Identification of a novel glyoxylate reductase supports phylogeny-based enzymatic substrate specificity prediction

Phylogenetic analysis of the superfamily of D-2-hydroxyacid dehydrogenases identified the previously unrecognized cluster of glyoxylate/hydroxypyruvate reductases (GHPR). Based on the genome sequence of Rhizobium etli, the nodulating endosymbiont of the common bean plant, we predicted a putative 3-phosphoglycerate dehydrogenase to exhibit GHPR activity instead. The protein was overexpressed and purified. The enzyme is homodimeric under native conditions and is indeed capable of reducing both glyoxylate and hydroxypyruvate. Other substrates are phenylpyruvate and ketobutyrate. The highest activity was observed with glyoxylate and phenylpyruvate, both having approximately the same kcat/Km ratio. This kind of substrate specificity has not been reported previously for a GHPR. The optimal pH for the reduction of phenylpyruvate to phenyllactate is pH 7. These data lend support to the idea of predicting enzymatic substrate specificity based on phylogenetic clustering.     

Human adipose tissue-derived multipotent stem cells differentiate in vitro and in vivo into osteocyte-like cells

Cell-based therapies are used to treat bone defects. We recently described that human multipotent adipose-derived stem (hMADS) cells, which exhibit a normal karyotype, self renewal, and the maintenance of their differentiation properties, are able to differentiate into different lineages. Herein, we show that hMADS cells can differentiate into osteocyte-like cells. In the presence of a low amount of serum and EGF, hMADS cells express specific molecular markers, among which alkaline phosphatase, CBFA-1, osteocalcin, DMP1, PHEX, and podoplanin and develop functional gap-junctions. When loaded on a hardening injectable bone substitute (HIBS) biomaterial and injected subcutaneously into nude mice, hMADS cells develop mineralized woven bone 4 weeks after implantation. Thus hMADS cells represent a valuable tool for pharmacological and biological studies of osteoblast differentiation in vitro and bone development in vivo.     

The uroprotection of mesna on cyclophosphamide cystitis in rats. Its consequences on behaviour and brain activities.

We studied the uroprotective effect of mesna, at doses of 40-300 mg/kg/i.p., in single or fractioned injections, on the development of cyclophosphamide (CP, 100 mg/kg/i.p.) cystitis in rats. The study concerns the histological, behavioural and nervous aspects of the disease. The specific effects of mesna, when injected alone, have also been considered. The mesna itself does not have specific deleterious effects, except at a dose of 300 mg/kg which provokes a moderate vesical inflammation although without consequence on the animal's behaviour. Mesna offers good protection against CP cystitis for only certain posologies. The uroprotective effects of mesna reach maxima at doses of 40-100 mg/kg and for fractioned injections given over the entire time frame of the urinary toxic release. The uroprotective effects of other posologies are only partial. The nervous activities were studied through the expression of Fos protein. The repetitive intraperitoneal injection of mesna induced a spinal activity and a preferential contralateral activity of the trigemino/reticular areas of the brainstem spinal cord junction--an effect which was reduced in the presence of CP. The prevention of cystitis by mesna was accompanied only by a reduction in spinal Fos activity, the supraspinal activities remaining high and in strict relationship with the vagal afferent activity. In conclusion, the uroprotective effect of mesna, which requires appropriate posologies, has led to the confirmation of the spinal actions of the CP cystitis, probably via the pelvic nerve, but did not allow a clear distinction between the consequences of the systemic (vagal) and local (spinal, pelvic) actions of CP at supraspinal level.     

Analysis of Cellular Mutants Resistant to Theiler’s Virus Infection: Differential Infection of L929 Cells by Persistent and Neurovirulent Strains

Theiler's murine encephalomyelitis virus (TMEV) is a natural pathogen of the mouse and belongs to the Picornaviridae family. TMEV strains are divided into two subgroups on the basis of their pathogenicity. The first group contains two neurovirulent strains, FA and GDVII, which cause a rapid fatal encephalitis. The second group includes persistent strains, like DA and BeAn, which produce a biphasic neurological disease in susceptible mice. Persistence of these viruses in the white matter of the spinal cord leads to chronic inflammatory demyelination. L929 cells, which are susceptible to TMEV infection, were subjected to physicochemical mutagenesis. Cellular clones that became resistant to TMEV infection were selected by viral infection. Three such mutants resistant to strain GDVII were characterized to determine the step of the virus cycle that was inhibited. The mutation present in one of these mutant cell lines inhibited, by more than 1,000-fold, the entry of strain GDVII but hardly decreased infection by strain DA. In the two other cellular mutants, replication of the viral genome was slowed down. Interestingly, one of these mutant cell lines resisted infection by both the persistent and neurovirulent strains while the second cell line resisted infection by strain GDVII but remained susceptible to the persistent virus. These results show that although they have 95% identity at the amino acid sequence level, neurovirulent and persistent viruses use partly distinct pathways for both entry into cells and genome replication.     

Role of Porins in Sensitivity of Escherichia coli to Antibacterial Activity of the Lactoperoxidase Enzyme System

Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN⁻) from thiocyanate (SCN⁻), using H₂O₂ as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN⁻.     

Induction of Shiga Toxin-Converting Prophage in Escherichia coli by High Hydrostatic Pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.     

IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo

Interferons (IFN) exert antiviral, immunomodulatory and cytostatic activities. IFN-alpha/beta (type I IFN) and IFN-lambda (type III IFN) bind distinct receptors, but regulate similar sets of genes and exhibit strikingly similar biological activities. We analyzed to what extent the IFN-alpha/beta and IFN-lambda systems overlap in vivo in terms of expression and response. We observed a certain degree of tissue specificity in the production of IFN-lambda. In the brain, IFN-alpha/beta was readily produced after infection with various RNA viruses, whereas expression of IFN-lambda was low in this organ. In the liver, virus infection induced the expression of both IFN-alpha/beta and IFN-lambda genes. Plasmid electrotransfer-mediated in vivo expression of individual IFN genes allowed the tissue and cell specificities of the responses to systemic IFN-alpha/beta and IFN-lambda to be compared. The response to IFN-lambda correlated with expression of the alpha subunit of the IFN-lambda receptor (IL-28R alpha). The IFN-lambda response was prominent in the stomach, intestine and lungs, but very low in the central nervous system and spleen. At the cellular level, the response to IFN-lambda in kidney and brain was restricted to epithelial cells. In contrast, the response to IFN-alpha/beta was observed in various cell types in these organs, and was most prominent in endothelial cells. Thus, the IFN-lambda system probably evolved to specifically protect epithelia. IFN-lambda might contribute to the prevention of viral invasion through skin and mucosal surfaces.     

Living on a surface: swarming and biofilm formation

Swarming is the fastest known bacterial mode of surface translocation and enables the rapid colonization of a nutrient-rich environment and host tissues. This complex multicellular behavior requires the integration of chemical and physical signals, which leads to the physiological and morphological differentiation of the bacteria into swarmer cells. Here, we provide a review of recent advances in the study of the regulatory pathways that lead to swarming behavior of different model bacteria. It has now become clear that many of these pathways also affect the formation of biofilms, surface-attached bacterial colonies. Decision-making between rapidly colonizing a surface and biofilm formation is central to bacterial survival among competitors. In the second part of this article, we review recent developments in the understanding of the transition between motile and sessile lifestyles of bacteria.