Bile acid conjugates of a nonsteroidal glucocorticoid receptor modulator

Bile acid conjugates of a selective nonsteroidal glucocorticoid receptor modulator were prepared and evaluated. Potent GR binding conjugates that showed improved metabolic stability were discovered. However, cellular potency and pharmacokinetics were not substantially improved.     

Alpha,alpha-trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6'-diamino-6,6'-dideoxy-alpha,alpha-trehalose was obtained from selective bromination of, alpha,alpha-trehalose at C-6,6', followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel alpha,alpha-trehalose derivatives. Their abilities to inhibit Tat-TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.     

Alpha-actinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and rapidly degraded cytoskeletal protein

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant alpha-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant alpha-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a "knockin" mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant alpha-actinin-4, mediated, at least in part, by the ubiquitin-proteasome pathway. We correlate these findings with studies of alpha-actinin-4 expression in human samples. "Knockin" mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 "knockin" and "knockout" mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.     

Bystander suppression of collagen-induced arthritis in mice fed ovalbumin

We wanted to assess whether B-cell and/or T-cell responses to collagen and thereby the course of collagen-induced arthritis could be suppressed by regulatory mechanisms associated with oral tolerance to an unrelated protein. DBA/1 mice were fed ovalbumin (OVA)-containing pellets ad libitum for 1 week and subsequently coimmunized twice, with a mixture of bovine collagen type II (BCII) and OVA in Freund's complete adjuvant. Mice fed OVA before coimmunization with BCII and OVA had significantly lower arthritic scores than mice immunized with BCII only. Their body weight increased during the study period and their anti-BCII antibody activity was significantly IgG_2a lower. The frequency of spleen cells producing IgG anti-BCII antibody was also reduced. Coimmunization per se slightly ameliorated the development of arthritis, resulting in an early, transient reduction. It resulted in significantly higher IgG₁ anti-BCII antibody activity and increased splenocyte secretion of IFN-γ and IL-10 in response to BCII. Our findings demonstrate that OVA-specific regulatory events induced by feeding OVA, i.e. bystander suppression, reduced the severity of arthritis in animals immunized with BCII and OVA. Anti-BCII specific antibody responses and cytokine secretion by types 1 and 2 T helper cells were also decreased.     

Phytoremediation of arsenic-contaminated groundwater by the arsenic hyperaccumulating fern Pteris vittata L

Arsenic concentrations in a much larger fraction of U.S. groundwater sources will exceed the maximum contaminant limit when the new 10 microg L(-1) EPA standard for drinking water takes effect in 2006. Thus, it is important to develop remediation technologies that can meet this new standard. Phytoremediation of arsenic-contaminated groundwater is a relatively new idea. In this research, an arsenic-hyperaccumulating fern, commonly known as Chinese Brake fern (Pteris vittata L.), was grown hydroponically to examine its effectiveness in arsenic removal from what is believed to be herbicide-contaminated groundwater. One plant grown in 600 mL of groundwater effectively reduced the arsenic concentration from 46 to less than 10 microg L(-1) in 3 days. Re-used plants continued to take up arsenic from the groundwater, albeit at a slower rate (from 46 to 20 microg L(-1) during the same time). Young fern plants were more efficient in removing arsenic than were older fern plants of similar size. The addition of a supplement of phosphate-free Hoagland nutrition to the groundwater had little effect on arsenic removal, but the addition of phosphate nutrition significantly reduced its arsenic affinity and, thus, inhibited the arsenic removal. This study suggested that Chinese Brake has some potential to remove arsenic from groundwater.     

Molecular mechanisms of relationships between the host organism and pathogenic enterobacteria

Modern data on the molecular mechanisms of relationships between the host organism and the pathogenic representatives of the family Enterobacteriaceae in the host-parasite system are presented. The process of cytokine and eicosanoid regulation of the immune process of the host in the norm and pathology states are analyzed. The examples of the mechanisms of immune suppression, false antigenic stimulation and the mimicry of pathogens are given.     

Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin,respectively

Heme oxygenase (HO) is implicated in protection against oxidative stress, proliferation and apoptosis in many cell types, including neurons. We utilized olfactory receptor neurons (ORNs) as a model to define the roles of HO-1 and HO-2 in neuronal development and survival, and to determine the mediators of these effects. The olfactory system is a useful model as ORNs display neurogenesis post-natally and do not contain nitric oxide synthase (NOS) activity, which could confound results. HO isoforms were expressed in ORNs during embryogenesis and post-natally. Mice null for either HO-1 or HO-2 displayed decreased proliferation of neuronal precursors. However, apoptosis was increased only in HO-2 null mice. Cyclic GMP immunostaining was reduced in ORNs in both genotypes, providing direct evidence that HO mediates cGMP production in vivo. Bilirubin immunostaining was reduced only in HO-2 null mice. These roles for HO-1 and HO-2 were confirmed using detergent ablation of the epithelium to observe increased neurogenesis of ORNs after target disruption in HO null mice. Primary cultures of ORNs revealed that proliferative and survival effects of HO were mediated through cGMP and bilirubin, respectively. These results support a role for HO, the CO-cGMP signaling system and bilirubin in neurodevelopment and in response to injury.     

Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.     

Gene knockdown of gamma-glutamylcysteine synthetase by RNAi in the parasitic protozoa Trypanosoma brucei demonstrates that it is an essential enzyme

The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNAi methods were used to knock down gene expression of gamma-GCS in procyclic T. brucei cells. Induction of gamma-GCS RNAi with tetracycline led to cell death within 4-6 days post-induction. Cell death was preceded by the depletion of the gamma-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH)2. The addition of GSH (80 microM) to cell cultures rescued the RNAi cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of gamma-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as gamma-GCS RNAi; however, addition of GSH did not restore the pools of GSH and T(SH)2. These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH)2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines.     

Inhibition of InhA activity,but not KasA activity,induces formation of a KasA-containing complex in mycobacteria

Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase InhA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of InhA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The InhA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of InhA inhibition. In addition, in vitro assays using purified InhA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited InhA but not KasA activity. Moreover, several thermosensitive InhA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of InhA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with InhA being the primary target of INH.