A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.     

Stabilization of the activated alphaMbeta2 integrin by a small molecule inhibits leukocyte migration and recruitment

Integrins are potential targets for the development of antiinflammatory agents. Here we develop a novel high-throughput assay by allowing a chemical library to compete with phage display peptide binding and identify a novel small-molecule ligand to the leukocyte-specific alpha(M)beta(2) integrin. The identified thioxothiazolidine-containing compound, IMB-10, had an unexpected activity in that it stabilized binding of alpha(M)beta(2) to its endogenous ligands proMMP-9 and fibrinogen. Single amino acid substitutions in the activity-regulating C-terminal helix and the underlying region in the ligand-binding I domain of the integrin suppressed the effect of IMB-10. A computational model indicated that IMB-10 occupies a distinct cavity present only in the activated form of the integrin I domain. IMB-10 inhibited alpha(M)beta(2)-dependent migration in vitro and inflammation-induced neutrophil emigration in vivo. Stabilization of integrin-mediated adhesion by a small molecule is a novel means to inhibit cell migration and may have a utility in treatment of inflammatory diseases involving leukocyte recruitment.     

Molecular biology of insect olfaction:recent progress and conceptual models

Insects have an enormous impact on global public health as disease vectors and as agricultural enablers as well as pests and olfaction is an important sensory input to their behavior. As such it is of great value to understand the interplay of the molecular components of the olfactory system which, in addition to fostering a better understanding of insect neurobiology, may ultimately aid in devising novel intervention strategies to reduce disease transmission or crop damage. Since the first discovery of odorant receptors in vertebrates over a decade ago, much of our view on how the insect olfactory system might work has been derived from observations made in vertebrates and other invertebrates, such as lobsters or nematodes. Together with the advantages of a wide range of genetic tools, the identification of the first insect odorant receptors in Drosophila melanogaster in 1999 paved the way for rapid progress in unraveling the question of how olfactory signal transduction and processing occurs in the fruitfly. This review intends to summarize much of this progress and to point out some areas where advances can be expected in the near future.     

Arresten,a Collagen-Derived Angiogenesis Inhibitor,Suppresses Invasion of Squamous Cell Carcinoma

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activity. Among these are the collagen-derived anti-angiogenic fragments, some of which are suggested to affect carcinoma cells also directly. Arresten is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of the basement membrane collagen IV α1 chain. As the mere prevention of tumor angiogenesis leads to hypoxia that can result in selection of more aggressive cell types and reduces the efficacy of chemotherapy, we aimed here to elucidate how arresten influences the aggressive human carcinoma cells. Arresten efficiently inhibited migration and invasion of HSC-3 tongue carcinoma cells in culture and in an organotypic model. Subcutaneous Arr-HSC xenografts grew markedly more slowly in nude mice and showed reduced tumor cell proliferation, vessel density and local invasiveness. In the organotypic assay, HSC-3 cells overproducing arresten (Arr-HSC) showed induction of cell death. In monolayer culture the Arr-HSC cells grew in aggregated cobblestone-like clusters and, relative to the control cells, showed increased expression and localization of epithelial marker E-cadherin in cell-cell contacts. Application of electric cell-substrate impedance sensing (ECIS) further supported our observations on altered morphology and motility of the Arr-HSC cells. Administration of a function-blocking α1 integrin antibody abolished the impedance difference between the Arr-HSC and control cells suggesting that the effect of arresten on promotion of HSC-3 cell-cell contacts and cell spreading is at least partly mediated by α1β1 integrin. Collectively, our data suggest novel roles for arresten in the regulation of oral squamous carcinoma cell proliferation, survival, motility and invasion through the modulation of cell differentiation state and integrin signaling.     

Single nucleotide polymorphism -799C/T in matrix metalloproteinase-8 promoter region in arterial disease

Arterial disease is associated with elevated serum matrix metalloproteinase (MMP)-8 concentration. We studied the role of two promoter region single nucleotide polymorphisms (SNPs) of MMP-8 gene in the arterial disease. The population comprised patients with arterial disease (n?=?124) and healthy blood donors (n?=?100) as a reference group for MMP-8 SNPs (-799C/T and -381A/G) genotypes and serum concentrations. Genotype frequencies for MMP-8 -799C/T SNP in arterial disease were C/C (43.5%), C/T (32.3%) and T/T (24.2%), and in the reference group they were C/C (50.0%), C/T (40.0%) and T/T (10.0%; P?=?0.012). The -799C allele frequency was lower in the patients (59.7%) than in the reference group (70.0%; P?=?0.023). The -799C allele showed protective effects against the arterial disease with an odds ratio [95% confidence interval (CI)] of 0.372 (0.141-0.980, P?=?0.045) after adjustment for age, gender, and serum MMP-8 and TIMP-1 concentrations. Only in the reference group and whole study population (n?=?224), the -799TT genotype significantly associated with an increase in serum MMP-8 concentrations (P?=?0.047, 0.025). The -799C allele appeared protective against the arterial disease. The genotype may have an effect on systemic MMP-8 levels which could not, however, be seen in the arterial disease patients probably as a result of the strong inflammation involved in the disease pathogenesis.     

Hyperchromic effect of collagen induced by human collagenase

Loss of the highly ordered triple-helix structure of native collagen on denaturation or enzymatic degradation involves a helix-to-coil transition, which can be seen as an increase at 227 nm in its ultraviolet difference absorption spectrum. We report here the successful use of this hyperchromic effect to quantify collagen in solution and to follow up the time-course of collagen degradation catalyzed by collagenase. Using 14C-labelled collagen substrate we show the excellent correlation between enzyme-induced increase in ultraviolet difference absorption and formation of specific cleavage products. The novel method was found to be suitable to characterize the enzymatic properties of human leukocyte collagenase. Activation of latent collagenase to the active enzyme could be followed continuously and an activation lag estimated.     

Examination of ras (P21) proteins in plasma from workers exposed to benzene emissions from petrochemical plants and healthy controls

Exposure of workers to benzene and polyaromatic hydrocarbons has been documented to be at relatively high levels in the production of benzene and in the coking process at a petrochemical plant in the oil shale area in Estonia. Altogether 97 plasma samples from workers and 40 from unexposed matched referents from two samplings in different seasons were analyzed for the presence of ras (P21) proteins; of the workers 50 were exposed to benzene in the benzene production plant and 47 to polyaromatic hydrocarbons and benzene in a cokery. Proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence, using a monoclonal antibody as the primary antibody. There were no statistically significant differences between the exposed and the referent groups. The results are thus in keeping with the lack of exposure related cytogenetic effects for this same workforce.     

Interaction of Mesna (2-mercaptoethane sulfonate) with the mutagenicity of cyclophosphamide in vitro and in vivo

The effects of sodium 2-mercaptoethane sulfonate (Mesna) on the mutagenicity of cyclophosphamide (CP) were assessed in vitro by the Ames test and in vivo in rats by analyzing micronuclei in bone marrow and mutagenic activity in urine. Mesna alone was negative in all test systems, while CP gave a positive response in all of them. In a combined treatment there was no significant reduction of the CP-induced mutagenicity in Salmonella. In rats the frequency of bone marrow micronuclei was not diminished when Mesna was given together with CP. May-Grunwald-Giemsa staining and Hoechst-Pyronin fluorescent staining techniques for micronuclei yielded similar results. The urine of rats treated with CP was mutagenic to Salmonella and no significant difference was observed when the rats had received both Mesna and CP. The results give support to the theory that Mesna acts primarily by reducing the toxicity of metabolites of CP, particularly acrolein, in the urinary tract and not by suppressing the mutagenicity of the active metabolites of CP.