Design and expression of peptides with antimicrobial activity against Salmonella typhimurium

We showed previously that insertion of Synechocystis Δ¹²‐desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ¹²‐desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α‐antimicrobial peptide (α‐AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg²⁺ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA‐Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α‐AMPs to a specific MLD generating live non‐virulent bacterial strains.     

Discovery of imidazo[1,2-a]-, [1,2,4]triazolo[4,3-a]-, and [1,2,4]triazolo[1,5-a]pyridine-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5

Based on a hypothesis that an intramolecular hydrogen bond was present in our lead series of picolinamide mGlu₅ NAMs, we reasoned that an inactive nicotinamide series could be modified through introduction of a fused heterocyclic core to generate potent mGlu₅ NAMs. In this Letter, we describe the synthesis and evaluation of compounds that demonstrate the viability of that approach. Selected analogs were profiled in a variety of in vitro assays, and two compounds were evaluated in rat pharmacokinetic studies and a mouse model of obsessive-compulsive disorder. Ancillary pharmacology screening revealed that members of this series exhibited moderate inhibition of the dopamine transporter (DAT), and SAR was developed that expanded the selectivity for mGlu₅ versus DAT.     

Human leptin induces angiogenesis in vivo

Leptin is an adipocyte-produced peptide, which plays a crucial role in the regulation of body weight. There is also evidence that leptin stimulates endothelial cell proliferation and the formation of capillary-like tubes in vitro. The disc angiogenesis system was used to measure the angiogenic effect of leptin in vivo. Discs containing 25, 50, 100 and 250 ng/ml of leptin were implanted subcutaneously in Wistar rats, removed after a growth period of 7 and 14 days, and compared with spontaneous growth controls and with positive controls containing equivalent doses of vascular endothelial growth factor (VEGF). Discs were examined morphologically for stroma and vessel development and by immunohistochemistry for quantitative evaluation of angiogenesis. The specificity of the angiogenic effect of leptin was tested by blocking leptin with a polyclonal anti-leptin antibody. Leptin induced a significant level of angiogenesis in a dose-dependent manner both at 7 and 14 days, with a peak at the dose of 100 ng/ml. The angiogenic activity of leptin was completely abolished by the anti-leptin neutralizing antibody. VEGF also induced significant dose-dependent angiogenesis at the same time points with a peak observed at a concentration of 100 ng/ml. The angiogenic response to leptin was significantly higher at 7 days compared with VEGF but not at the 14-day time point. In conclusion, leptin has a specific angiogenic effect in vivo, which is dose- and time-dependent in a concentration range of 25–250 ng/ml. This effect is equivalent to the angiogenic effect of VEGF but is evident earlier compared with VEGF.     

RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation

In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.     

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.     

Estimating dataset size requirements for classifying DNA microarray data.

A statistical methodology for estimating dataset size requirements for classifying microarray data using learning curves is introduced. The goal is to use existing classification results to estimate dataset size requirements for future classification experiments and to evaluate the gain in accuracy and significance of classifiers built with additional data. The method is based on fitting inverse power-law models to construct empirical learning curves. It also includes a permutation test procedure to assess the statistical significance of classification performance for a given dataset size. This procedure is applied to several molecular classification problems representing a broad spectrum of levels of complexity.     

Plant chitinases--regulation and function

The aim of this review is to present the current state of knowledge on plant chitinases and their regulation and function. Chitinases are up-regulated by a variety of stress conditions, both biotic and abiotic, and by such phytohormones as ethylene, jasmonic acid, and salicylic acid. Like other PR proteins, chitinases play a role in plant resistance against distinct pathogens. Moreover, by reducing the defence reaction of the plant, chitinases allow symbiotic interaction with nitrogen-fixing bacteria or mycorrhizal fungi. However, recent investigations have shown that these enzymes are also involved in numerous physiological events. The involvement of chitinases in development and growth processes is also described.     

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.