Inhibition of human blood coagulation factor XIa by C-1 inhibitor

The inactivation of activated factor XI (factor XIa) and of its isolated light chain by C-1 inhibitor was studied. Irreversible inhibition was observed in a reaction in which no reversible enzyme-inhibitor complex was formed. The second-order rate constants for the inactivation of factor XIa or its light chain by C-1 inhibitor were 2.3 X 10(3) and 2.7 X 10(3) M-1 s-1, respectively. High molecular weight kininogen did not affect the rate of inactivation. The nature of the complexes formed between factor XIa or its light chain and C-1 inhibitor was studied by using sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis. Under nonreducing conditions, two factor XIa-C-1 inhibitor complexes were observed with apparent molecular weights of 230,000 and 300,000. Reduction of these complexes resulted in the formation of a single band with a molecular weight of 130,000. This band is also formed in the reaction of the isolated light chain of factor XIa with C-1 inhibitor. These results demonstrate that two C-1 inhibitor molecules can become bound to the light chains of a factor XIa molecule. In addition, the mechanism of interaction of factor XIa or its isolated light chain with C-1 inhibitor appears identical, and the rate of inactivation of the enzyme by C-1 inhibitor is very similar. Neither the heavy chain of factor XIa nor high molecular weight kininogen is significantly involved in the inactivation of factor XIa by C-1 inhibitor.     

加拿大红豆杉扦插苗中的化学成分研究

从加拿大红豆杉(Taxus canadensis)的扦插苗中分离出三个紫杉烷二萜化合物1~3,用一维和二维核磁共振技术鉴定它们的化合物结构,并根据HMBC实验结果修订了化合物3中H-9,H-10和C-9,C-10的化学位移的归属。这三个化合物均为首次从加拿大红豆杉中分离得到。     

An enzymically catalysed reaction between d-glucose and the protein cyst coat of the ciliate Colpoda steinii

1. The isolated protein cyst coat of Colpoda steinii reacted with [(14)C]glucose to bind (14)C label in a reaction that was not an artifact of bacterial contamination or of adsorption. 2. The reaction was enzymically catalysed, had optimum pH7.0-7.4 and a temperature optimum of 36-38 degrees C, was non-competitively and reversibly inhibited by 10mum-p-hydroxymercuribenzoate and was specific for d-glucose. It had K(m)8.2x10(-6)m. 3. With the exception of C-1, all the carbon atoms of glucose were bound to the same extent and, measured relative to C-2 or C-6, the binding of C-1 varied between 0.45 and 0.82. The lost C-1 was not accounted for as carbon dioxide. 4. On prolonged incubation a coat preparation reacted with 3.6% of its own weight of glucose. 5. The label was tightly bound, but after acid treatment a variable proportion was recovered as glucose and there was no evidence for the release of any other (14)C-containing compound. 6. Even after dissolution of the coat protein, bound label was not removed by treatment with periodate or lead tetra-acetate.     

Novel taxa-4(20),12-diene and 2(3→20)abeotaxane from needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Phosphorylation of Charge Isomers (Components) of Human Myelin Basic Protein: Identification of Phosphorylated Sites

Myelin basic protein isolated from normal human brain was resolved into its various components (charge isomers) by CM-52 column chromatography. Two of the components C-1 and C-4, were phosphorylated in vitro with a soluble preparation of brain protein kinase C. For each component, the peptides phosphorylated were identified. In both components a major site of phosphorylation was found at Ser7 in the N-terminal portion of the protein. Both the specific activity and the rate of phosphorylation were greatest at this site in both components when compared with the other sites. The rate of phosphorylation of peptide 5-13 was approximately 10 times greater than that of any of the other peptides derived from C-1, while the rate of phosphorylation of peptide 5-13 derived from C-4 was 10-20 times greater than that of any of the other peptides derived from C-4. In addition, peptide 5-13, which contained a major phosphorylation site in both C-1 and C-4, was phosphorylated at a faster rate in C-4 (460 cpm/nM/min) compared with C-1 (285 cpm/nM/min). Both the specific activity and the rate data presented in the present communication were correlated with the proportion of beta-structure in a previous study. In that study, C-1, which contained about 13% beta-structure before phosphorylation, increased to approximately 40% after phosphorylation. Construction of a model peptide of this N-terminal region, which included the phosphorylation site at Ser7, demonstrated that the beta-structure was stabilized by electrostatic interactions between the phosphate on Ser7 and the guanidyl groups of Arg5 and Arg9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata

Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic data and chemical evidence. These isolated compounds were assayed for their immunological activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activities in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing activities.     

螺卷毛壳霉代谢产物的抗菌活性研究(英文)

采用管碟法首次对螺卷毛壳霉（Chaetomium cochloides）的含硫次生代谢产物（1—5）进行了体外抗细菌和抗真菌活性测定,并对其构效关系进行了研究。活性测定显示化合物1—5对革兰氏阳性细菌（Staphylococcus aureus）具有较强的抑制活性,其中化合物1的抑菌活性最强。构效关系研究表明位于C-3’和C-6’之间的二硫桥为化合物的抑菌活性中心,C-3与C-11a之间的二硫桥和N-6与O—3’之间的大环结构能够增强活性。     

Germacranolides from Viguiera microphylla

Three new germacranolides, including two heliangolides (niveusin C-2′,3′-epoxide and 1,2-dehydroniveusin C-2′,3′-epoxide) and a germacrolide (3β-hydroxy-8β-epoxyangeloyloxycostunolide-1β,10α-epoxide) were isolated from Viguiera microphylla. Their structures were established by spectroscopic analyses, including extensive ¹H NMR and ¹³C NMR decoupling experiments and chemical transformations. X-ray diffraction analysis confirmed the structure of niveusin C-2′,3′-epoxide.     

Further evidence for the classical pentose phosphate cycle in the liver

Isolated rat hepatocytes were incubated with [3-(14)C]xylitol or d-[3-(14)C]xylulose plus xylitol or glucose at substrate concentrations. The glucose formed was isolated and degraded to give the relative specific radioactivities in each carbon atom. C-4 of glucose had the highest specific radioactivity, followed by C-3, with half to one-fifth that of C-4. Only about 1% of the total radioactivity was in C-1. The data are compared with the predictions of the classical pentose phosphate cycle [Horecker, Gibbs, Klenow & Smyrniotis (1954) J. Biol. Chem.207, 393-403], and the proposed new version of the pentose phosphate cycle in liver [Longenecker & Williams (1980) Biochem. J.188, 847-857], which they denoted as the ;L-type pentose cycle'. The Williams pathway predicts that the specific radioactivity of C-1 of glucose should be half that of C-4 (after correction for approximately equal labelling on C-3 and C-4 of hexose phosphate in the pathway involving fructose 1,6-bisphosphatase). The actual labelling in C-1 is 20-350-fold less than this. When the hepatocytes are incubated with phenazine methosulphate, to stimulate the oxidative branch of the pentose phosphate cycle, the predicted relationship between (C-2/C-3) and (C-1/C-3) ratios of specific radio-activities is nearly exactly in accord with the classical pentose phosphate cycle. Glucose and glucose 6-phosphate were isolated and degraded from an incubation of hepatocytes from starved/re-fed rats with [3-(14)C]xylitol. Although the patterns were of the classical type, there was more randomization of (14)C into C-2 and C-1 in the glucose 6-phosphate isolated at the end of the incubation than in the glucose which was continuously produced.     

New steroidal glycosides from rhizomes of Clintonia udensis

A total of 10 steroidal glycosides, together with three new spirostanol glycosides (6-8), a new furostanol glycoside (9), and a new cholestane glycoside (10), were isolated from the rhizomes of Clintonia udensis (Liliaceae). The structures of the new compounds were determined on the basis of extensive spectroscopic analyses, including 2-D nuclear magnetic resonance (NMR) data, and of hydrolytic cleavage followed by chromatographic or spectroscopic analyses. The isolated glycosides were evaluated for their cytotoxic activity against HL-60 leukemia cells. Spirostanol glycosides 1 and 2, and furostanol glycoside 4 showed cytotoxic activity with IC(50) values of 3.2+/-0.02, 2.2+/-0.12, and 2.2+/-0.06 microg/ml, respectively. Neither the spirostanol and furostanol saponins with a hydroxy group at C-1 (6 and 9) and C-12 (7 and 8) nor cholestane glycosides (5 and 10) exhibited apparent cytotoxic activity at a sample concentration of 10 microg/ml.     

Characterization of Mexican Bacillus thuringiensis strains toxic for lepidopteran and coleopteran larvae

Bacillus thuringiensis strains C-4, C-9, GM-7, and GM-10, isolated from northeast Mexico and selected for their high toxicity against lepidopteran and coleopteran pests, were characterized following United States Environmental Protection Agency (EPA)'s guidelines. Flagellar serotyping revealed that GM-7 and GM-10 belonged to serotype aizawai, whereas C-4, C-9 corresponded to the kumamotoensis serotype. GM-10 and C-9 were also shown to be the most effective against lepidoptera and coleoptera larvae, respectively. None of the tested strains produced beta-exotoxin or showed activity against mosquitoes. GM-7 and GM-10 were sensitive to R-41 and CP-51 phages. All strains synthesized crystal proteins of 130-140 kDa. PCR analysis showed that C-4, GM-7, and GM-10 strains expressed cry1 genes, and C-9 expressed cry3 and cry7/8 genes, but not cry1. However, the C-9 strain had no cross-reaction with antisera raised against Cry3A and Cry7A proteins. GM-7 and GM-10 were sensitive to R-41 and CP-51 phages. When the delta-endotoxin (crystal) from the four strains was subcutaneously injected to Balb/c mice, alone or in combination with spores, only C-4 and C-9 provoked tissue necrosis similar to that caused by the beta-exotoxin producer HD-41. Tissue necrosis was prevented with the injection of pentoxifylline, an inhibitor of tumor necrosis factor alpha (TNF-alpha) production, suggesting a role of this cytokine in the observed effect. Our results demonstrated that GM-7 and GM-10 strains are effective and suitable for control of lepidopteran pests and safe for mammals under EPA regulations. The potential of the C-9 strain for the control of several coleopteran pests, and the induction of tissue necrosis in mice by C-4 and C-9 strains, are discussed.     

Desaturation,chain scission,and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase

Stearoyl acyl carrier protein Delta(9) desaturase catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k(cat)/K(M) values for the O-8- and O-11-substituted acyloxy-ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k(cat) values were decreased to approximately 3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an omega-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate "register-shift" of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Delta9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered.     

Green tea catechins as a BACE1 (beta-secretase) inhibitor

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of green tea, which was suspected to be rich in catechin content, showed potent inhibitory activity. (-)-Epigallocatechin gallate, (-)-epicatechin gallate, and (-)-gallocatechin gallate were isolated with IC(50) values of 1.6 x 10(-6), 4.5 x 10(-6), and 1.8 x 10(-6) M, respectively. Seven additional authentic catechins were tested for a fundamental structure-activity relationship. (-)-Catechin gallate, (-)-gallocatechin, and (-)-epigallocatechin significantly inhibited BACE1 activity with IC(50) values of 6.0 x 10(-6), 2.5 x 10(-6), and 2.4 x 10(-6) M, respectively. However, (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin exhibited about ten times less inhibitory activity. The stronger activity seemed to be related to the pyrogallol moiety on C-2 and/or C-3 of catechin skeleton, while the stereochemistry of C-2 and C-3 did not have an effect on the inhibitory activity. The active catechins inhibited BACE1 activity in a non-competitive manner with a substrate in Dixon plots.     

Characterization of vacuolar membrane proteins changed in rice root treated with gibberellin

Rice vacuolar membrane proteins changed by gibberellin (GA) were analyzed using a proteome approach. Vacuolar membrane fractions were isolated using a discontinuous sucrose/sorbitol system and 10 proteins increased in vacuolar membrane of the root, treated with GA(3) as compared with control. Fructose-1,6-bisphosphate aldolase C-1 and vacuolar H(+)-ATPase (V-ATPase) increased in root vacuolar membrane by GA(3) interacted in rice roots. It suggests that aldolase C-1 regulates the V-ATPase mediated control of cell elongation that determines root growth.     

Novel Taxa-4(20),12-diene and 2(3→20)Abeotaxane from Needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Halomonas aidingensis sp. nov., a moderately halophilic bacterium isolated from Aiding salt lake in Xinjiang, China

Two Gram-negative moderately halophilic bacterial strains, designated Ad-1(T) and C-12, were isolated from Aiding salt lake of Xinjiang in China. The novel isolates were subjected to a polyphasic taxonomic study. Cells of these strains were cocci or short rods and motile with polar flagella. Colonies produced brown-red pigment. The isolates grew in the range of 0.5-25% (w/v) NaCl, pH 5.5-10.5 and 4-45°C. Analysis of their 16S rRNA gene sequences indicated that strains Ad-1(T) and C-12 belonged to the genus Halomonas showing 92.7-98.4% similarity with the type species. The isoprenoid quinones of the isolates were Q-9 and Q-8. The major cellular fatty acids were C18:1ω7c, C16:1ω7c/6c, C16:0, C12:0-3OH and C10:0. The DNA G + C contents of strains Ad-1(T) and C-12 were 64.6 and 63.9 mol%, respectively. The DNA relatedness between the two isolates was 89.2%. The similarities of these newly isolated strains with closely related type strains were lower than 35% at the genetic level. Based on phenotypic, chemotaxonomic and genetic characteristics, the representative strain Ad-1(T) is considered to be a novel species of the genus Halomonas, for which the name Halomonas aidingensis sp. nov. is proposed, with Ad-1(T) (= CGMCC 1.10191(T) = NBRC 106173(T)) as the type strain.     

Structure of dicarboxyl malto-oligomers isolated from hypochlorite-oxidised potato starch studied by 1H and 13C NMR spectroscopy.

The main oxidised component in hypochlorite-oxidised potato starch was isolated by anion-exchange chromatography after enzymatic hydrolysis. The primary structure of the isolated oligosaccharides was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The isolated pentamer and hexamer contained one glucose unit oxidised to a dicarboxyl residue. As the hypochlorite oxidation has occurred at positions C-2 and C-3 of a glucose unit, the introduced carboxyl groups caused ring cleavage between the carbons C-2 and C-3. The ring-cleaved dicarboxyl residue had glycosidic linkages on both sides, implying that this oxidation pathway does not result in depolymerisation. The vicinal coupling constant between H-4 and H-5 in the ring-cleaved dicarboxyl residue was 3.2 Hz, showing that the gauche orientations are preferred. As a result, a different bending of the starch chain is observed and is probably, therefore, one of the reasons why hypochlorite oxidation reduces the tendency to retrogradation. The pKa values (3.0) were determined from the pH-dependent chemical shifts of H-1, H-4 and H-5 of the dicarboxylic residue.     

Cyanidin-3-O-beta-glucopyranoside protects myocardium and erythrocytes from oxygen radical-mediated damages

The cyanidin-3- O - β-glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 μM C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 μM C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 μM C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 μM C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 μM was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 μM). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Localization within the heavy chain sequence of tyrosyl peptides from the combining sites in a rabbit anti-3-azopyridine antibody preparation

A relatively homogeneous rabbit heavy chain was cleaved by CNBr. Fragment C-1 (the N-terminal half of the heavy chain) was isolated. Reduction and alkylation of C-1 liberated three fragments and partial sequence analysis of these isolated fragments showed that C-1 had been split on the carboxyl-side of Met 84. Similar results were obtained with another anti-hapten antibody preparation in which tyrosyl residues in the combining sites had been labeled. The labeled tyrosyl residues were found in the fragment representing residues 85–253. Since the constant region begins at about residue 120 and the sequences of the tyrosyl peptides from the combining sites are not present in published constant region sequences, these peptides appear to be derived from a variable region between residues 85 and 120.     

NMR structural determination of dissolved O-acetylated galactoglucomannan isolated from spruce thermomechanical pulp

Water-soluble O-acetylated galactoglucomannan (GGM) isolated from spruce thermomechanical pulp (TMP) by hot-water extraction was characterized by 1D and 2D (homo- and heteronuclear) NMR analysis. The backbone was found to consist of (1-->4)-linked mannopyranosyl and glucopyranosyl units in a ratio of 10:1.9-2.6. The mannopyranosyl units were acetylated at C-2 and C-3 with a degree of acetylation around 0.28-0.37 as determined by NMR. A slightly larger amount of 2-O-acetylated mannopyranosyl was detected when compared to the 3-O-acetylated component. Approximately every 10th mannopyranosyl unit was substituted at C-6 by a single alpha-galactopyranosyl unit. Fine structure determination based on sequence-specific chemical shift variations showed that the distribution of glycosyl residues is random. Small amounts of other minor polysaccharide species including xylans and galactans could also be identified by NMR.