Inhibition of the sole type I signal peptidase of Mycobacterium tuberculosis is bactericidal under replicating and nonreplicating conditions

Proteins secreted by bacteria perform functions vital for cell survival and play a role in virulence in Mycobacterium tuberculosis. M. tuberculosis lepB (Rv2903c) encodes the sole homolog of the type I signal peptidase (SPase). The lepB gene is essential in M. tuberculosis, since we could delete the chromosomal copy only when a second functional copy was provided elsewhere. By placing expression under the control of an anhydrotetracycline-inducible promoter, we confirmed that reduced lepB expression was detrimental to growth. Furthermore, we demonstrated that a serine-lysine catalytic dyad, characteristic for SPase function, is required for LepB function. We confirmed the involvement of LepB in the secretion of a reporter protein fused to an M. tuberculosis signal peptide. An inhibitor of LepB (MD3; a beta-aminoketone) was active against M. tuberculosis, exhibiting growth inhibition and bactericidal activity. Overexpression of lepB reduced the susceptibility of M. tuberculosis to MD3, and downregulation resulted in increased susceptibility, suggesting that LepB is the true target of MD3. MD3 lead to a rapid loss of viability and cell lysis. Interestingly, the compound had increased potency in nonreplicating cells, causing a reduction in viable cell numbers below the detection limit after 24 h. These data suggest that protein secretion is required to maintain viability under starvation conditions and that secreted proteins play a critical role in generating and surviving the persistent state. We conclude that LepB is a promising novel target for drug discovery in M. tuberculosis, since its inhibition results in rapid killing of persistent and replicating organisms.     

Urate oxidase in peroxisomes from maize root tips

Summary Peroxisomes isolated from maize root tips contained urate oxidase, although the supplementary enzymes allantoinase, allantoicase and NADH-glyoxylate reductase were not detected. Some glutamate-oxalacetate transaminase was present in peroxisomes. Enzymes of two other pathways occuring in plant peroxisomes, namely glycolate metabolism and the glyoxylate cycle, were not present. The root peroxisome thus resembles peroxisomes of the Arum spadix and supports the concept that peroxisomes constitute a dynamic and differentiating system.     

Regulation of the immune response by antibody. II. The effects of different classes of passive guinea pig antibody on humoral and cell-mediated immunity in rats

The ability of different classes of passively administered guinea pig antibody (γ1, γ2, and IgM) to regulate humoral and cell-mediated immunity to flagellin, polymerized flagellin (POL), and sheep red blood cells (SRBC) was investigated in rats. It was found that at high concentrations, all classes of antibody suppressed the primary antibody responses and usually enhanced the delayed-type hypersensitivity induced by the three antigens. With flagellin and SRBC, the different classes of passive antibody varied in their suppressing and enhancing properties, being in the order: γ2 > γ1 = IgM. At low concentrations, γ1 and IgM enhanced the primary antibody response and suppressed the delayed hypersensitivity induced by flagellin. Such an effect was not observed with either POL or SRBC. Priming for a secondary antibody response was less readily suppressed by all classes of passive antibody. The removal of macrophage cytophilic antibody from γ2 converted this antibody to a preparation (γ2 absorbed) which had effects on humoral and cell-mediated immunity approaching that of γ1 antibody.     

Lymphocytes express a diverse array of specific receptors for sulfated polysaccharides

Lymphocyte receptors for sulfated polysaccharides were detected in two ways, namely, by the ability of lymphocytes to form rosettes with sheep red blood cells (SRBC) coupled with one of fourteen different sulfated polysaccharides, and by the ability of cholate extracts of lymphocytes to hemagglutinate the same sulfated polysaccharide-coupled SRBC. It was found that murine lymphocytes lacked receptors for a number of glycosaminoglycans, such as hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate, but reacted strongly with heparin, arteparon, and a number of sulfated polysaccharides of plant and bacterial origin. In each case receptor activity was demonstrated by rosetting and by the ability of lymphocyte lysates to strongly agglutinate sulfated polysaccharide-coupled SRBC. The receptors exhibited a high degree of diversity as evidenced by (a) only subpopulations of lymphocytes, particularly splenic B cells, expressing receptors for some of the sulfated polysaccharides and (b) hemagglutination-inhibition analyses revealing numerous subsets of receptors with different binding specificities. Receptor diversity was further highlighted by a 48% difference in the hemagglutination-inhibiton results between thymus and spleen. It is proposed that these receptors are involved in cell-cell communication and lymphocyte homing and recirculation. The likely target structures for the receptors in vivo are the heparan sulfates, a ubiquitous and structurally diverse family of sulfated glycosaminoglycans.     

Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.     

Studies of the oxysterol inhibition of tumor cell growth

The oxysterols 3 beta-hydroxy-5 alpha-cholest-8-en-11-one, 3 beta-hydroxy-5 alpha-cholest-8-en-7-one, 3 beta-hydroxy-5 alpha-cholest-8(14)-en-7-one, 3 beta-hydroxy-4,4'-dimethylcholest-5-ene-7 one, 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol, lanost-8-ene-3 beta, 25-diol, 25-hydroxylanost-8-en-3-one, 9 alpha, 11 alpha-epoxy-5 alpha-cholest-7-en-3 beta-ol, 3 beta-hydroxycholest-5 alpha-en-22-one, and 3 beta-hydroxycholest-5-en-22-one oxime were evaluated with respect to their ability to inhibit cell growth. All of the sterols were found to possess cytotoxicity when incubated with hepatoma (HTC) and lymphoma (RDM-4) cells in culture at 10-30 microM concentrations.     

Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pH_i) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pH_i, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152–163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1. Received: 23 December 1997 / Accepted: 16 January 1998     

A polyanion binding site on the CD4 molecule. Proximity to the HIV-gp120 binding region

Recent studies have demonstrated that sulfated polyanions (SP) are potent inhibitors of HIV infection in vitro, appearing to inhibit virus attachment. To understand the mode of action of these compounds a large panel of SP were examined for their ability to inhibit HIV infection, block anti-CD4 mAb binding and, when immobilized, bind soluble CD4 and virion gp120. Based on anti-CD4 mAb binding-inhibition studies a SP binding site was identified on the CD4 molecule. Dextran sulfate (DXS)-500 kDa, polyvinylsulfate (PVS), and polyanethole sulfonate were particularly potent SP inhibitors, blocking the binding of 11 of the 12 anti-CD4 mAb tested. These 11 mAb are known to interact with the two amino-terminal Ig-like domains of CD4. In fact, DXS-500 kDa exhibited an hierarchy of inhibition of anti-CD4 mAb which suggests that SP bind to a conformational site incorporating the first two Ig-like domains of CD4. This SP binding site is clearly distinct but closely associated with the gp120 binding region of CD4. In terms of anti-HIV activity there was no evidence that SP act at the virion level as rgp120 did not bind to immobilized SP and preincubation of virions with SP did not affect infectivity. In contrast, many of the SP tested showed some affinity for CD4 based on anti-CD4 mAb blocking studies and binding of soluble CD4 to immobilized SP. The most active in this regard were DXS-500 kDa and PVS, whose anti-HIV activity could be entirely due to disruption of the CD4-gp120 interaction. However, with SP such as heparin, fucoidan, the carrageenans, and polyanethole sulfonate, although CD4 blocking may contribute to anti-HIV activity, some other anti-viral mechanism is also operating. Finally, pentosan sulfate, a SP with anti-HIV activity comparable to DXS-500 kDa and PVS, showed little or no reactivity with CD4 and must inhibit HIV infection by a totally CD4-independent mechanism.