分别占2.12%和0.68%).与外层丝相比,卵袋内层丝纤维丝氨酸和苏氨酸的含量显著增加,分别为19.1%和6.11%左右,同时甘氨酸的含量显著减少,为7.2%左右,谷氨酸和脯氨酸的含量较低.大小侧链氨基酸比值(LC:SC)分析表明卵袋内层丝(1.03)﹥卵袋框丝(0.85)＞卵袋外层丝(0.66)≈拖牵丝(0.64)＞蚕丝(0.34),似乎表明卵袋框丝是不同于同纺自大壶状腺的典型蛛丝(拖牵丝)的新型蛛丝纤维.卵袋内层的主要构成成分是另一种具有截然不同氨基酸组成的柱状腺丝.棒络新妇能调节卵袋层丝的颜色、直径、丝纤维的组成及覆盖层的氨基酸组成,使卵袋获得各方面最优化的功能,为卵或若蛛提供支持和保护.     

High nitrate removal from synthetic wastewater with the mixed bacterial culture

The applicability of the mixed bacterial culture, originated from two-stage anaerobic-aerobic industrial yeasts production wastewater treatment plant for high rate denitrification processes was investigated. After acclimation to nitrate, the dominant strains were Pseudomonas and Paracoccus sp. Complete denitrification with low accumulation of nitrite-N (0.1 mg/l) was found in synthetic wastewater, obeying a zero-order reaction with respect to nitrate and a first-order reaction with respect to biomass concentration. Denitrification was then monitored in the continuous-flow stirred reactor at different hydraulic retention time, HRT (62-28 h) in order to achieve the optimal HRT. Nitrate was completely removed during following 45 days, at 25 degrees C with HRT, which we reduced from 62 to 28 h. Yet still, at 28 h HRT, high average specific denitrification rate of 142 mg NO3- -N/g VSS h was obtained.