Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: Effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK_a values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CγH, whereas the deviation of pK_a from the reference value for Glu4 and Glu8 CγH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.     

Deimination of Human Myelin Basic Protein by a Peptidylarginine Deiminase from Bovine Brain

Abstract: A peptidylarginine deiminase (PAD; EC 3.5.3.15) has been isolated from bovine brain and some of its characteristics have been studied. The enzyme showed an absolute requirement for Ca²⁺, a temperature optimum at ～50°C, and two K_mvalues when benzoylarginine ethyl ester was used as substrate, 0.78 mMand 11.2 mM.The higher K_mhas not been reported previously. Protein substrates for the enzyme included polyarginine and myelin basic protein but not histones. Because one of the components of MBP contains six citrullinyl residues per mole, enzymic deimination appeared to be a likely mechanism. When the most cationic component (C-1) was subjected to PAD in solution, 17 of the 19 arginyl residues were modified. From sequence analyses we concluded that the nature of the amino acid residues adjacent to the deiminated arginine were not modifiers of the reaction as arginyl residues in a variety of environments were deiminated. This deimination was reflected in a large increase in random structure, as measured by [θ]₂₀₀. At 5°C, the [θ]₂₀₀of the deiminated protein was -70 × 10³ compared with -30 × 10³ deg cm²/dmol for the native protein. When the temperature was increased to 70°C, the [θ]₂₀₀ was -44 × 10³ for the deiminated protein and -20 × 10⁷ deg cm²/ dmol for the native C-1. When plotted as a function of temperature, [θ]₂₀₀ decreased linearly from 5°C to 50°C for both proteins and did not change from 50°C to 70°C. PAD provides a mechanism for deimination of arginyl residues of myelin basic protein. The selective deimination of the six arginyl residues that are consistently found deiminated in C-8 may be determined by the orientation of the protein in the membrane and/or the more complex lipid composition of myelin may affect the selectivity of the deimination.     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.     

Renal sodium-d-glucose cotransport system. Involvement of tyrosine residues in sodium-transporter interaction

Using brush-border membrane vesicles isolated from calf kidney cortex the effect of tyrosine-reactive reagents on sodium-dependent d-glucose transport was investigated. Treatment of the membranes for 60 min with NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole), N-acetylimidazole or tetranitromethane decreased d-glucose uptake 50, 70 and 40%, respectively. Tracer exchange experiments revealed that the inhibition of transport is due to a direct modification of the sodium-d-glucose cotransport system. The modification by NBD-Cl decreases the apparent V_max of the transport system with respect to its interaction with sodium. In addition, the rate of inactivation of the transport system by NBD-Cl is reduced in the presence of high concentrations of sodium. The results indicate that tyrosine residues play an essential role in sodium-d-glucose cotransport and are probably involved in the binding and/or transport of sodium by the sodium-d-glucose cotransport system.     

The locations of the three cysteine residues in the primary structure of the intrinsic segments of band 3 protein,and implications concerning the arrangement of band 3 protein in the bilayer

The intrinsic domains of band 3 protein contain three cysteine residues, one in a 17 kDa middle segment and two in a 35 kDa C-terminal segment. The latter are retained in an 8 kDa fragment produced by chymotrypsin treatment of ghosts. Cleavage of cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) allows localization of this amino acid in the primary structure of the 8, 17, 35 and 52 (17 plus 35) kDa segments of band 3 protein. The mapping of these residues taken with other information concerning accessibility of various sites at the two sides of the membrane leads to the conclusion that band 3 protein crosses the membrane at least five times, or ten times in a dimer structure. The implications of this conclusion in terms of band 3 protein structure and function are briefly discussed.     

Conjugation morphology of Zygogonium ericetorum (Zygnematophyceae,Charophyta) from a high alpine habitat

Reproductive characteristics are important for defining taxonomic groups of filamentous Zygnematophyceae, but they have not been fully observed in the genus Zygogonium. Specimens of Z. ericetorum previously studied and used to clarify the generic concept lacked fertile material, which was obtained recently. This study illustrates for the first time, using color light microscopic and fluorescence images, a consequent conjugation stage in Z. ericetorum, including completely developed zygospores and purple cytoplasmic residue content left outside the zygospores, similar to aplanospore formation. Structures confirmed earlier reports and provided new observation informative regarding phylogenetically relevant reproductive characters of Z. ericetorum.     

氰戊菊酯在茶叶和茶汤中的残留动态及最大残留限量评价

在生长均匀的茶园喷施氰戊菊酯(fenvalerate),采摘施药后2 h和1 h、2 h、3 h、5 h、7 h、9 h、14 h、21天的茶树鲜叶加工成绿茶,用气相色谱法测定成茶、茶汤和茶渣中反式氰戊菊酯和顺式氰戊菊酯的含量,研究了氰戊菊酯在成茶、茶汤中的残留动态。结果表明:反式氰戊菊酯和顺式氰戊菊酯在成茶中的残留水平随施药间隔天数的增加呈下降趋势,20 mL/667 m2施药剂量下,分别由施药当天2 h的9.40 mg/kg和17.51 mg/kg减小到第21天的1.07 mg/kg和1.53 mg/kg,消解幅度为88.62%和91.26%,40 mL/667 m2施药剂量下,分别由施药当天2 h的20.37 mg/kg和38.67 mg/kg减小到第21天的1.94 mg/kg和3.06 mg/kg,消解幅度为90.49%和92.09%。茶汤中氰戊菊酯含量(y)与成茶中氰戊菊酯含量(x)呈二项式函数关系,反式氰戊菊酯的函数关系为y=-0.0007x2 0.0242x,顺式氰戊菊酯的函数关系为y=-0.0002x2 0.0114x。按我国标准饮茶摄入的氰戊菊酯占每日允许摄入量的0.049%,足以达到保护人体健康水平的要求。而按欧盟的标准,饮茶摄入的氰戊菊酯占每日允许摄入量的0.0019%,即在10-5水平上,这样的风险水平已接近对非阈效应化学物质的风险控制水平(10-6)。     

Pyrolysis of olive residue and sugar cane bagasse: non-isothermal thermogravimetric kinetic analysis

Thermal degradation and kinetics for olive residue and sugar cane bagasse have been evaluated under dynamic conditions in the presence of nitrogen atmosphere, using a non-isothermal thermogravimetric method (TGA). The effect of heating rate was evaluated in the range of 2-50 K min(-1) providing significant parameters for the fingerprinting of the biomass. The DTG plot for the olive residue and sugar cane bagasse clearly shows that the bagasse begins to degrade at 473 K and exhibits two major peaks. The initial mass-loss was associated with hemicellulose pyrolysis and responsible for the first peak (538-543 K) whereas cellulose pyrolysis was initiated at higher temperatures and responsible for the second peak (600-607 K). The two biomass mainly devolatilized around 473-673 K, with total volatile yield of about 70-75%. The char in final residue was about 19-26%. Mass loss and mass loss rates were strongly affected by heating rate. It was found that an increase in heating rate resulted in a shift of thermograms to higher temperatures. Ozawa-Flynn-Wall and Vyazovkin methods were applied to determine apparent activation energy to the olive residue and sugar cane bagasse. Two different steps were detected with apparent activation energies in the 10-40% conversion range have a value of 153-162 kJ mol(-1) and 168-180 kJ mol(-1) for the hemicellulose degradation of olive residue and sugar cane bagasse, respectively. In the 50-80% conversion range, this value is 204-215 kJ mol(-1) and 231-240 kJ mol(-1) for the cellulose degradation of olive residue and sugar cane bagasse, respectively.     

川西北泡沙参种群根系生长动态及其与环境因素关系研究

对川西北地区泡沙参种群根系生物量累积和形态发育过程及其与环境因素的关系进行了研究.结果表明,泡沙参种群根系生物量累积一般规律符合Logistic增长过程.较高的泡沙参根系生物量累积和形态发育时期可以持续到15～17年生以后,根系采收直径应在1.7cm以上.动物啃食、人为采挖、土壤、气候等环境条件与不同海拔各种群的根系生物量累积等有着密切联系,中海拔地区(2800～3300m)人为干扰少,土壤和水热条件适宜,根系生物量和形态发育达到较高水平,适合高产栽培.而低海拔和高海拔地区的不利环境条件限制了泡沙参根系生长.在进行野生资源保护利用以及人工栽培泡沙参时应充分考虑环境因素,努力减少放牧、采挖等人为破坏,有条件的地区应实行分区禁牧、禁采挖,为泡沙参种群恢复和药材品质提高创造条件.     

消炎药药渣堆放过程中真菌多样性分析

中药药渣的处理是中药制药的难题,本研究利用PacBio Sequel高通量测序对堆放2年与5年的消炎药药渣进行理化性质及真菌多样性研究,为重要药渣的利用提供依据。分别对堆放2年(New 1-New 3)及5年(Old 1-Old 3)各3个消炎药渣高通量测序,共检测到43 753条有效序列,堆放2年的药渣中共检测到4个门、86个属、126个种,节担菌纲、小丛壳目、曲霉属为优势类群。堆放5年的药渣中检测到3个门、49个属、59个种,Chaetomium novozelandicum及小囊菌科为优势类群。消炎药药渣功能组成分析发现,堆放2年的药渣中腐生型真菌占比67.7%、致病真菌占比32.3%;堆放5年的药渣中腐生型真菌占比96.8%,仅检测到极少数其他型真菌。堆放2年的药渣pH为4.9±0.2,含水量为49.2%;堆放5年的药渣pH为3.6±0.1,含水量为20.8%。结果表明,随着堆放时间的增加,药渣pH及含水量下降,真菌营养类型由腐生、致病与共生三者丰度相当转变为单一的腐生型,说明随着堆放时间的增加,腐生真菌丰度增加,药渣降解加快,研究结果为药渣的利用提供了一定依据。