Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication

Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.     

Role for PADI6 and the cytoplasmic lattices in ribosomal storage in oocytes and translational control in the early mouse embryo

The mechanisms that mediate the establishment of totipotency during the egg-to-embryo transition in mammals remain poorly understood. However, it is clear that unique factors stored in the oocyte cytoplasm are crucial for orchestrating this complex cellular transition. The oocyte cytoplasmic lattices (CPLs) have long been predicted to function as a storage form for the maternal contribution of ribosomes to the early embryo. We recently demonstrated that the CPLs cannot be visualized in Padi6-/- oocytes and that Padi6-/- embryos arrest at the two-cell stage. Here, we present evidence further supporting the association of ribosomes with the CPLs by demonstrating that the sedimentation properties of the small ribosomal subunit protein, S6, are dramatically altered in Padi6-/- oocytes. We also show that the abundance and localization of ribosomal components is dramatically affected in Padi6-/- two-cell embryos and that de novo protein synthesis is also dysregulated in these embryos. Finally, we demonstrate that embryonic genome activation (EGA) is defective in Padi6-/- two-cell embryos. These results suggest that, in mammals, ribosomal components are stored in the oocyte CPLs and are required for protein translation during early development.     

Effects of weight loss on liver and erythrocyte polyunsaturated fatty acid pattern and oxidative stress status in obese patients with non-alcoholic fatty liver disease

Our aim was to study the influence of weight loss on the fatty acid (FA) composition of liver and erythrocyte phospholipids and oxidative stress status in obese, non-alcoholic, fatty liver disease (NAFLD) patients. Seven obese NAFLD patients who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in roux and Y were studied immediately and 3 months after surgery. Seven non-obese patients who underwent anti-reflux surgery constituted the control group. Serum F2-isoprostane levels were measured by GS/NICI-MS/MS and FA composition was determined by GC. At the time of surgery, controls and obese patients exhibited a hepatic polyunsaturated fatty acid (PUFA) pattern that correlated with that of erythrocytes. Three months after surgery, NAFLD patients lost 21% of initial body weight; serum F2-isoprostane levels decreased by 76%; total PUFA, long-chain PUFA (LCPUFA), n-3 PUFA, and n-3 LCPUFA increased by 22, 29, 81, and 93%, respectively; n-6/n-3 LCPUFA ratio decreased by 51%; docosahexaenoic acid/docosapentaenoic acid ratio increased by 19-fold; and the n-3 product/precursor ratio (20: 5 + 22: 5 + 22: 6)/18: 3 increased by 164% (p<0.05). It is concluded that weight loss improves the n-3 LCPUFA status of obese patients in association with significant amelioration in the biomarkers of oxidative stress, membrane FA insaturation, and n-3 LCPUFA biosynthesis capacity, thus representing a central therapeutic issue in the improvement of obesity-related metabolic alterations involved in the mechanism of hepatic steatosis.     

Munc 18-1 and granuphilin collaborate during insulin granule exocytosis

Munc 18-1 is a member of the Sec/Munc family of syntaxin-binding proteins known to bind to the plasma membrane Q-SNARE syntaxin1 and whose precise role in regulated exocytosis remains controversial. Here, we show that Munc 18-1 plays a positive role in regulated insulin secretion from pancreatic beta cells. Munc 18-1 depletion caused a loss in the secretory capacity of both transiently transfected INS 1E cells and a stable clone with tetracycline-regulated Munc 18-1 RNA interference. In addition, Munc 18-1-depleted cells exhibited defective docking of insulin granules to the plasma membrane and accumulated insulin in the trans Golgi network. Furthermore, glucose stimulation after Munc 18-1 depletion resulted in the rapid formation of autophagosomes. In contrast, overexpression of Munc 18-1 had no effect on insulin secretion. Although there was no detectable interaction between Munc 18-1 and Munc-18-interacting protein 1 or calcium/calmodulin-dependent serine protein kinase, Munc 18-1 associated with the granular protein granuphilin. This association was regulated by glucose and was required for the specific interaction of insulin granules with syntaxin1. We conclude that Munc 18-1 and granuphilin collaborate in the docking of insulin granules to the plasma membrane in an initial fusion-incompetent state, with Munc 18-1 subsequently playing a positive role in a later stage of insulin granule exocytosis.     

Widespread distribution of a unique marine protistan lineage

Unicellular eukaryotes (protists) are key components of marine food webs, yet knowledge of their diversity, distributions and respective ecologies is limited. We investigated uncultured protists using 18S rRNA gene sequencing, phylogenetic analyses, specific fluorescence in situ hybridization (FISH) probes and other methods. Because few studies have been conducted in warm water systems, we focused on two Atlantic subtropical regions, the Sargasso Sea and the Florida Current. Cold temperate waters were also sampled. Gene sequences comprising a unique eukaryotic lineage, herein termed 'biliphytes', were identified in most samples, whether from high- (30 degrees C) or from low- (5 degrees C) temperature waters. Sequences within this uncultured group have previously been retrieved from high latitudes. Phylogenetic analyses suggest biliphytes are a sister group to the cryptophytes and katablepharids, although the relationship is not statistically supported. Bootstrap-supported subclades were delineated but coherence was not obvious with respect to geography or physicochemical parameters. Unlike results from the initial publication on these organisms (therein 'picobiliphytes'), we could not detect a nucleomorph, either visually, or by targeted primers. Phycobilin-like fluorescence associated with biliphyte-specific FISH-probed cells supports the hypothesis that they are photosynthetic. Our data indicate the biliphytes are nanoplanktonic in size, averaging 4.1 +/- 1.0 x 3.5 +/- 0.8 microm (+/-SD) for one probed group, and 3.5 +/- 0.9 x 3.0 +/- 0.9 microm (+/-SD) for another. We estimate biliphytes contributed 28 (+/-6)% of the phytoplanktonic biomass in tropical eddy-influenced surface waters. Given their broad thermal and geographic distribution, understanding the role these protists play in biogeochemical cycling within different habitats is essential.     

Toward a global view of alcohol, tobacco, cannabis, and cocaine use: findings from the WHO World Mental Health Surveys

Background

Alcohol, tobacco, and illegal drug use cause considerable morbidity and mortality, but good cross-national epidemiological data are limited. This paper describes such data from the first 17 countries participating in the World Health Organization''s (WHO''s) World Mental Health (WMH) Survey Initiative.

Methods and Findings

Household surveys with a combined sample size of 85,052 were carried out in the Americas (Colombia, Mexico, United States), Europe (Belgium, France, Germany, Italy, Netherlands, Spain, Ukraine), Middle East and Africa (Israel, Lebanon, Nigeria, South Africa), Asia (Japan, People''s Republic of China), and Oceania (New Zealand). The WHO Composite International Diagnostic Interview (CIDI) was used to assess the prevalence and correlates of a wide variety of mental and substance disorders. This paper focuses on lifetime use and age of initiation of tobacco, alcohol, cannabis, and cocaine. Alcohol had been used by most in the Americas, Europe, Japan, and New Zealand, with smaller proportions in the Middle East, Africa, and China. Cannabis use in the US and New Zealand (both 42%) was far higher than in any other country. The US was also an outlier in cocaine use (16%). Males were more likely than females to have used drugs; and a sex–cohort interaction was observed, whereby not only were younger cohorts more likely to use all drugs, but the male–female gap was closing in more recent cohorts. The period of risk for drug initiation also appears to be lengthening longer into adulthood among more recent cohorts. Associations with sociodemographic variables were consistent across countries, as were the curves of incidence of lifetime use.

Conclusions

Globally, drug use is not distributed evenly and is not simply related to drug policy, since countries with stringent user-level illegal drug policies did not have lower levels of use than countries with liberal ones. Sex differences were consistently documented, but are decreasing in more recent cohorts, who also have higher levels of illegal drug use and extensions in the period of risk for initiation.     

Heteroallyl-containing 5-nitrofuranes as new anti-Trypanosoma cruzi agents with a dual mechanism of action

New heteroallyl-containing 5-nitrofuranes were synthesized as potential anti-Trypanosoma cruzi agents with a dual mechanism of action, oxidative stress and inhibition of membrane sterol biosynthesis. Some of the derivatives were found to have high and selective activity against the proliferative stages of the parasite, with IC(50) values against the clinically relevant intracellular amastigote forms in the low micromolar to sub-micromolar range. Oxidative stress was verified measuring cyanide dependent respiration. Inhibition of the de novo sterol biosynthesis at the level of squalene epoxidase was confirmed, using high-resolution gas-liquid chromatography coupled to mass spectrometry, by the disappearance of the parasite's mature sterols and the concomitant accumulation of squalene. The in vitro activities of these novel compounds were superior to that of nifurtimox, a nitrofuran currently used in the treatment of human Chagas' disease, and terbinafine, a commercially available allylamine-based squalene epoxidase inhibitor. The results support further in vivo studies of some of these nitrofuran derivatives.     

Intraluminal-restricted 17 beta-estradiol exerts the same myocardial protection against ischemia/reperfusion injury in vivo as free 17 beta-estradiol

Several in vitro studies show that in animals and isolated cells, 17 beta-estradiol induces cardiovascular protective effects and it has also been observed that it reduces coronary heart disease risk. However, the use of estrogens to improve or protect cardiovascular function in humans has been controversial, this might be explained by the wide variety of effects, because estrogen receptors (ER) are expressed ubiquitously. Therefore, a cell-specific targeting therapeutic approach might be necessary. 17 beta-Estradiol was coupled to a large modified dextran through an aminocaproic spacer. For this study we used intact and gonadectomized male Wistar rats, 15 days after surgical procedure. Intravascular administration of 17 beta-estradiol-macromolecular conjugate, prior to coronary reperfusion diminishes the area of damage induced by coronary ischemia reperfusion (I/R) injury on an in vivo model. This effect was observed at 17 beta-estradiol sub-physiological concentrations [0.01 nmol/L], it is mediated by luminal endothelial ER alpha activation. 17 beta-Estradiol-macromolecular conjugate decreases phosphorylation level of PKC alpha and Akt, as part of the process to induce myocardial protection against coronary I/R. We proved that the hormone-macromolecular conjugate labeled with [3H]estradiol remained confined in the intravascular space the conjugate was not internalized into organs like heart, lung or liver. It is noteworthy that the 17 beta-estradiol-macromolecular conjugate has a slow renal elimination, which might increase its pharmacological advantage. We concluded that the stimulus of endothelial estrogen receptors is enough to decrease the myocardial damage induced by coronary reperfusion.     

Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia lyase

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.     

Airway smooth muscle relaxation induced by 5-HT(2A) receptors: role of Na(+)/K(+)-ATPase pump and Ca(2+)-activated K(+) channels

AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 muM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 muM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 muM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.