Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.

Characterization of mouse neuronal Ca2+/calmodulin kinase II inhibitor alpha

We have overexpressed an 8.5-kDa mouse Ca2+/calmodulin kinase II inhibitor a protein (mCaMKIIN alpha) in Escherichia coli and demonstrate that the recombinant protein is a potent inhibitor of Ca2+/calmodulin kinase 11 (CaMKII) in vitro. However, antibodies raised against recombinant mCaMKIIN alpha. react with an similar to 37-kDa protein present in mouse brain. The pattern of expression of the similar to 37-kDa protein is similar to that of mCaMKIIN alpha mRNA as both are expressed in normal but not Japanese encephalitis virus (JEV)-infected mouse brain. Subcellular localization studies indicate that the similar to 37-kDa protein is present in the post-synaptic density (PSD) where mCaMKII alpha is known to perform key regulatory functions. We conclude that the similar to 37-kDa protein identified in this study is mCaMKIIN alpha. and its localization in the PSD indicates a novel role for this protein in the regulation of neuronal CaMKII alpha. (c) 2007 Elsevier B.V. All rights reserved.

Epigallocatechin gallate is a slow-tight binding inhibitor of enoyl-ACP reductase from Plasmodium falciparum

Epigallocatechin gallate (EGCG) is known to have numerous pharmacological properties. In the present study, we have shown that EGCG inhibits enoyl–acyl carrier protein reductase of Plasmodium falciparum (PfENR) by following a two-step, slow, tight-binding inhibition mechanism. The association/isomerization rate constant (k5) of the reversible and loose PfENR–EGCG binary complex to a tight [PfENR–EGCG]* or EI* complex was calculated to be 4.0 × 10−2 s−1. The low dissociation rate constant (k6) of the [PfENR–EGCG]* complex confirms the tight-binding nature of EGCG. EGCG inhibited PfENR with the overall inhibition constant (Ki*) of 7.0 ± 0.8 nM. Further, we also studied the effect of triclosan on the inhibitory activity of EGCG. Triclosan lowered the k6 of the EI* complex by 100 times, lowering the overall Ki* of EGCG to 97.5 ± 12.5 pM. The results support EGCG as a promising candidate for the development of tea catechin based antimalarial drugs.

Functional interaction of diphenols with polyphenol oxidase - Molecular determinants of substrate/inhibitor specificity SO FEBS JOURNAL

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones. The quinones autopolymerize to form dark pigments, an undesired effect. PPO is therefore the target for the development of antibrowning and antimelanization agents. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Docking studies suggested two distinct modes of binding, dividing the docked ligands into two groups. Remarkably, the first group corresponds to ligands determined to be substrates and the second group corresponds to reversible inhibitors. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on o-diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His 109 of the four coordinates of the trigonal pyramidal coordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity, leading to inhibition, or because of a steric clash flips the molecule vertically, facilitating oxidation. This is the first study to explain, at the molecular level, the determinants Of substrate and inhibitor specificity of a catechol oxidase, thereby providing a platform for the design of selective inhibitors useful to both the food and pharmaceutical industries.

Synthesis of neoglycopeptides and analyses of their biodistributionin vivo to identify tissue specific uptake and novel putative membrane lectins

Complex typeN-linked oligosaccharides derived from fetuin, fibrinogen and thyroglobulin were coupled to acetyltyrosine affording a series of neoglycopeptides with retention of terminal structures and the beta-anomeric configuration of their reducing endN-acetylglycosamine residue. The neoglycopeptides thus synthesized could be labelled to high specific activities with125I in the aromatic side chain of tyrosine. Analysis of the fate of these neoglycopeptides in conjunction with inhibition with asialofetuin and oligosaccharides of defined structure in micein vivo revealed the uptake of galactosylated biantennary compound by kidneys, in addition to the known itinerary of triantennary galactosylated complex oligosaccharide from fetuin to liver and the galactosylated biantennary chain with fucosylation in the core to bone marrows. On the other hand, the agalacto, aglucosamino biantennary chains with and without fucosylation in the core region are taken up by submaxillary glands while the conserved trimannosyl core with fucose is primarily concentrated in stomach tissue. These studies thus define new routes for the uptake of complexN-linked glycans and also subserve to identify lectins presumably involved in their recognition.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Activation of succinate dehydrogenase by 2,4-dinitrophenol-a competitive inhibitor

1.The reported inhibition of the succinate oxidase system at high concentrations of dinitrophenol, considered to be at the primary dehydrogenase level, is now confirmed by measuring the activity of succinate dehydrogenase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1) in the presence of dinitrophenol, using the dye reduction method. 2. 2. The results indicate that the inhibition of substrate-activated succinate dehydrogenase by dinitrophenol is competitive. 3. 3. Low concentrations of dinitrophenol inhibited the basal activity, while at higher concentrations the kinetics were complicated by an apparent activation. 4. 4. Preincubation of mitochondria with dinitrophenol stimulated the enzyme activity, a phenomenon shown by succinate and competitive inhibitors. This activation was very rapid at 37°, compared to that by succinate; activation by dinitrophenol was observed even at 25°, under conditions where succinate had no effect. 5. 5. Repeated washing of the activated mitochondrial samples with the sucrose homogenizing medium reduced the succinate-stimulated activity to the basal level, but only partially reversed the dinitrophenol activation. 6. 6. The relevance of this activation phenomenon to the physiological modulation of this enzyme system is discussed.

Chloromycetin in the nutrition of the silkworm Bombyx mori L.-III. Influence on gut weight, oxygen uptake, and glucose absorption

Administration of chloromycetin has been found to enhance the oxygen uptake of the gut of the silkworm. The possibility that this increase might have been due to a thinning of the gut wall has been ruled out since the reduction in gut weight set in much later. Although glucose ultilization by the gut has been found to be increased in vitro, increase in oxygen uptake has not been affected in the presence of glucose. The possibility of a hormonal stimulation has been discussed.

Chloromycetin in the nutrition of the silkworm Bombyx mori L.-III. Influence on gut weight, oxygen uptake, and glucose absorption

Administration of chloromycetin has been found to enhance the oxygen uptake of the gut of the silkworm. The possibility that this increase might have been due to a thinning of the gut wall has been ruled out since the reduction in gut weight set in much later. Although glucose ultilization by the gut has been found to be increased in vitro, increase in oxygen uptake has not been affected in the presence of glucose. The possibility of a hormonal stimulation has been discussed.

Mechanism of glycine uptake by the silk glands of the silkworm Bombyx mori L.

The transport of glycine in vitro into the silk glands of the silkworm has been studied. Glycine accumulates inside the tissue to a concentration higher than that present outside, indicating an active transport mechanism. The kinetics of uptake show a biphasic curve and two apparent Km values for accumulation, 0.33 mM and 5.00 mM. The effect of inhibitors on the energy metabolism of glycine transport is inconclusive. Exchange studies indicate the existence of two pools inside the gland, one that is easily removed by exchange and osmotic shock, and the other which is not. The results obtained conform with the carrier model of Britten and McClure concerning the amino-acid pool in E. coli.

Hormones and Cuscuta development: IAA uptake transport and metabolism in relation to growth in the absence and presence of applied cytokinin

Transport of 1-14C-IAA in successive stem segments of Cuscuta was strictly basipetal in growing and non growing regions of the vine with a flux velocity of 10-12 mm/h (intercept method). This transport showed a distinct peaked profile, increasing from a low value at 10 mm from the apex to a maximum between 50 and 90 mm before declining to a low value again around 160 mm at which elongation growth ceased. The IAA transport profile paralleled the in vivo growth rate profile, though the latter peaked ahead of transport. A better correlation was observed between the profile of growth responsiveness of the vine to exogenous IAA application and the profile of IAA transport. Growth responsiveness was determined as the differential in growth rate of stem segments in vitro in the absence and presence of growth optimal concentration of IAA (10 μm). Retention of exogenous IAA in the stem was maximal where transport decreased, and this coincided with the region of maximal conjugation of applied 1-14C-IAA to aspartic acid to form indoleacetylaspartate (IAAsp). In addition to aspartate, IAA was conjugated to a small extent to an unidentified compound. IAA destruction by decarboxylation was greatest where transport was low, particularly in the nongrowing region, where lignification occurred (i.e., beyond 180 mm). At concentrations up to 20 μM, a pulse of 1-14C-IAA chased by "cold" IAA moved as a peak (with a peak displacement velocity of 12-18 mm/h) in the "growth" region of the vine, but became diffusionlike where growth either fell off steeply or ceased. At a higher (50 μM) IAA concentration, though uptake was not saturated, transport in the growth region became diffusionlike, indicating saturation of the system. Reduced IAA flux in the region where growth responsiveness to IAA declined coincided with the region of increased IAA conjugation. However, it cannot be concluded whether increased IAA conjugation was the cause or effect of decreased IAA flux. Application of benzyladenine to the vines in vivo, a treatment that elicited haustoria formation by 72 h, resulted in the inhibition of both IAA transport and elongation growth rate in the subapical region. In vitro treatment of vine segments with BA similarly increased IAA retention and decreased IAA transport. IAA loss was suppressed, and conjugation to IAAsp was enhanced. © 1989 Springer-Verlag New York Inc.

Isolation and characterization of a gonadotropin receptor binding inhibitor from porcine follicular fluid

The presence of a gonadotropin receptor binding inhibitor in pooled porcine follicular fluid has been demonstrated. Porcine follicular fluid fractionation on DE-32 at near neutral pH, followed by a cation exchange chromatography on SPC-50 and Cibacron blue affinity chromatography, yielded a partially purified gonadotropin receptor binding inhibitor (GI-4). The partially purified GI binding inhibitor inhibited the binding of both 125I labelled hFSH and hCG to rat ovarian receptor preparation. SDS electrophoresis of radioiodinated partially purified GI followed by autoradiography made it possible to identify the binding component as a protein of molecular weight of 80000. Subjecting 125I labelled GI-4 to chromatography on Sephadex G-100 helped obtain a homogeneous material, Gl-5. The 125I labelled GI-5 exhibited in its binding to ovarian membrane preparations characteristics typical of a ligand-receptor interaction such as saturability, sensitivity to reaction conditions as time, ligand and receptor concentrations and finally displaceability by unlabelled inhibitor as well as FSH and hCG in a dose dependent manner. This material could bind ovarian receptors for both FSH and LH, its binding being inhibited by added FSH or hCG in a dose dependent manner.