Novel urea and thiourea derivatives of thiazole-glutamic acid conjugate as potential inhibitors of microbes and fungi.

Since discovery and development of effective as well as safe drugs has brought a progressive era in human healthcare that is accompanied by the appearance of drug resistant bacterial strains, there is constant need of new antibacterial agent having novel mechanisms of action to act against the harmful pathogens. In the present study, several N-terminal substituted urea/thiourea derivatives were synthesized by the reaction of glutamic acid and 3-(1-piperazinyl)-1,2-benzisothiazole with various substituted phenyl isocyanates/isothiocyanates. Elemental analysis, IR, 1H NMR, 13C NMR and mass spectral data confirmed the structure of the newly synthesized compounds. The derivatives were investigated for their antibacterial and antifungal activities against various pathogens of human origin by agar well diffusion method and microdilution method. The preliminary antimicrobial bioassay reveals that the compounds containing fluoro and bromo as substituents showed promising antimicrobial activity.

CD36 modulates proinflammatory cytokine responses to Plasmodium falciparum glycosylphosphatidylinositols and merozoites by dendritic cells.

Studies have shown that glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum activate macrophages mainly through Toll-like receptor 2 (TLR2)-mediated signalling and to certain extent through TLR4-mediated signalling to induce proinflammatory cytokine production. However, the ability of parasite GPIs to activate dendritic cells (DCs) has not been reported. Here, we show that parasite GPIs efficiently activate DCs through TLR2-mediated signalling mechanism and induce the production of TNF-α and IL-12. We also studied the role of scavenger receptor CD36 in P. falciparum GPI- and merozoite-induced cytokine responses by DCs. The results indicate that CD36 modulates the cytokine-inducing activity of the parasite GPIs by collaborating with TLR2 in DCs. Furthermore, our data reveal that CD36 modulates the activity of P. falciparum merozoites, likely by the contribution of phagocytosis-coupled CD36-mediated signalling to the signalling induced by merozoites. Altogether, these results contribute towards understanding of signalling mechanisms in malaria parasite-induced activation of the innate immune system.

Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis.

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn-2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor alpha release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

Effect of GPI anchor moiety on the immunogenicity of DNA plasmids encoding the 19-kDa C-terminal portion of Plasmodium falciparum MSP-1.

The glycosylphosphatidylinositol (GPI)-anchored Plasmodium falciparum merozoite surface protein 1 (MSP-1) is a widely studied malaria vaccine candidate. The C-terminal 19-kDa portion of MSP-1 (MSP-1(19)) is of particular interest because this polypeptide moiety remains bound to the parasite even after erythrocyte invasion, while the remainder of MSP-1 is shed during invasion. Studies have shown that antibodies against MSP-1(19) inhibit merozoite invasion of erythrocytes efficiently, and that MSP-1(19) produces protective immunity in mice and monkeys. To investigate the efficacy of MSP-1(19 )DNA vaccine and role of GPI anchor moiety in the immunogenicity of MSP-1(19), we constructed expression vectors that produce MSP-1(19) as either secretory or GPI-anchored polypeptide. Both constructs efficiently expressed MSP-1(19) in transfected HEK-293 cells. While the recombinant plasmid lacking GPI anchor signal sequence expressed MSP-1(19) mainly as secreted polypeptide, that containing GPI anchor signal sequence produced GPI-anchored MSP-1(19 )on cell surface. In immunized mice, both constructs produced substantial levels of MSP-1(19)-specific IgG1, IgG2a, IgG2b, IgG3, IgA and IgM antibodies. In both cases, the IgG1 level was significantly higher than other isotypes. Interestingly, the plasmid containing GPI anchor signal sequence produced significantly higher levels of IgG2a and IgG2b than the plasmid that lacks GPI signal sequence.

Structure of the major oligosaccharide of cobra venom factor.

Cobra venom factor (CVF), the complement-activating glycoprotein in cobra venom, contains three or possibly four N-linked oligosaccharide chains per molecule and is devoid of O-linked saccharides. Analysis by lectin-affinity staining revealed the presence of complex-type oligosaccharides containing non-reducing terminal alpha-galactosyl residues and fucose residues linked to the proximal N-acetylglucosamine. Sialic acid residues could not be detected. For their structural analysis, the oligosaccharides were released by hydrazinolysis and fractionated on Bio-Gel P-4. Approximately 80% of the eluted oligosaccharides have a size equivalent of 17 +/- 2 glucose units. The major oligosaccharide representing about 45% of the total carbohydrate present in CVF was purified to homogeneity by MicroPak AX-5 HPLC and its structure was analyzed by sequential exoglycosidase digestion. The positions of the glycosidic linkages of the sugar residues were established by methylation analysis of CVF-derived glycopeptides. The data of these analyses indicated that the major oligosaccharide has a symmetrical fucosylated biantennary complex-type structure terminating with unusual alpha-galactosyl residues.

Occurrence of O-linked Xyl-GlcNAc and Xyl-Glc disaccharides in trocarin, a factor Xa homolog from snake venom.

Trocarin is a 46515-Da group D prothrombin-activating glycoprotein from the venom of the Australian elapid, Tropidechis carinatus. Amino acid sequencing and functional characterization of trocarin demonstrated that it is a structural and functional homolog of mammalian blood coagulation factor (F)Xa. In this study we show that, in contrast to mammalian Xa, which is not glycosylated, trocarin contains an O-linked carbohydrate moiety in its light chain and an N-linked carbohydrate oligosaccharide in its heavy chain. Mass spectrometry and sugar compositional analysis indicate that the O-linked carbohydrate moiety is a mixture of Xyl-GlcNAc-, GlcNAc-, Xyl-Glc- and Glc- structures linked to Ser 52. The N-linked carbohydrate on Asn 45 of the heavy chain is a sialylated, diantennary oligosaccharide that is located at the lip of the active site of the prothrombin activator.

Glycobiology of Plasmodium falciparum.

The human malaria parasite, Plasmodium falciparum, has as its only glycoconjugate GPI anchors. These structures, present in essentially all parasite surface proteins, are associated with disease pathology. In contrast, the parasite depends for essential recognition events on saccharides associated with host cell glycoproteins and proteoglycans.

Adherence of Plasmodium falciparum-infected erythrocytes to chondroitin 4-sulfate.

Adherence of Plasmodium falciparum-infected erythrocytes (PRBCs) to the microvascular endothelium of specific organs and consequent sequestration is believed to be responsible for the development of malaria pathology. A number of studies have shown that cell adhesion molecules expressed on the surface of endothelial cells mediate the adherence. Recent studies indicate that a subpopulation of PRBCs adhere to chondroitin 4-sulfate (C4S). This adhesion can be effectively inhibited by C4S oligosaccharides. In pregnant women, the placenta specifically selects C4S-adherent PRBCs, and thus these phenotypes multiply and sequester in the intervillous spaces. Over successive pregnancies, women develop a protective humoral response to the C4S-adhesion phenotype. Disruption of C4S-mediated PRBCs adhesion using either a C4S oligosaccharide mimetic or an antiC4S-adhesion vaccine can be an efficient strategy for the treatment of malaria caused by C4S-adherent P. falciparum.

A novel L-fuco-4-O-methyl-D-glucurono-D-xylan from Hyptis suaveolens.

The acidic polysaccharide from the seed-coat mucilage of Hyptis suaveolens is a highly branched L-fuco-4-O-methyl-D-glucurono-D-xylan for which a structure is proposed having a 4-linked beta-D-xylan backbone carrying side chains of single 4-O-methyl-alpha-D-glucuronic acid residues at O-2 and 2-O-L-fucopyranosyl-D-xylopyranose units at O-3. The structural analysis involves base-catalyzed beta-elimination of uronic acid residues from the methylated glycan followed by degradation using a modified Svensson oxidation-elimination sequence.

Kinetics and mechanism of oxidation of erythro-series pentoses and hexoses by N-chloro-p-toluenesulfonamide.

The kinetics and mechanism of oxidation of D-glucose, D-mannose, D-fructose, D-arabinose, and D-ribose with chloramine-T in alkaline medium were studied. The rate law, rate = k [Chloramine-T] [Sugar] [HO-]2, was observed. The rate of the reaction was influenced by a change in ionic strength of the medium, and the dielectric effect was found to be negative. The latter enabled the computation of dAB, the size of the activated complex. The reaction rate was almost doubled in deuterium oxide. Activation energies were calculated from the Arrhenius plots. HPLC and GLC-MS analyses of the products indicated that the sugars were oxidized to a mixture of aldonic acids, consisting of arabinonic, ribonic, erythronic, and glyceric acids. Based on these data, a plausible mechanism involving the aldo-enolic anions of pentoses and keto-enolic anions of hexoses is suggested.

Synthetic polypeptide sleeve for strabismus surgery.

A synthetic polypentapeptide sleeve was placed around the superior rectus muscle of five New Zealand white rabbits in hopes of preventing postoperative fibrous scarring. Two forms of the polypentapeptide were used. No significant inflammation or scarring occurred with either form of the polypentapeptide when compared to controls. One form elicited a fibrous membrane surrounding the sleeve within 2 weeks. The other elicited no such reaction after 2 months. The latter form of the polypentapeptide may be useful in preventing scarring following strabismus surgery.

Modification at C6 of the terminal galactosyl residues of cobra venom factor abolishes anti-alpha-Gal antibody immunoreactivity without affecting functional activity.

The N-linked oligosaccharides of cobra venom factor (CVF) contain unique terminal alpha-galactosylated Lewis X structures. We have previously shown that CVF immobilized on nylon membranes binds naturally occurring human anti-alpha-Gal antibody. The present study shows that soluble CVF can effectively inhibit the binding of anti-alpha-Gal antibody to CVF-coated microtiter plates, indicating that the terminal alpha-galactosyl residues of the functionally active CVF are accessible to anti-alpha-Gal antibody binding. Modification of the terminal galactosyl residues of CVF by treatment with galactose oxidase and in situ derivatization of the generated aldehyde groups with hydrazides abolished the human anti-alpha-Gal antibody immunoreactivity without affecting the complement-activating activity.

Isolation and characterization of novel mucin-like glycoproteins from cobra venom.

A high molecular weight, heavily glycosylated protein fraction was isolated from cobra venom. It consists of mucin-like glycoproteins (designated as cobra venom mucin) in noncovalent association with several lower molecular weight proteins and glycoproteins. The mucin was purified by CsBr density gradient centrifugation under dissociative conditions. The purified venom mucin comprised about 85% carbohydrate and 15% protein and was rich in Thr, Ser, Pro, Gly, Glu, Asp, and Ala. The mucin was resolved into two or more distinct classes of mucin-like glycoproteins which differ in their amino acid compositions and/or carbohydrate content. Unlike other mucins, cobra venom mucin does not form highly viscous solutions. It appears to keep several venom proteins and glycoproteins soluble by noncovalent interactions. Cobra venom mucin contains both O- and N-linked oligosaccharides; 1 N-linked chain for every 8-10 O-linked oligosaccharides. The O-linked chains are novel structures with high molar proportions of fucose, galactose, and N-acetylglucosamine relative to N-acetylgalactosamine; they have a very low sialic acid content and lack sulfate esters. The majority of the O-linked oligosaccharides are unusually large and contain 15 to as many as 50 sugar residues. The O-linked oligosaccharides are poly-N-acetyllactosaminyl chains consisting of -3Gal beta 1-4GlcNAc beta 1- and -3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1- repeats and thus they contain inner Le(X) antigenic determinants. These oligosaccharides terminate with novel alpha-galactosylated Le(X) and Le(a) epitopes. Due to the abundance of terminal alpha-galactosyl residues, cobra venom mucin reacts with anti-alpha-Gal antibodies that are normally present in human serum.

Structural features of carbohydrate moieties in snake venom glycoproteins.

The structures of the carbohydrate moieties of glycoproteins in snake venoms are largely unknown. In the present study, we have analyzed venoms of several species of snakes as well as plasma and tissue glycoproteins from one species of cobra (Naja naja kaouthia) by lectin affinity staining of Western blots. The data demonstrate that glycoproteins in cobra venom invariably contain terminal alpha-galactosyl residues with negligible proportions of sialic acids. Interestingly, however, terminal alpha-galactosyl residues are present in significantly lower proportions in cobra tissues such as brain, liver, lung, kidney, spleen, muscle, and totally absent in cobra plasma glycoproteins. In sharp contrast to cobras, venom glycoproteins of other snakes do not contain terminal alpha-galactosyl residues but do contain terminal 2,3- and/or 2,6-linked sialic acids as well as beta-galactosyl residues. Cobra venom also contains high molecular weight heavily glycosylated proteins bearing poly-N-acetyllactosaminyl oligosaccharides, the majority of which appear to be linked to the protein core via O-glycosidic bonds.

Protein glycosylation in the malaria parasite.

The nature and extent of glycosylation in Plasmodium falciparum has long been controversial. It has been widely believed that O-glycosylation is the major carbohydrate modification in the intraerythrocytic stage of P. falciparum and that the parasite has no N-glycosylation capacity. Contrary to this, recent studies have demonstrated that P. falciparum has a low N-glycosylation capability, and O-glycosylation is either absent or present at an extremely low level, whereas glycosylphosphatidylinositol (GPI) anchor modification is common and is the major carbohydrate modification in parasite proteins. The GPI anchor moieties are essential for parasite survival. The parasite GPI anchors can activate signaling pathways in host cells, and thereby induce the expression of inflammatory cytokines, adhesion molecules and induced nitric oxide synthase (iNOS). This might cause erythrocyte sequestration, hypoglycemia, triglyceride lipogenesis and immune dysregulation. Thus, the parasite GPI anchor structure and biosynthetic pathways are attractive targets for antimalarial and/or antiparasite drug development, as discussed here by Channe Gowda and Eugene Davidson.

Carbohydrate-directed conjugation of cobra venom factor to antibody by selective derivatization of the terminal galactose residues.

Cobra venom factor (CVF) can cause cell death by complement-mediated bystander cell lysis. Several studies have investigated CVF for application in cancer therapy by conjugating CVF to antibodies against tumor cell surface-specific antigens via the side-chain amino acid residues. In most cases, the activity of CVF was markedly impaired, presumably by modification of the factor B binding domain due to random derivatization. Since CVF is a glycoprotein and its oligosaccharide chains are distal to the factor B binding domain, coupling of CVF to antibodies through its oligosaccharide chains is expected to yield immunoconjugates with retention of CVF activity and elimination of the immunoreactivity of the terminal alpha-galactosyl residues. In this study, we investigated the carbohydrate site-directed conjugation of CVF to a monoclonal IgG specific to a cell-surface antigen of human ovarian cancer cells. The terminal galactosyl residues of CVF were selectively modified at C-6 by treatment with galactose oxidase, and the generated aldehyde groups were derivatized in situ with hydrazides containing either protected thiol or maleimide functional groups. The CVF derivatives were allowed to react with thiol groups introduced to the antibody by derivatization with 2-iminothiolane to yield carbohydrate site-directed CVF-antibody conjugates. In both cases, 30-40% of the antibody cross-linked to CVF to yield predominantly monovalent CVF-antibody conjugates. The purified immunoconjugates retained 70-75% of CVF activity and significant level of antigen-binding capacity. This is the first study to exploit the oligosaccharide chains of CVF for the preparation of active immunoconjugates.

Electromechanical transduction: reduction-driven hydrophobic folding demonstrated in a model protein to perform mechanical work.

It has long been appreciated that hydrophobic folding is an important element of protein structure formation. Here it is demonstrated for the first time that the electrochemical or chemical reduction of a nicotinamide in a model protein, which increases hydrophobicity, can drive hydrophobic folding and assembly in such a way as to lift a weight or otherwise contract against a constant tensional force. The model protein, poly[0.73(GVGVP),0.27(GK(NMeN)GVP], can be gamma-irradiation cross-linked to form an elastic matrix which contracts on raising the temperature from below to above the transition range for hydrophobic folding and assembly. On reduction of the N-methyl nicotinamide, (NMeN), the transition temperature range is lowered from above to below 20 degrees C to drive contraction due to hydrophobic folding with the performance of mechanical work.

Phosphorylation and dephosphorylation modulation of an inverse temperature transition.

Poly[15(IPGVG),(RGYSLG)], where RGYSLG is a protein kinase site, was synthesized. On raising the temperature of a 5 mg/ml solution, this polypeptide undergoes an inverse temperature transition at 18 degrees C in which it folds into a contracted state by optimizing intramolecular hydrophobic interactions. Averaging the data of five experiments, phosphorylation by means of a 3':5' cyclic AMP dependent protein kinase to the extent of one phosphate in 360 residues raises the temperature of the folding transition to 32 degrees C. The shift is completely reversed on dephosphorylation by alkaline phosphatase. Phosphorylation is hereby shown to be the most potent chemical perturbation known for shifting the temperature of an inverse temperature transition, which has been shown to be an efficient mechanism for achieving chemomechanical transduction (mechanochemical coupling).

Structures of O-linked oligosaccharides present in the proteoglycans secreted by human mammary epithelial cells.

The structures of O-glycosidically linked oligosaccharides present in the heparan sulfate and chondroitin sulfate proteoglycans isolated from the culture medium of a normal (HBL-100) and a malignant (MDA-MB-231) human mammary epithelial cell line have been determined. Both proteoglycan types from the two cell lines contain a series of O-linked oligosaccharides ranging in size from di- to hexasaccharide. Cells were grown in the presence of either [3H]glucosamine or [3H]galactose and Na2 35SO4, and the proteoglycans were isolated as described (Gowda, D. C., Bhavanandan, V. P., and Davidson, E. A. (1986) J. Biol. Chem. 261, 4926-4934). The O-linked oligosaccharides were released from the proteoglycans by alkaline borohydride treatment and purified by a combination of gel filtration and high voltage paper electrophoresis. The structures of two neutral and seven acidic oligosaccharides were established based on sugar composition, the results of periodate oxidation, sequential exoglycosidase treatment, and methylation analysis. Periodate oxidation, taking advantage of tritium label at specific positions of constituent sugars, proved to be a valuable tool in establishing the structure of isomeric components in the mixture. The structures of the oligosaccharides were assigned as follows: (Formula: see text) The oligosaccharide containing both sialic acid and ester sulfate is novel and has not been reported previously.