GLycol-EThanol-WAter (3:3:4); A SOlvent for SUdan BLack B STaining

The previously used supersaturated 60% isopropanol method (Lillie 1954, p. 303) protected the more readily soluble lipids from the solvent action of 70% alcohol but precipitation of dye particles within and around sections is sometimes a disturbing complication (cf. Vlachos 1959). The propylene glycol method of Chiffelle and Putt (Lillie, p. 304) has been found to remove much of the birefringent lipid from the sections. Glacial acetic acid dissolves most tissue lipids except those of the solvent-resistant ceroid or lipofuscin type. While solutions in 60–70% acetic acid conserve ordinary fats well (Sills and Marsh 1959) their effect on the more easily soluble lipids should be further explored.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotriene B4 has been developed. The assay is sensitive; 5 pg LTB4 caused significant inhibition of binding of [3H]-LTB4 and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B2 and arachidonic acid do not exhibit detectable cross-reactions (less than 0.03%). However, some non-cyclic dihydroxy- and monohydroxy-eicosatetraenoic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic and 12-hydroxy-5,8,10,14-eicosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB4 from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB4 by reverse phase high pressure liquid chromatography following solvent extraction and silicic acid chromatography.     

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.     

Bacillus thuringiensis Cry4A and Cry4B mosquito-larvicidal proteins: homology-based 3D model and implications for toxin activity

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B delta-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel beta-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting alpha4 and alpha5 of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.     

2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF); antimicrobial compound with cell cycle arrest in nosocomial pathogens

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF), an aroma compound found in a number of fruits and foods, has shown various biological properties in animal models, but its antimicrobial effect remains poorly understood. The current study investigated the antimicrobial effect of DMHF using human pathogenic microorganisms including clinically isolated antibiotics-resistant strains. The results indicated that DMHF exhibited broad spectrum antimicrobial activities in an energy-dependent manner without hemolytic effect on human erythrocytes. To confirm antifungal effect of DMHF, we investigated the effect on dimorphism of Candida albicans induced by FBS, which plays a key role for pathogenesis in host invasion. The result showed that DMHF exerted a potent antifungal activity on the serum-induced mycelia of C. albicans. To elucidate the physiological changes of the fungal cells induced by DMHF, cell cycle analysis was performed, and the results showed that DMHF arrested the cell cycle at the S and G2/M phase in yeast. Therefore, it could be expected that DMHF may have potential as an anti-infective agent in human microbial infections.     

大豆11S球蛋白Gy5(A3B4)的基因克隆和序列分析

大豆11Ｓ球蛋白(Ｇｌｙｃｉｎｉｎ)是大豆种子的主要贮藏蛋白,分子量为360ｋＤ,由6对相同的蛋白亚基(每对亚基的分子量约60ｋＤ)构成。每对亚基又是由一个酸性Ａ肽(35～45ｋＤ)和一个碱性Ｂ肽(22ｋＤ)通过二硫键连接而成。Ａ肽和Ｂ肽源自同一个基因,即首先由一个大的ｍＲ?..     

Sturgeons (Pisces: Acipenseridae); threats and prospects

Life histories of 24 species of sturgeons are briefly summarized and threats to their existence are reviewed. The most important of these are fishing and physical disturbance of rivers (barriers; fluctuating water levels; gravel extraction). Conservation measures to limit and compensate for these impacts are reviewed. In Europe the common sturgeon, Acipenser sturio , is an endangered species; it has been the object of a conservation programme in France since 1982.     

Splice variants DNMT3B4 and DNMT3B7 overexpression inhibit cell proliferation in 293A cell line

DNA methyltransferase 3B (DNMT3B) is critical in abnormal DNA methylation patterns in cancer cells. Nearly 40 alternatively spliced variants of DNMT3B have been reported. DNMT3B4 and DNMT3B7 are two kinds of splice variants of DNMT3B lacking the conserved methyltransferase motif. In this study, the effect of inactivation of DNMT3B variants, DNMT3B4 and DNMT3B7, on cell proliferation was assessed. pCMV-DNMT3B4 and pCMV-DNMT3B7 recombinant plasmids were developed and stably transfected into 293A cells. 293A cells transfected with plasmid pCMV-DNMT3B4 or pCMV-2B were then treated with G418 to the stable cell lines. After that, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method was used for testing the proliferation level, and flow cytometry was used to test cell cycle distribution of the cell line. The expression of p21 was detected by real-time PCR and Western blot. The methylation status of p21 promoter was detected by methylation-specific PCR (MS-PCR). It was found that DNMT3B4 and DNMT3B7 overexpression could inhibit cell proliferation and increase the expression of p21. Cell cycle analysis demonstrated that inactivation of DNMT3B variants overexpression inhibited cell cycle progression. Inactivation of DNMT3B variants overexpression facilitated p21 expression to delay 293A cell proliferation. These findings indicate that inactivation of DNMT3B variants might play an important role in cell proliferation correlating with the change of p21.     

Hexadecanedionic acid-sepharose 4B: A new tool for preparation of albumin-depleted plasma

Serum and plasma are the major sources of human material for clinical molecular diagnostics and drug discovery. However, due to the high abundance of some proteins, of which serum albumin (SA) is most prominent, lower-abundance proteins often remain undetectable in proteomic analysis of these body fluids. We have used hexadecanedionic acid (HDA) immobilized to Sepharose 4B to develop an affinity resin that is effective in the removal of SA from plasma. Two-dimensional gel analysis of the SA-depleted samples shows a significant enhancement of the low-abundance proteins and highly specific capture of serum albumin. The HDA resin shows better performance in terms of specificity than dye-based resins.     

Enzymatic synthesis of 3-O-methyl-4-O-β--galactopyranosyl--glucose (3-O-methyl-lactose); a potential agent for the assessment of intestinal lactase activity

Enzymatic synthesis of 3-O-methyl-4-O-β--galactopyranosyl--glucose (3-O-methyl-lactose) has been attempted using both galactosyltransferase and galactosidase activities. The transferase-catalysed reaction produces exclusively the desired product in β-1,4-glycosidic linkage whereas the galactosidase-catalysed reactions predominantly form a 1,6-linked disaccharide. With galactosidase, in order to change the regioselectivity, blocking of the 6-position of 3-O-methyl--glucose and anomeric modification of the acceptor structure were investigated. Although acetylation of the 6-position of 3-O-methyl glucose catalysed by lipase was successful, the synthesis of the desired disaccharide did not occur.     

A reevaluation of the chemotactic potency of leukotriene B4 (LTB4)

The chemotactic potency of leukotriene B4 (LTB4) was reevaluated based on an improved purification procedure which combines reversed phase and straight phase high pressure liquid chromatography (RP-HPLC and SP-HPLC). Socalled LTB4 isomer III prepared by RP-HPLC contains two double oxygenated 5,12-dihydroxy acids in addition to LTB4. On a molar basis, the chemotactic activity of LTB4 repurified by SP-HPLC was far greater than that of the other two 5,12-dihydroxy acids and comparable to that of formyl-methionyl-leucyl-phenylalanine (fMLP). The chemotactic activity of LTB4 isomer III is dependent upon the relative concentrations of the double oxygenated 5,12-dihydroxy acids and LTB4. Further purification of peak III by SP-HPLC is required before assessing the biologic activity of LTB4.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB4 (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB4 and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo-LTB4. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB4 was at 10(-7)M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB4 was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at c-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB4. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta6 double bond are all important structural features for chemotactic activity with delta6 stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies.(ABSTRACT TRUNCATED AT 250 WORDS)     

A GABA(B) mystery: the search for pharmacologically distinct GABA(B) receptors

The classification of neurotransmitter receptors into distinct pharmacological subtypes is of major importance in drug discovery. This quest is particularly important for neurotransmitter systems that are widely distributed. Because gamma-aminobutyric acid (GABA) receptors, both GABA(A) and GABA(B), are found throughout the neuroaxis, they are likely involved in all central nervous system functions. Accordingly, the therapeutic promise of GABA(B) receptor manipulation depends upon the identification of subtypes than can be specifically targeted.     

P alpha-chiral phosphorothioate analogues of bis(5''-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation.

Synthesis of Sp and Rp diastereomers of Ap4A alpha S has been characterized in two enzymatic systems, the lysyl-tRNA synthetase from Escherichia coli and the Ap4A alpha, beta-phosphorylase from Saccharomyces cerevisiae. The synthetase was able to use both (Sp)ATP alpha S and (Rp)ATP alpha S as acceptors of adenylate thus yielding corresponding monothioanalogues of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S. No dithiophosphate analogue was formed. Relative synthetase velocities of the formation of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S were 1:0.38:0.15, and the computed Km values for (Sp)ATP alpha S and (Rp)ATP alpha S were 0.48 and 1.34 mM, respectively. The yeast Ap4A phosphorylase synthesized (Sp)Ap4A alpha S and (Rp)Ap4A alpha S using adenosine 5'-phosphosulfate (APS) as source of adenylate. The adenylate was accepted by corresponding thioanalogues of ATP. In that system, relative velocities of Ap4A, (Sp)Ap4A alpha S and (Rp)Ap4A alpha S formation were 1:0.15:0.60. The two isomeric phosphorothioate analogues of Ap4A were tested as substrates for the following specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow lupin (Lupinus luteus) seeds hydrolyzed each of the analogues to AMP and the corresponding isomer of ATP alpha S; (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from E. coli produced ADP and the corresponding diastereomer of ADP alpha S; and Ap4A phosphorylase (EC 2.7.7.53) from S. cerevisiae cleaved the Rp isomer only at the unmodified end yielding ADP and (Rp)ATP alpha S whereas the Sp isomer was degraded non-specifically yielding a mixture of ADP, (Sp)ADP alpha S, ATP and (Sp)ATP alpha S. For all the Ap4A-degrading enzymes, the Rp isomer of Ap4A alpha S appeared to be a better substrate than its Sp counterpart; stereoselectivity of the three enzymes for the Ap4A alpha S diastereomers is 51, 6 and 2.5, respectively. Basic kinetic parameters of the degradation reactions are presented and structural requirements of the Ap4A-metabolizing enzymes with respect to the potential substrates modified at the Ap4A-P alpha are discussed.