Design and synthesis of indole and tetrahydroisoquinoline hydantoin derivatives as human chymase inhibitors

The synthesis of new potential inhibitors of human chymase is described. Treatment of dihydroimidazo[1,5-a]indole and [1,5-b]isoquinoline-dione with thioaryl followed by oxidation gave the N-arylsulfonylmethyl of polycyclic hydantoin derivatives 3, 5 and 6.     

Allosteric-type control of synaptobrevin cleavage by protect tetanus toxin light chain

Summary The light chain of tetanus neurotoxin (TeNTL chain) has been shown to be endowed with zine endopeptidase activity, selectively directed towards the Gln⁷⁶-Phe⁷⁷ bond of synaptobrevin, a vesicle-associated membrane protein critically involved in neuroexocytosis. In previous reports, truncations at the NH₂- and COOH-terminus of synaptobrevin have shown that the sequence 39–88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well-defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH₂- and COOH-terminal peptides of synaptobrevin, S 27–55 (S₁) and S 82–93 (S₂), to the synaptobrevin fragment S 56–81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S₁ and S₂ with complementary sites present on TeNT as shown by surface plasmon resonance experiments. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT probably involving a conformational change of the toxin. This could accound for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.     

The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.     

PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.     

Assignment by in situ hybridization of a fibroblast growth factor receptor gene to human chromosome band 10q26

Summary A 2.3-kb cDNA probe for the human bek fibroblast growth factor receptor was used to determine the chromosomal localization of the corresponding gene by in situ hybridization. The results show that this gene, a form of which is amplified in some poorly differentiated stomach cancers, is localized on chromosome region 10q26. The two previously identified fibroblast growth factor receptor genes are thus not on the same chromosome, as the related fig (fms-like gene) fibrovblast growth factor receptor gene has previously been mapped to human chromosome region 8p12.     

Polysialylation increases lateral diffusion of neural cell adhesion molecule in the cell membrane

Polysialic acid (PSA) is a polymer of N-acetylneuraminic acid residues added post-translationally to the membrane-bound neural cell adhesion molecule (NCAM). The large excluded volume created by PSA polymer is thought to facilitate cell migration by decreasing cell adhesion. Here we used live cell imaging (spot fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) combined with biochemical approaches in an attempt to uncover a link between cell motility and the impact of polysialylation on NCAM dynamics. We show that PSA regulates specifically NCAM lateral diffusion and this is dependent on the integrity of the cytoskeleton. However, whereas the glial-derivative neurotrophic factor chemotactic effect is dependent on PSA, the molecular dynamics of PSA-NCAM is not directly affected by glial-derivative neurotrophic factor. These findings reveal a new intrinsic mechanism by which polysialylation regulates NCAM dynamics and thereby a biological function like cell migration.     

Serum and Lymphocytic Neurotrophins Profiles in Systemic Lupus Erythematosus: a Case-Control Study

Background

Neurotrophins play a central role in the development and maintenance of the nervous system. However, neurotrophins can also modulate B and T cell proliferation and activation, especially via autocrine loops. We hypothesized that both serum and lymphocytic neurotrophin levels may be deregulated in systemic Lupus erythematosus (SLE) and may reflect clinical symptoms of the disease.

Methods

Neurotrophins in the serum (ELISA tests) and lymphocytes (flow cytometry) were measured in 26 SLE patients and 26 control subjects. Th1 (interferon-γ) and Th2 (IL-10) profiles and serum concentration of BAFF were assessed by ELISA in the SLE and control subjects.

Findings

We have demonstrated that both NGF and BDNF serum levels are higher in SLE patients than healthy controls (p=0.003 and p<0.001), independently of Th1 or Th2 profiles. Enhanced serum NT-3 levels (p=0.003) were only found in severe lupus flares (i.e. SLEDAI ≥ 10) and significantly correlated with complement activation (decreased CH 50, Γ=-0.28, p=0.03). Furthermore, there was a negative correlation between serum NGF levels and the number of circulating T regulatory cells (Γ=0.48, p=0.01). In circulating B cells, production of both NGF and BDNF was greater in SLE patients than in healthy controls. In particular, the number of NGF-secreting B cells correlated with decreased complement levels (p=0.05). One month after SLE flare treatment, BDNF levels decreased; in contrast, NGF and NT-3 levels remained unchanged.

Conclusion

This study demonstrates that serum and B cell levels of both NGF and BDNF are increased in SLE, suggesting that the neurotrophin production pathway is deregulated in this disease. These results must be confirmed in a larger study with naive SLE patients, in order to avoid the potential confounding influence of prior immune-modulating treatments on neurotrophin levels.     

Polymorphisms,de novo lipogenesis,and plasma triglyceride response following fish oil supplementation

Interindividual variability in the response of plasma triglyceride concentrations (TG) following fish oil consumption has been observed. Our objective was to examine the associations between single-nucleotide polymorphisms (SNPs) within genes encoding proteins involved in de novo lipogenesis and the relative change in plasma TG levels following a fish oil supplementation. Two hundred and eight participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g eicosapentaenoic acid and 1.1 g docosahexaenoic acid. SNPs within SREBF1, ACLY, and ACACA genes were genotyped using TAQMAN methodology. After correction for multiple comparison, only two SNPs, rs8071753 (ACLY) and rs1714987 (ACACA), were associated with the relative change in plasma TG concentrations (P = 0.004 and P = 0.005, respectively). These two SNPs explained 7.73% of the variance in plasma TG relative change following fish oil consumption. Genotype frequencies of rs8071753 according to the TG response groups (responders versus nonresponders) were different (P = 0.02). We conclude that the presence of certain SNPs within genes, such as ACLY and ACACA, encoding proteins involved in de novo lipogenesis seem to influence the plasma TG response following fish oil consumption.     

Sleeping site selection and presleep behavior in wild pigtailed macaques

Several factors are likely to control sleeping site selection and presleep behavior in nonhuman primates, including predation risk and location of food resources. We examined the effects of these factors on the sleeping behavior of northern pigtailed macaques (Macaca leonina). While following a troop living in the surroundings of the Visitor Center of Khao Yai National Park (Thailand), we recorded the physical characteristics and location of each sleeping site, tree, the individuals' place in the tree, posture, and behavior. We collected data for 154 nights between April 2009 and November 2010. The monkeys preferred tall sleeping trees (20.9 ± SD 4.9 m) and high sleeping places (15.8 ± SD 4.3 m), which may be an antipredator strategy. The choice of sleeping trees close to the last (146.7 ± SD 167.9 m) or to the first (150.4 ± SD 113.0 m) feeding tree of the day may save energy and decrease predation risk when monkeys are searching for food. Similarly, the choice of sleeping sites close to human settlements eases the access to human food during periods of fruit scarcity. Finally, the temporal pattern of use of sleeping sites, with a preference for four of the sleeping sites but few reuses during consecutive nights, may be a trade‐off between the need to have several sleeping sites (decreasing detection by predators and travel costs to feeding sites), and the need to sleep in well‐known sites (guaranteeing a faster escape in case of predator attack). Am. J. Primatol. 73:1222–1230, 2011. © 2011 Wiley Periodicals, Inc.     

Src-family kinase signaling,actin-mediated membrane trafficking and organellar dynamics in the control of cell fate: Lessons to be learned from the adenovirus E4orf4 death factor

Evidence has accumulated that there are different modes of regulated cell death, which share overlapping signaling pathways. Cytoskeletal-dependent inter-organellar communication as a result of protein and lipid trafficking in and out of organelles has emerged as a common, key issue in the regulation of cell death modalities. The movement of proteins and lipids between cell compartments is believed to relay death signals in part through modifications of organelles dynamics. Little is known, however, regarding how trafficking is integrated within stress signaling pathways directing organelle-specific remodeling events. In this review, we discuss emerging evidence supporting a role for regulated changes in actin dynamics and intracellular membrane flow. Based on recent findings using the adenovirus E4orf4 death factor as a probing tool to tackle the mechanistic underpinnings that control alternative modes of cell death, we propose the existence of multifunctional platforms at the endosome-Golgi interface regulated by SFK-signaling. These endosomal platforms could be mobilized during cell activation processes to reorganize cellular membranes and promote inter-organelle signaling.