Response surface methodology, an approach to predict the effects of a lactoperoxidase system, Nisin, alone or in combination, on Listeria monocytogenes in skim milk

Experimental designs using Response Surface Methodology (RSM) were used to determine effects and interactions of Nisin (0-200 i.u. ml-1), pH values (5.4-6.6), incubation time (0-36 h or 0-144 h) and the lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS) on Listeria monocytogenes CIP 82110 in skim milk, at 25 degrees C. The LPS varied from level 0-2; LPS at level 1 consisted of lactoperoxidase (35 mg l-1), thiocyanate (25 mg l-1) and H2O2, which was supplied exogenously by glucose-oxidase (1 mg l-1) and glucose (0.2 g l-1); LPS activity was dependent on LPS level and incubation time. In the presence of LPS at level 1, a bacteriostatic phase was followed by growth, whereas at a higher level, a bactericidic phase was observed. Nisin response was time- and pH-dependent. Nisin was bactericidic at acidic pH values and for a short incubation time (12 h) only; then, a re-growth phase was observed. Nisin and LPS in combination gave an original response which lacked the transitory bactericidal effect of Nisin and had a continuously bactericidal affect, leading to 10 cfu ml-1 of L. monocytogenes at 144 h; the response was greatly affected by incubation time. Predicted values were in good agreement with experimental values. Response Surface Methodology is a useful experimental approach for rapid testing of the effects of inhibitors.     

Fetal toxicity of valsartan and possible reversible adverse side effects

BACKGROUND: Published cases suggest that the use of angiotensin II receptor antagonists is fetotoxic during the third trimester, but not in early pregnancy. CASE: We report a case in which the adverse fetal effect of angiotensin II receptor antagonist treatment was reversed. A woman with chronic hypertension was treated with valsartan until gestation week (GW) 20, when a complete anhydramnios was observed. Six days after interruption of the treatment, amniotic fluid reappeared. It reached a normal level at GW 23.5. The plasmatic creatinine level and the renal ultrasound examination were within normal limits at the six-month follow-up. CONCLUSIONS: Whereas angiotensin-II-receptor antagonist generates a severe renal toxicity, this case suggests that, at least in the first half of pregnancy, these effects can be reversed.     

Mitochondrial requirement for endothelial responses to cyclic strain: implications for mechanotransduction

Mechanical strain triggers a variety of cellular responses, but the underlying mechanotransduction process has not been established. Endothelial cells (EC) respond to mechanical strain by upregulating adhesion molecule expression through a signaling process involving reactive oxygen species (ROS), but the site of their generation is unknown. Mitochondria anchor to the cytoskeleton and could function as mechanotransducers by releasing ROS during cytoskeletal strain. In human umbilical vein EC (HUVEC), ROS production increased 221 +/- 17% during 6 h of cyclic strain vs. unstrained controls. Mitochondrial inhibitors diphenylene iodonium or rotenone abrogated this response, whereas inhibitors of nitric oxide (NO) synthase (L-nitroarginine), xanthine oxidase (allopurinol), or NAD(P)H oxidase (apocynin) had no effect. The antioxidants ebselen and diethyldithiocarbamate inhibited the increase in ROS, but the NO scavenger Hb had no effect. Thus strain induces ROS release from mitochondria. In other studies, HUVEC were rendered mitochondria deficient (rho0 EC) to determine the requirement for electron transport in the response to strain. Strain-induced 2'7'-dichlorofluorescein fluorescence was attenuated by >80% in rho0 EC compared with HUVEC (43 +/- 7 vs. 221 +/- 17%). Treatment with cytochalasin D abrogated strain-induced ROS production, indicating a requirement for the actin cytoskeleton. Cyclic strain (6 h) increased VCAM-1 expression in wild-type but not rho0 EC. Increases in NF-kappaB activation and VCAM-1 mRNA expression during strain were prevented by antioxidants. These findings demonstrate that mitochondria function as mechanotransducers in endothelium by increasing ROS signaling, which is required for strain-induced increase in VCAM-1 expression via NF-kappaB.     

Horizontal Gene Transfer of “Prototype” Nramp in Bacteria

Eukaryotic Nramp genes encode divalent metal ion permeases important for nutrition and resistance to microbial infection. Bacterial homologs encode proton-dependent transporters of manganese (MntH), and other divalent metal ions. Bacterial MntH were classified in three homology groups (A, B, C) and MntH C further subdivided in C, C, C. The proteins from C. tepidum (MntH B) and E. faecalis (MntH C1, 2), divergent in sequence and hydropathy profile, conferred increased metal sensitivity when expressed in E. coli, suggesting conservation of divalent metal transport function in MntH B and C. Several genomic evidence suggest horizontal gene transfer (HGT) of mntH C genes: (i) The enterobacteria Wigglesworthia mntH C gene is linked to an Asn t-RNA, and its sequence most conserved with Gram positive bacteria homologs; (ii) all the C genes identified in oral streptococcaceae are associated with different potentially mobile DNA elements; (iii) Lactococcus lactis and Burkholderia mallei genomes contain an mntH gene prematurely terminated and a novel full-length mntH C gene; (iv) remarkable sequence relatedness between the unicellular alga C. reinhardtii prototype Nramp and some MntH C (e.g., Nostoc spp., Listeria spp.) suggests HGT between Eukarya and Bacteria. Other prototype Nramp genes (intronless, encoding proteins strongly conserved with MntH A and B proteins) identified in invertebrates represent a possible source for transfer of Nramp genes toward opportunistic bacteria. This study demonstrates complex evolution of MntH in Bacteria. It is proposed that prototype Nramp are ancestors of bacterial MntH C proteins, which could facilitate bacterial infection. Equally contributing authors (Etienne Richer and Pascal Courville).     

Human cytomegalovirus US3 chimeras containing US2 cytosolic residues acquire major histocompatibility class I and II protein degradation properties

Human cytomegalovirus (HCMV) glycoprotein US2 increases the proteasome-mediated degradation of major histocompatibility complex (MHC) class I heavy chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. US2-initiated degradation of MHC proteins apparently involves the recruitment of cellular proteins that participate in a process known as endoplasmic reticulum (ER)-associated degradation. ER-associated degradation is a normal process by which misfolded proteins are recognized and translocated into the cytoplasm for degradation by proteasomes. It has been demonstrated that truncated forms of US2, especially those lacking the cytoplasmic domain (CT), can bind MHC proteins but do not cause their degradation. To further assess how the US2 CT domain interacts with the cellular components of the ER-associated degradation pathway, we constructed chimeric proteins in which the US2 CT domain or the CT and transmembrane (TM) domains replaced those of the HCMV glycoprotein US3. US3 also binds both class I and II proteins but does not cause their degradation. Remarkably, chimeras containing the US2 CT domain caused the degradation of both MHC class I and II proteins although this degradation was less than that by wild-type US2. Therefore, the US2 CT and TM domains can confer on US3 the capacity to degrade MHC proteins. We also analyzed complexes containing MHC proteins and US2, US3, US11, or US3/US2 chimeras for the presence of cdc48/p97 ATPase, a protein that binds polyubiquitinated proteins and likely functions in the extraction of substrates from the ER membrane before the substrates meet proteasomes. p97 ATPase was present in immunoprecipitates containing US2, US11, and two chimeras that included the US2 CT domain, but not in US3 complexes. Therefore, it appears that the CT domain of US2 participates in recruiting p97 ATPase into ER-associated degradation complexes.     

Assessing the role of the T cell receptor beta gene enhancer in regulating coding joint formation during V(D)J recombination

To assess the role of the T cell receptor (TCR) beta gene enhancer (Ebeta) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRbeta rearrangement of Ebeta-deleted (DeltaEbeta) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with DeltaEbeta, DeltaEbeta Bcl-2 thymocytes show a small accumulation of TCRbeta standard recombination products, including coding ends, that involves the proximal Dbeta-Jbeta and Vbeta14 loci but not the distal 5' Vbeta genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Ebeta, by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Ebeta-dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRbeta allelic exclusion depending on the position within the locus.     

Cloning of a pine germin-like protein (GLP) gene promoter and analysis of its activity in transgenic tobacco Bright Yellow 2 cells

Germins and germin-like proteins (GLPs) constitute a large and highly diverse family of ubiquitous plant cell wall proteins. These proteins seem to be involved in many developmental stages and stress-related processes, but their exact participation in these processes generally remains obscure. In Pinus caribaea Morelet, the PcGER1 gene is expressed uniquely in embryo tissues, and encodes a GLP ionically bound to the walls of pine embryo cells maintained in 2,4-D-containing medium. We have cloned a genomic fragment including the 1520 bp 5'-upstream promoter region of PcGER1 . This sequence contains, in its 1200 bp distal part, several cis elements (e.g. SEF4, 60 kDa protein, ABA RE and Dof recognition sites) present in genes responding to hormones and/or expressed in embryo or seed tissues, or during germination. The PcGER1 promoter sequence was cloned upstream of the GUS ( β -glucuronidase) reporter gene and transferred to tobacco Bright Yellow 2 (BY-2) cells via Agrobacterium tumefaciens -mediated transformation. Promoter activity and growth performances of transgenic asynchronous cell suspensions were analysed in the presence or absence of 2,4-D and/or BA. Optimal growth, maximum cell-wall yield and PcGER1 promoter activity were observed in the presence of 2,4-D and BA at day 4, the end of the exponential growth phase where 70–75% cells have a 2C DNA content. Analysis of promoter activity during the cell cycle in an aphidicoline-synchronized culture suggested that the expression is maximum in G1 cells. We also showed that under optimal growth conditions, 5' promoter deletions decreased the activity of the reporter gene. We discuss the function of this gene with regards to cell growth.
Accession number : The PcGER1 promoter sequence was submitted to the genbank database under the accession number AY077704 .     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.