Mannose-binding hemagglutinins in extracts of Pseudomonas aeruginosa.

Mannose-binding hemagglutinins were found in the extracts of a pyocyanin-forming Pseudomonas aeruginosa, which contain galactose-specific hemagglutinins. They were purified simultaneously with the latter proteins by heating to 70 degrees C, precipitating with ammonium sulfate, application to a Sepharose 4B column, and elution from it by 0.05 M mannose. The mannose-specific hemagglutinins were shown to be similar to the galactophilic ones in (a) being glycoproteins of very low molecular weight (about 11 000 by SDS gel electrophoresis), (b) their tendency to aggregate, and (c) their ability to effect stronger agglutination of erythrocytes treated with papain than of untreated ones. They were found to resemble them also in their reaction with simple sugars and interactions with divalent cations, which are essential for their activity. In these properties, as well as in their relative resistance to heat and to proteolytic enzymes, these two types of bacterial hemagglutinins are like most of the plant, contrasted with the animal, hemagglutinins. The reactions with mannose and mannose-bearing compounds (yeast mannan, horseradish peroxidase (EC 1.11.1.7), and serum globulins), which are not shared with the galactophilic Pseudomonas hemagglutinins, indicate a relationship of the mannose-binding protein of Pseudomonas to the plant lectin concanavalin A. The mannose-binding hemagglutinins do not exhibit identical cell-agglutinating spectra owing to difference in profiles of sugar specificity and relative affinity to mannose derivatives compared with free mannose.     

α-葡萄糖苷酶抑制剂的构效关系

为建立抑制剂与α 葡萄糖苷酶之间的构效关系 ,按照Dixon的方法测定了多种糖及糖衍生物的抑酶性 .共出现 3种Dixon曲线 ,分别代表了 3种不同的酶结合方式 .这些糖和糖衍生物的结构变化 (如羟基构象、C 1位取代基、聚合度等的变化 )都会影响其与酶的结合能力 .结果表明 ,与酶有高结合力的物质需要满足一定的结构要求 ,如恰当的羟基构象、阳离子、共价连接的环形成的半椅状或椅状构型等     

Linker and/or transmembrane regions of influenza A/Group-1, A/Group-2, and type B virus hemagglutinins are packed differently within trimers

Influenza virus hemagglutinin is a homotrimeric spike glycoprotein crucial for virions' attachment, membrane fusion, and assembly reactions. X-ray crystallography data are available for hemagglutinin ectodomains of various types/subtypes but not for anchoring segments. To get structural information for the linker and transmembrane regions of hemagglutinin, influenza A (H1-H16 subtypes except H8 and H15) and B viruses were digested with bromelain or subtilisin Carlsberg, either within virions or in non-ionic detergent micelles. Proteolytical fragments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Within virions, hemagglutinins of most influenza A/Group-1 and type B virus strains were more susceptible to digestion with bromelain and/or subtilisin compared to A/Group-2 hemagglutinins. The cleavage sites were always located in the hemagglutinin linker sequence. In detergent, 1) bromelain cleaved hemagglutinin of every influenza A subtype in the linker region; 2) subtilisin cleaved Group-2 hemagglutinins in the linker region; 3) subtilisin cleaved Group-1 hemagglutinins in the transmembrane region; 4) both enzymes cleaved influenza B virus hemagglutinin in the transmembrane region. We propose that the A/Group-2 hemagglutinin linker and/or transmembrane regions are more tightly associated within trimers than type A/Group-1 and particularly type B ones. This hypothesis is underpinned by spatial trimeric structure modeling performed for transmembrane regions of both Group-1 and Group-2 hemagglutinin representatives. Differential S-acylation of the hemagglutinin C-terminal anchoring segment with palmitate/stearate residues possibly contributes to fine tuning of transmembrane trimer packing and stabilization since decreased stearate amount correlated with deeper digestion of influenza B and some A/Group-1 hemagglutinins.     

Formylchromone derivatives as irreversible and selective inhibitors of human protein tyrosine phosphatase 1B. Kinetic and modeling studies

A series of formylchromone derivatives were synthesized as PTP1B inhibitors and some of them were potent against PTP1B with IC50 values as low as 1.0 microM. They exhibited remarkable selectivity for PTP1B over other human PTPases. Kinetic studies revealed that formylchromone derivatives are irreversible and active site-directed inhibitors. Molecular modeling study identified the orientation of the inhibitor bound at the active site of PTP1B.     

Monoamine oxidase inhibition by N-alkyloxycarbonyl derivatives of ethylene diamine

Urethane type derivatives of ethylene diamine (EDA) were synthesized and tested as inhibitors of rat liver mitochondrial monoamine oxidase (MAO) A and B. The nature of the aromatic ring and the position of substituents in it were crucial for manifestation of the inhibitory activity. 3,4- and 2,4-Chlorobenzyloxycarbonyl-EDA derivatives were the most potent MAO A inhibitors. The inhibition of both MAO A and to a lesser extent MAO B depended on preincubation time with these inhibitors. The activity of both enzymes did not recover completely after repeated sedimentation and resuspension of inhibitor-treated mitochondria. The data suggest that these compounds exhibit properties of tight-binding reversible inhibitors of MAO A and B. The development of a new generation of MAO inhibitors causing simultaneous reversible nonselective inhibition of MAO A and B must meet one important criterion, the same type of inhibition of both the enzymes.     

萘查耳酮类化合物的设计合成及对CYP1B1酶的抑制活性评价

目的:设计并合成几类新型的萘查耳酮衍生物,初步测定其对CYP1B1酶的抑制活性,筛选具有良好抗癌作用的CYP1B1抑制剂。方法:以1,5-二羟基萘为原料,首先合成两个重要的中间体2-乙酰基-1,4,5,8-四甲氧基萘、1,5,6-三甲氧基-2-萘乙酮,然后利用羟醛缩合反应合成所需要的化合物。结果:合成了19个目标化合物,其结构均经过核磁共振氢谱确证,所有化合物均为全新化合物。对所合成的新化合物均进行了EROD酶实验测定。结论:所合成的化合物均具有较强的CYP1B1酶一抑制活性,其中4-1、4-2、5'-1对CYP1B1的抑制活性强于CYP1B1强抑制剂α-萘黄酮(IC50=11 nmol/L),他们的IC50分别为6.5、0.47、8nmol/L。     

Inhibition of cell-mediated lympholysis by cloned and uncloned lines of natural killer cells and cytotoxic T lymphocytes with sugars and lectins

This study was designed to further understand the nature of the interaction between natural killer (NK) cells and their susceptible targets. To do this, a panel of sugars and two lectins was tested for the ability to inhibit the lysis of NK-sensitive targets by cloned and uncloned lines of human NK cells. Six of these sugars (beta-gentiobiose, sucrose, alpha-lactose, beta-lactose, N-acetylglucosamine, and N-acetylgalactosamine) and one lectin, wheat germ agglutinin (WGA), proved to be potent inhibitors of the lytic activity of NK cells as well as of cytotoxic T lymphocytes activated in mixed lymphocyte cultures. Both beta-gentiobiose and WGA were shown to inhibit lysis at the level of the killer cell. Finally, the inhibitory effect of WGA could be reversed by addition of its sugar ligand, N-acetylglucosamine, which is itself an inhibitor of lytic function. From these findings it is concluded that these inhibitors probably do not act at the recognition stage of lysis since all of the NK and CTL lines tested, regardless of specificity, were inhibited by the same panel of sugars and lectins. Instead, it appears more likely that these inhibitors block some postrecognition stage of the lytic mechanism. The common inhibition profile by these sugars on NK and CTL activity further suggests that these two cell types may share, at least partially, a common lytic mechanism.     

Inhibition of glycoprotein processing by L-fructose and L-xylulose

A number of unusual and rare carbohydrates were tested as potentialinhibitors of various glycosidases, as well as inhibitors ofN-linked oligosaccharide processing. The best inhibitors ofseveral arylglycosidases and of glucosidase I were L-xyluloseand L-fructose. Both of these sugars showed some inhibitoryactivity towards yeast     

An o-toluidine method for detection of carbohydrates in protein hydrolysates

The o-toluidine high-performance thin-layer chromatography (HPTLC) method for detection of reducing sugars has been demonstrated to be a facile method for composition analysis of protein hydrolysates with a maximum sensitivity range of 50-100 pmol. The solution phase reaction of o-toluidine with reducing sugars has been previously used for spectrophotometric detection of glucose at 480-630 nm. In contrast, the heterogeneous reaction of o-toluidine with reducing sugars resolved by thin-layer chromatography produces chromophoric derivatives which have a broad absorbance at 295 nm. Detection of these chromophoric derivatives is achieved by uv diffuse reflectance scanning densitometry. It is demonstrated that detection limits of less than 10 ng can be achieved by using HPTLC plates and is therefore equal or more sensitive for some sugars than recently reported high-pressure liquid chromatography methods using amperometric or fluorescence detection.     

Transport of 4-deoxy- and 6-deoxy-D-glucose in baker's yeast

Tritium-labelled 4-deoxy-D-glucose (4-dglc) and 6-deoxy-D-glucose (6-dgcl) were prepared by catalytic hydrogenolysis of the corresponding deoxyiodo derivatives with gaseous tritium. The two sugars are transported into Saccharomyces cerevisiae by both the constitutive glucose and the inducible galactose carrier. Uranyl ions are powerful inhibitors. The pH optimum in uninduced cells lies at 5.5 for both sugars, the apparent activation energies (between 15 and 35 degrees C) are 25.1 kJ/mol and 16.5 kJ/mol, respectively. The steady-state intracellular concentration of both sugars is less than the extracellular one (no uphill transport). Neither of them is a substrate of yeast hexokinase. 4-Deoxy-D-glucose undergoes a dinitrophenol-sensitive conversion to an unknown metabolite which is not phosphorylated and may represent one of its oxidation products.     

Identification of aryl dihydrouracil derivatives as palm initiation site inhibitors of HCV NS5B polymerase

Aryl dihydrouracil derivatives were identified from high throughput screening as potent inhibitors of HCV NS5B polymerase. The aryl dihydrouracil derivatives were shown to be non-competitive with respect to template RNA and elongation nucleotide substrates. They demonstrated genotype 1 specific activity towards HCV NS5B polymerases. Structure activity relationships and genotype specific activities of aryl dihydrouracil derivatives suggested that they bind to the palm initiation nucleotide pocket, a hypothesis which was confirmed by studies with polymerases containing mutations in various inhibitor binding sites. Therefore, aryl dihydrouracil derivatives represent a novel class of palm initiation site inhibitors of HCV NS5B polymerase.     

On the specificity of the D-galactose-binding lectin (PA-I) of Pseudomonas aeruginosa and its strong binding to hydrophobic derivatives of D-galactose and thiogalactose.

The D-galactose-binding lectin (PA-I) from the bacterium Pseudomonas aeruginosa, isolated by affinity chromatography on Sepharose, was examined for its relative affinities for simple sugars and their derivatives using equilibrium dialysis and hemagglutination inhibition tests. The lectin, which was found to bind 0.68 mol of D-galactose per subunit of 12.8 kDa, exhibited an association constant (Ka) of 3.4 x 10(4) M-1 for D-galactose and higher affinities for hydrophobic and thio derivatives of D-galactose (with highest affinity for the hydrophobic thio derivatives). alpha-Methyl-galactoside was a stronger inhibitor than the beta-methyl derivative and alpha-lactose was a weak inhibitor but the hydrophobic phenylated derivatives of the beta-configuration of D-galactose were more potent inhibitors than the respective alpha-galactosides.     

Acquired B antigen: further studies using synthetic oligosaccharides

The reactivity of acquired-B red cells with various antisera has been investigated by agglutination inhibition assays using four trisaccharides obtained by chemical synthesis. Two of these had the structure GalNAc alpha 1-3 (LFuc alpha 1-2). Gal and Gal alpha 1-3 (LFuc alpha 1-2) Gal which are characteristic of the A and B determinants respectively and were indeed strong inhibitors of human anti-A and -B antibodies. The other two sugars denoted B-OAc and B-NH2 are derivatives of the B-trisaccharide by substitution of the hydroxyl group on carbon-2 of the alpha-galactose residue with the O-acetyl group (B-OAc) or the amino group (B-NH2) respectively. We have shown that both B and B-NH2 trisaccharides inhibited strongly the agglutination of acquired B red cells by the anti-B reagents (crude or affinity purified) whereas sera containing "anti-acquired B" agglutinins were specifically inhibited by B-NH2 but not by the A, B or B-OAc structures. We have also shown that the agglutination of Tk-activated erythrocytes by the BS-II lectin is specifically inhibited by N-acetylglucosamine but not by B, B-OAc or B-NH2 structures. These results and the observation that anti-acquired B agglutinins cannot be adsorbed on Tk red cells suggest that (i) the "B-like" and the "acquired-B" determinants share a common structure best represented by B-NH2 and (ii) Tk and "acquired-B" antigens are not identical.     

New developments in the synthesis of phosphopolyprenols and their glycosyl esters.

Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of alpha- and beta-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.     

Hemagglutinin activity in Nereis coelomic fluid

1. Nereis coelomic fluid agglutinates rat, mouse, chicken, guinea pig and rhesus monkey erythrocytes (RBC). 2. Lipid fractions of the particulate matter from coelomic fluid are hemagglutinins exhibiting different activity inhibition profiles with complex polysaccharides. 3. The high mol. wt hemagglutinin from coelomic fluid supernatant is not a protein and is inhibited by bovine submaxillary mucin (BSM), thyroglobulin, transferrin and their asialo derivatives. 4. Coelomic fluid supernatant has a population of low mol. wt protein hemagglutinins inhibited by BSM, fetuin, antiserum to coelomic fluid and some mannan preparations. 5. Hemagglutination by lipids characterized by RBC specificity and specificity for inhibition by carbohydrate is noteworthy and may be significant in studies of cellular interactions and immunity in invertebrates.     

Sugar specificity of anti-B hemagglutinin produced by Streptomyces sp

A hemagglutinin specific for blood group B antigen has been purified to 190-fold from the culture fluid of a strain of $\text{Streptomyces}$ sp. by conventional procedure involving ammonium sulfate fractionation and column chromatography. The molecular weight of the partially purified preparation was estimated to be approximately 5000±1000; this value is extremely small as compared with those of hemagglutinins which have been so far isolated from various sources.Hemagglutination-inhibition tests revealed that the $\text{Streptomyces}$ agglutinin has a specificity to combine with D-galactose and several saccharides having D-galactose residues at the non-reducing terminal, and that the special configuration of the hydroxyl groups at C-2 and C-4, particularly the hydroxyl group at C-2, is essential for binding of the sugars to the hemagglutinin.     

Comparative immunochemical study of the hemagglutinins of Cl. botulinum A and B]

The authors demonstrated an incomplete indentity of Cl. botulinum hemagglutinins of types A and B in the double diffusion reaction in agar gel, and their difference by electrophoretic mobility. Some differences in the interaction of hemagglutinins A and B with human erythrocytes were found by the hemagglutination inhibition method; apparently, of the principal significance in the relization of the reaction of human erythrocyte hemagglutination with hemagglutinins of Cl. botulinum, types A and B, was the OH-group position in the C4 galactose of the mucopolysaccharides of the erythrocyte cell wall. Apart from C4, apparently, for hemagglutinin of types A of significance was the reactive capacity of C1 and C2 galactose atoms, whereas for hemagglutinin of type B--free OH-group in C2 galactose atom.     

Amino acids and peptides. XV. Synthesis of Gln-Val-Val-Ala-Gly and derivatives, a common sequence of thiol proteinase inhibitors and their effects on thiol proteinase

The Gln-Val-Val-Ala-Gly sequence, which occurs frequently in several natural thiol proteinase inhibitors, and derivatives were synthesized by conventional solution methods and their effect on thiol proteinases were examined. The studies led us to the conclusion that certain of these peptides exhibited a weak inhibitory effect on the thiol proteinase, papain. One of them, Z-Gln-Val-Val-Ala-Gly-OMe, showed a protective effect on papain from natural thiol proteinase inhibitor-induced inactivation. The relationship between structure and activity of these derivatives was studied and certain conclusions were derived on possible mode of action of these inhibitors. From these studies, it was concluded that Z-Gln-Val-Val-OMe was the smallest peptide to exhibit some effect on papain.     

Novel 1-alkynyl substituted 1,2-dihydroquinoline derivatives from nimesulide (and their 2-oxo analogues): a new strategy to identify inhibitors of PDE4B

A number of novel 1-(3-arylprop-2-ynyl) substituted 1,2-dihydroquinoline derivatives related to nimesulide and their 2-oxo analogues have been designed as potential inhibitors of PDE4. All these compounds were synthesized by using Sonogashira coupling as a key step. In vitro PDE4B inhibitory properties and molecular modeling studies of some of the compounds synthesized are presented.