Opiate regulation of IL-1beta and TNF-alpha in cultured human articular chondrocytes

In order to investigate if beta-endorphins anti-inflammatory effect in cartilage-damaging states is mediated via tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we examined its influence on these two cytokines in vitro. Human articular chondrocytes were obtained from patients undergoing total knee arthroplasty and stimulated with beta-endorphin (60-6000 ng/ml). Protein levels of TNF-alpha and IL-1 beta were measured by ELISA in supernatants from articular chondrocyte cultures. beta-Endorphin significantly increased the levels of IL-1 beta for all concentrations used after 15 min incubation, and when stimulated with 600 and 6000 ng/ml after 24 h incubation. The opioid-induced increase in IL-1 beta was blocked by naltrexone in the group tested. TNF-alpha expression was also significantly stimulated by 60 and 600 ng/ml beta-endorphin after 15 min, an effect blocked by naltrexone in the group tested. These findings indicate that the mechanism of beta-endorphins anti-inflammatory influence in cartilage-damaging states is not apparently mediated via these two cytokines modulation.     

Carnosine and anserine act as effective transglycating agents in decomposition of aldose-derived Schiff bases

There are numerous publications describing the positive effects of carnosine (beta-alanyl-histidine) and anserine (beta-alanyl-1-N-methyl-histidine) on cell and organ function. Of special interest to us is the fact that these dipeptides act to retard and (in one instance) reverse non-enzymatic glycation. To date, the primary explanation for these anti-glycating effects has been the fact that carnosine and anserine can serve as alternative and competitive glycation targets, thereby protecting proteins from this deleterious process. In this paper, we document another mechanism by which these two peptides can retard or reverse glycation. The process involves decomposition of the very first intermediates of the non-enzymatic glycation cascade (aldosamines a.k.a. Schiff bases) by nucleophilic attack of carnosine and/or anserine on the preformed aldosamine such as glucosyl-lysine. If future research shows this reaction is to be physiologically important, this mechanism could explain some of the beneficial effects of carnosine and anserine as anti-glycating agents.     

Adamantoylated monosaccharides: new compounds for modification of the properties of cyclodextrin-containing materials

Adamantoyl glycosides were obtained in good yields by coupling adamantanecarboxylic acid with monosaccharides. They form very stable inclusion complexes with beta-cyclodextrin, as shown by (1)H NMR measurements.     

Regioselective synthesis of long-chain ethers and their sulfates derived from methyl beta-D-galactopyranoside and derivatives via dibutylstannylene acetal intermediates

A number of different conditions were investigated for the alkylation of the dibutylstannylene acetals of methyl beta-d-galactopyranoside with long-chain primary alkyl bromides, decyl, dodecyl, and tetradecyl bromide. The best yields of the major products, the 3-O-alkyl ethers, were obtained by reaction of the alkyl bromide with the monodibutylstannylene acetal in DMF in the presence of cesium fluoride for extended periods of time at moderate temperatures (65 degrees C). These products were always accompanied by minor amounts of the 3,6-di-O-alkyl derivative. Performing the reaction with excess alkyl halide on the bis(dibutylstannylene) acetal resulted in more of the 3,6-di-O-alkyl derivative, particularly for the shorter alkyl bromides, but this product was never predominant. Sulfation of the dibutylstannylene acetal of methyl 3-O-tetradecyl-beta-D-galactopyranoside resulted in the 6-sulfate in 96% yield.     

beta-Microseminoprotein binds CRISP-3 in human seminal plasma

beta-Microseminoprotein (MSP) and cysteine-rich secretory protein 3 (CRISP-3) are abundant constituents of human seminal plasma. Immunoprecipitation and gel filtration of seminal plasma proteins combined with examination of the proteins in their pure form showed that MSP and CRISP-3 form stable, non-covalent complexes. CRISP-3 binds MSP with very high affinity, as evidenced by surface plasmon resonance. Due to far higher abundance of MSP in prostatic fluid, it manifests large overcapacity for CRISP-3 binding. Structural similarity with an MSP-binding protein from blood plasma suggests that CRISP-3 binds MSP through its aminoterminal SCP-domain.     

A new beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Helicobacter pylori: Molecular cloning, enzymatic characterization, and structural modeling

Helicobacter pylori is a gram-negative pathogenic bacterium that causes peptic ulcer disease and gastric cancer, and studies of the related potent enzymes associated with this bacterium are urgent for the discovery of novel drug targets. In bacteria, beta-hydroxyacyl-acyl carrier protein (ACP) dehydratase (FabZ) is a potent enzyme in fatty acid biosynthesis and catalyzes the dehydration of beta-hydroxyacyl-ACP to trans-2-acyl-ACP. In this study, the cloning and enzymatic characterization of FabZ from H. pylori strain SS1 (HpFabZ) were reported, and the gene sequence of HpfabZ was deposited in the GenBank database. Enzyme dynamic analysis showed that HpFabZ had a K(m) of 82.6+/-4.3 microM toward its substrate analog crotonoyl-CoA. Dynamic light scattering and native-PAGE investigations suggested that HpFabZ exists as hexamer in native state. Enzymatic characterization and thermal-induced unfolding analysis based on circular dichroism spectral measurements indicated that HpFabZ is very stable against high temperature (90 degrees C). Such a high stability of HpFabZ was well elucidated by the strong H-bonds and hydrophobic interactions among the HpFabZ hexamer as investigated in the modeled HpFabZ hexamer structure. Our current study is hoped to provide useful information in better understanding the FabZ of H. pylori strain and further supply possible hints in the discovery of anti-bacterial compounds using HpFabZ as target.     

The first crystal structure of a Mimosoideae lectin reveals a novel quaternary arrangement of a widespread domain

The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of beta-prism domains, and highlight the adaptability of the beta-prism fold as a building block in the evolution of plant lectins. The P.platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroid-shaped structure of the P.platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P.platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms.     

Unclosed beta-propellers display stable structures: implications for substrate access to the active site of prolyl oligopeptidase

Prolyl oligopeptidase is implicated in the metabolism of neuropeptides and is involved in amnesia and depression. It contains a peptidase and an unusual beta-propeller domain that excludes large peptides and proteins from the active site. The propeller consists of seven blades not closed by a "Velcro" between the first and last blades. The propeller domain was expressed as a stable, soluble protein, P(7). Its conformational identity with that of the native propeller was verified by circular dichroism and digestion with trypsin. Differential scanning calorimetry, kinetic denaturation with urea and equilibrium denaturation with guanidinium chloride have shown that the propeller is more stable than the parent prolyl oligopeptidase. The deletion of the seventh blade of P(7) led to a stable structure, a six-bladed propeller, P(6), which immediately dimerized, in contrast with the monomeric P(7). Addition of an 11 amino acid residue extension to the C terminus of P(6) also produced a dimer, whereas the P(6) labelled with a His-tag at the N terminus displayed a monomer structure. The stability of P(6) and its variants was lower than that of P(7). The denatured propellers refolded readily. This study shows that the unclosed P(7) is a stable structure, and suggests that an opening between the peptidase and the propeller domains is more important for the substrate entry than is the putative opening between the first and seventh blades. Our results suggest that the propellers are simple, versatile structures, which can be prepared artificially.     

Enzyme-substrate complex structures of a GH39 beta-xylosidase from Geobacillus stearothermophilus

Beta-D-Xylosidases are glycoside hydrolases that catalyse the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicelluloses. beta-D-Xylosidases are found in glycoside hydrolase families 3, 39, 43, 52 and 54. The first crystal structure of a GH39 beta-xylosidase revealed a multi-domain organization with the catalytic domain having the canonical (beta/alpha)8 barrel fold. Here, we report the crystal structure of the GH39 Geobacillus stearothermophilus beta-D-xylosidase, inactivated by a point mutation of the general acid-base residue E160A, in complex with the chromogenic substrate molecule 2,5-dinitrophenyl-beta-D-xyloside. Surprisingly, six of the eight active sites present in the crystallographic asymmetric unit contain the trapped covalent glycosyl-enzyme intermediate, while two of them still contain the uncleaved substrate. The structural characterization of these two critical species along the reaction coordinate of this enzyme identifies the residues forming its xyloside-binding pocket as well as those essential for its aglycone recognition.     

Divergent effects of estrogen and nicotine on Rho-kinase expression in human coronary vascular smooth muscle cells

Recent studies have demonstrated that up-regulated Rho-kinase plays an important role in the pathogenesis of coronary arteriosclerosis and vasospasm. We have shown that inflammatory stimuli, such as angiotensin II and interleukin-1beta, up-regulate Rho-kinase expression and activity in human coronary vascular smooth muscle cells, for which intracellular signal transduction mediated by protein kinase C and NF-kappaB is involved. Here, we show that estrogen down-regulates while nicotine up-regulates Rho-kinase and that nicotine counteracts the inhibitory effect of estrogen on angiotensin II-induced Rho-kinase expression. Furthermore, we demonstrated that the intracellular signal transduction of the inhibitory effect of estrogen is mediated by an estrogen receptor. These results demonstrate that inflammatory stimuli up-regulate Rho-kinase, for which estrogen (mediated by an estrogen receptor) and nicotine exert divergent inhibitory and stimulatory effects on the Rho-kinase expression, respectively, and may explain in part why the incidence of arteriosclerotic and vasospastic disorders is increased in postmenopausal women and smokers.