R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile

Clostridium difficile induces antibiotic-associated diarrhea through the production of toxin A and toxin B; the former toxin has been assumed to be responsible for the symptoms of the disease. Several toxin A-negative strains from C. difficile have recently been isolated from clinical cases and have been reported to produce toxin B variants eliciting an atypical cytopathic effect. Ultrastructural analysis indicated these toxins induce a rounding cytopathic effect and filopodia-like structures. Toxin B variants glucosylated R-Ras, and transfection with a constitutively active mutant of this GTPase protected cells against their cytopathic effect. Treatment of cells with toxin B variants induced detachment from the extracellular matrix and blockade of the epidermal growth factor-mediated phosphorylation of extracellular-regulated protein kinases, demonstrating a deleterious effect on the R-Ras-controlled avidity of integrins. Treatment with toxin B variants also induced a transient activation of RhoA probably because of inactivation of Rac1. Altogether, these data indicate that the cytopathic effect induced by toxin B variants is because of cell rounding and detachment mediated by R-Ras glucosylation, and the induction of filopodia-like structures is mediated by RhoA activation. Implications for the pathophysiology of C. difficile-induced diarrhea are discussed.     

The influence of an intramolecular hydrogen bond in differential recognition of inhibitory acceptor analogs by human ABO(H) blood group A and B glycosyltransferases

Human ABO(H) blood group glycosyltransferases GTA and GTB catalyze the final monosaccharide addition in the biosynthesis of the human A and B blood group antigens. GTA and GTB utilize a common acceptor, the H antigen disaccharide alpha-l-Fucp-(1-->2)-beta-d-Galp-OR, but different donors, where GTA transfers GalNAc from UDP-GalNAc and GTB transfers Gal from UDP-Gal. GTA and GTB are two of the most homologous enzymes known to transfer different donors and differ in only 4 amino acid residues, but one in particular (Leu/Met-266) has been shown to dominate the selection between donor sugars. The structures of the A and B glycosyltransferases have been determined to high resolution in complex with two inhibitory acceptor analogs alpha-l-Fucp(1-->2)-beta-d-(3-deoxy)-Galp-OR and alpha-l-Fucp-(1-->2)-beta-d-(3-amino)-Galp-OR, in which the 3-hydroxyl moiety of the Gal ring has been replaced by hydrogen or an amino group, respectively. Remarkably, although the 3-deoxy inhibitor occupies the same conformation and position observed for the native H antigen in GTA and GTB, the 3-amino analog is recognized differently by the two enzymes. The 3-amino substitution introduces a novel intramolecular hydrogen bond between O2' on Fuc and N3' on Gal, which alters the minimum-energy conformation of the inhibitor. In the absence of UDP, the 3-amino analog can be accommodated by either GTA or GTB with the l-Fuc residue partially occupying the vacant UDP binding site. However, in the presence of UDP, the analog is forced to abandon the intramolecular hydrogen bond, and the l-Fuc residue is shifted to a less ordered conformation. Further, the residue Leu/Met-266 that was thought important only in distinguishing between donor substrates is observed to interact differently with the 3-amino acceptor analog in GTA and GTB. These observations explain why the 3-deoxy analog acts as a competitive inhibitor of the glycosyltransferase reaction, whereas the 3-amino analog displays complex modes of inhibition.     

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.     

Mutagenesis of putative catalytic and regulatory residues of Streptomyces chromofuscus phospholipase D differentially modifies phosphatase and phosphodiesterase activities

Phospholipase D from Streptomyces chromofuscus (sc-PLD) is a member of the diverse family of metallo-phosphodiesterase/phosphatase enzymes that also includes purple acid phosphatases, protein phosphatases, and nucleotide phosphodiesterases. Whereas iron is an essential cofactor for scPLD activity, Mn2+ is also found in the enzyme. A third metal ion, Ca2+, has been shown to enhance scPLD catalytic activity although it is not an essential cofactor. Sequence alignment of scPLD with known phosphodiesterases and phosphatases requiring metal ions suggested that His-212, Glu-213, and Asp-389 could be involved in Mn2+ binding. H212A, E213A, and D389A were prepared to test this hypothesis. These three mutant enzymes and wild type scPLD show similar metal content but considerably different catalytic properties, suggesting different roles for each residue. His-212 appears involved in binding the phosphate group of substrates, whereas Glu-213 acts as a ligand for Ca2+. D389A showed a greatly reduced phosphodiesterase activity but almost unaltered ability to hydrolyze the phosphate group in p-nitrophenyl phosphate suggesting it had a critical role in aligning groups at the active site to control phosphodiesterase versus phosphatase activities. We propose a model for substrate and cofactor binding to the catalytic site of scPLD based on these results and on sequence alignment to purple acid phosphatases of known structure.     

Angiogenesis and nerve regeneration in a model of human skin equivalent transplant

The angiogenesis and reinnervation were studied in a porcine model of human skin equivalent (SE) graft and the relationship between the two processes was investigated. Confocal laser scanning microscopy was used to monitor, during the healing process, the pattern of vascularization and reinnervation at different time points. The SE was obtained by co-culturing fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan biopolymer and grafted on dorsal wounds generated by full-thickness resection in 25/30 Kg Large white pigs. Frozen sections were obtained from biopsies performed in autograft and xenograft, then were immunolabeled by using the endothelial marker lectin Lactifolia and with the neuronal marker gene product PGP9.5. Cajal staining was also used to visualize the nerve fibers. The results show that the vascularization precedes the innervation process. These data are consistent with the view that the development of nervous tissue is driven by nutritional and trophic factors provided by the vascular system. The arborization of the two systems observed during the third week from the graft might play a key role in maintaining the healing process and the graft survival.     

Implementation of a molecular epidemiology approach to human pleural malignant mesothelioma

The carcinogenic effect of asbestos has been reported in the literature since 40 years, and early studies describing the epidemic occurrence of malignant mesothelioma (MM) in asbestos workers, have become a paradigm of occupational cancer research. Research on MM was abandoned for many years since MM was considered as an asbestos-related disease, interesting only from a perspective of disease control and preventive policies. The introduction of new biological endpoints in the epidemiological studies has boosted research in the field, providing new tools for the study of emerging priority in cancer research and in public health. This approach, known as molecular epidemiology has a great potential in the study of MM, contributing to the understanding of susceptibility factors, to the evaluation of cancer risk in people occupationally or environmentally exposed to carcinogens, and to the enhancement of diagnosis and therapy. A comprehensive approach based on the use of banks of biological samples is presented and its advantages discussed here. The application of innovative endpoints, such as oncoproteins in biologic fluids, genetic polimorphisms, or gene function is discussed, and relevant literature reviewed.     

Micronuclei frequency in children exposed to environmental mutagens: a review

Cytogenetic monitoring has been traditionally used for the surveillance of populations exposed to genotoxic agents. In recent years sensitivity problems emerged in surveys of populations exposed to low levels of mutagens, and therefore alternative approaches have been explored. Biomonitoring studies in children are a promising field, since because of evident differences in the uptake, metabolism, distribution and excretion of mutagens this population seems to be more susceptible than adults. Further, the effect of major confounders such as cigarettes smoking, occupation, life-style, and dietary factors plays a minor role. Among cytogenetic assays, the micronucleus assay (MN) has several advantages and is increasingly used. A review was then carried out to synthesize the published data on the occurrence of MN in children and adolescents (age range 0-18 years), and to assess the impact of genotoxic exposure on MN frequency. Overall, 20 papers from international literature and 8 Russian papers were included. An effect of age was found within this age range, while the influence of gender on MN frequency was irrelevant. These results were confirmed by the re-analysis of data for 448 children selected from the HUMN database. An effect of chronic and infectious diseases on MN levels has been reported by various authors. Most studies describing the effect of exposure to genotoxic agents (ionizing radiation, chemicals, drugs, environmental tobacco smoke) found an increase of MN in exposed children. The limited number of published papers indicates that the conduct of properly designed studies on the effect of environmental pollutants in children may be difficult. This review confirmed the usefulness of MN assay in biomonitoring studies conducted in children, revealing that in many circumstances investigating children increases the sensitivity of the study, even with low dose exposures.     

Inhibition of Moloney murine leukemia virus integration using polyamides targeting the long-terminal repeat sequences

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.     

Acetohydroxyacid synthase from Mycobacterium avium and its inhibition by sulfonylureas and imidazolinones

Tuberculosis (TB) remains one of the world's leading causes of death from infectious disease. It is caused by infection with Mycobacterium tuberculosis or sometimes, particularly in immune-compromised patients, Mycobacterium avium. The aim of this study was to create a tool that could be used in the search for new anti-TB drugs that inhibit branched-chain amino acid (BCAA) biosynthesis, as these are essential amino acids that are not available to a mycobacterium during growth in an infected organism. To this end, we cloned, overexpressed, purified and characterised for the first time an acetohydroxyacid synthase (AHAS), a key enzyme in the pathway to the biosynthesis of the BCAAs, from the genus Mycobacterium. Nine commercial herbicides of the sulfonylurea and imidazolinone classes were tested for their influence on this enzyme. Four of the sulfonylureas were potent inhibitors of the enzyme. The relative potency of the different inhibitors towards the M. avium enzyme was unlike their potency towards other AHASs whose inhibitor profile has been reported, emphasising the advantage of using a mycobacterial enzyme as a tool in the search for new anti-TB drugs.     

Molecular requirements for the regulation of the renal outer medullary K(+) channel ROMK1 by the serum- and glucocorticoid-inducible kinase SGK1

The serum- and glucocorticoid- inducible kinase SGK1 stimulates the renal outer medullary K(+) channel ROMK1 in the presence of the Na(+)/H(+) exchanger regulating factor NHERF2. SGK1/NHERF2 are effective through enhancement of ROMK1 abundance within the cell membrane. The present study aims to define the molecular requirements for the interaction of ROMK1 with SGK1/NHERF2. Pull down assays reveal that SGK1 interacts with NHERF2 through the second PDZ domain of NHERF2. According to chemiluminescence and electrophysiology, deletion of the second PDZ domain of NHERF2 or the putative PDZ binding motif on ROMK1 abrogates the stimulating effect of SGK1 on ROMK1 protein abundance in the plasma membrane and K(+) current.