Neuropeptide Y: a missing link?

Yet another 'orphan' molecule that had to find its place in life after isolation and sequencing, neuropeptide Y appears to be an important cardiovascular neuroregulator and also links the sympathetic and renin-angiotensin systems. The peptide's physiologic and pathophysiologic roles, as well as its potential therapeutic value, are examined.

Antibacterial burst-release from minimal Ag-containing plasma polymer coatings.

Biomaterials releasing silver (Ag) are of interest because of their ability to inhibit pathogenic bacteria including antibiotic-resistant strains. In order to investigate the potential of nanometre-thick Ag polymer (Ag/amino-hydrocarbon) nanocomposite plasma coatings, we studied a comprehensive range of factors such as the plasma deposition process and Ag cation release as well as the antibacterial and cytocompatible properties. The nanocomposite coatings released most bound Ag within the first day of immersion in water yielding an antibacterial burst. The release kinetics correlated with the inhibitory effects on the pathogens Pseudomonas aeruginosa or Staphylococcus aureus and on animal cells that were in contact with these coatings. We identified a unique range of Ag content that provided an effective antibacterial peak release, followed by cytocompatible conditions soon thereafter. The control of the in situ growth conditions for Ag nanoparticles in the polymer matrix offers the possibility to produce customized coatings that initially release sufficient quantities of Ag ions to produce a strong adjacent antibacterial effect, and at the same time exhibit a rapidly decaying Ag content to provide surface cytocompatibility within hours/days. This approach seems to be favourable with respect to implant surfaces and possible Ag-resistance/tolerance built-up.

S100A1 deficiency results in prolonged ventricular repolarization in response to sympathetic activation.

S100A1 is a Ca(2+)-binding protein and predominantly expressed in the heart. We have generated a mouse line of S100A1 deficiency by gene trap mutagenesis to investigate the impact of S100A1 ablation on heart function. Electrocardiogram recordings revealed that after beta-adrenergic stimulation S100A1-deficient mice had prolonged QT, QTc and ST intervals and intraventricular conduction disturbances reminiscent of 2 : 1 bundle branch block. In order to identify genes affected by the loss of S100A1, we profiled the mutant and wild type cardiac transcriptomes by gene array analysis. The expression of several genes functioning to the electrical activity of the heart were found to be significantly altered. Although the default prediction would be that mRNA and protein levels are highly correlated, comprehensive immunoblot analyses of salient up- or down-regulated candidate genes of any cellular network revealed no significant changes on protein level. Taken together, we found that S100A1 deficiency results in cardiac repolarization delay and alternating ventricular conduction defects in response to sympathetic activation accompanied by a significantly different transcriptional regulation.

Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

The vitamin D receptor gene bAt (CCA) haplotype impairs the response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C patients.

BACKGROUND: Chronic hepatitis C infection is a major cause of end-stage liver disease. Therapy outcome is influenced by 25-OH vitamin D deficiency. To further address this observation, our study investigates the impact of the vitamin D receptor (NR1I1) haplotype and combined effects of plasma vitamin D levels in a well-described cohort of hepatitis C patients. METHODS: A total of 155 chronic hepatitis C patients were recruited from the Swiss Hepatitis C Cohort Study for NR1I1 genotyping and plasma 25-OH vitamin D level measurement. NR1I1 genotype data and combined effects of plasma 25-OH vitamin D level were analysed regarding therapy response (sustained virological response). RESULTS: A strong association was observed between therapy non-response and the NR1I1 CCA (bAt) haplotype consisting of rs1544410 (BsmI) C, rs7975232 (ApaI) C and rs731236 (TaqI) A alleles. Of the HCV patients carrying the CCA haplotype, 50.3% were non-responders (odds ratio [OR] 1.69, 95% CI 1.07, 2.67; P=0.028). A similar association was observed for the combinational CCCCAA genotype (OR 2.94, 95% CI 1.36, 6.37; P=0.007). The combinational CCCCAA genotype was confirmed as an independent risk factor for non-response in multivariate analysis (OR 2.50, 95% CI 1.07, 5.87; P=0.034). Analysing combined effects, a significant impact of low 25-OH vitamin D levels on sustained virological response were only seen in patients with the unfavourable NR1I1 CCA (bAt) haplotype (OR for non-SVR 3.55; 95% CI 1.005, 12.57; P=0.049). CONCLUSIONS: NR1I1 vitamin D receptor polymorphisms influence response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C and exert an additive genetic predisposition to previously described low 25-OH vitamin D serum levels.

Estrogen-dependent in vitro transcription from the vitellogenin promoter in liver nuclear extracts.

One approach to analyzing the molecular mechanisms of gene expression in vivo is to reconstitute these events in cell-free systems in vitro. Although there is some evidence for tissue-specific transcription in vitro, transcriptionally active extracts that mimic a steroid hormone-dependent enhancement of transcription have not been described. In the study reported here, nuclear extracts of liver from the frog Xenopus laevis were capable of estrogen-dependent induction of a homologous vitellogenin promoter that contained the estrogen-responsive element.

Blood pressure, cardiac, and renal responses to salt and deoxycorticosterone acetate in mice: role of Renin genes.

Several studies have demonstrated that mice are polymorphic for the number of renin genes, with some inbred strains harboring one gene (Ren-1(c)) and other strains containing two genes (Ren-1(d) and Ren-2). In this study, the effects of 1% salt and deoxycorticosterone acetate (DOCA)/salt were investigated in one- and two-renin gene mice, for elucidation of the role of renin in the modulation of BP, cardiac, and renal responses to salt and DOCA. The results demonstrated that, under baseline conditions, mice with two renin genes exhibited 10-fold higher plasma renin activity, 100-fold higher plasma renin concentrations, elevated BP (which was angiotensin II-dependent), and an increased cardiac weight index, compared with one-renin gene mice (all P < 0.01). The presence of two renin genes markedly increased the BP, cardiac, and renal responses to salt. The number of renin genes also modulated the responses to DOCA/salt. In one-renin gene mice, DOCA/salt induced significant renal and cardiac hypertrophy (P < 0.01) even in the absence of any increase in BP. Treatment with losartan, an angiotensin II AT(1) receptor antagonist, decreased BP in two-renin gene mice but not in one-renin gene mice. However, losartan prevented the development of cardiac hypertrophy in both groups of mice. In conclusion, these data demonstrate that renin genes are important determinants of BP and of the responses to salt and DOCA in mice. The results confirm that the Ren-2 gene, which controls renin production mainly in the submaxillary gland, is physiologically active in mice and is not subject to the usual negative feedback control. Finally, these data provide further evidence that mineralocorticoids promote cardiac hypertrophy even in the absence of BP changes. This hypertrophic process is mediated in part by the activation of angiotensin II AT(1) receptors.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Treatment implications of the emerging molecular classification system for melanoma.

Melanoma is an aggressive disease with few standard treatment options. The conventional classification system for this disease is based on histological growth patterns, with division into four subtypes: superficial spreading, lentigo maligna, nodular, and acral lentiginous. Major limitations of this classification system are absence of prognostic importance and little correlation with treatment outcomes. Recent preclinical and clinical findings support the notion that melanoma is not one malignant disorder but rather a family of distinct molecular diseases. Incorporation of genetic signatures into the conventional histopathological classification of melanoma has great implications for development of new and effective treatments. Genes of the mitogen-associated protein kinase (MAPK) pathway harbour alterations sometimes identified in people with melanoma. The mutation Val600Glu in the BRAF oncogene (designated BRAF(V600E)) has been associated with sensitivity in vitro and in vivo to agents that inhibit BRAF(V600E) or MEK (a kinase in the MAPK pathway). Melanomas arising from mucosal, acral, chronically sun-damaged surfaces sometimes have oncogenic mutations in KIT, against which several inhibitors have shown clinical efficacy. Some uveal melanomas have activating mutations in GNAQ and GNA11, rendering them potentially susceptible to MEK inhibition. These findings suggest that prospective genotyping of patients with melanoma should be used increasingly as we work to develop new and effective treatments for this disease.

Caveolin-1 opens endothelial cell junctions by targeting catenins.

AIMS: A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown.¦METHODS AND RESULTS: Impedance spectroscopy, biochemistry, and morphology were used to investigate the role of caveolin-1 in the regulation of thrombin-induced opening of cell junctions in cultured human and mouse endothelial cells. Here, we demonstrate that the vascular endothelial (VE) cadherin/catenin complex targets caveolin-1 to endothelial cell junctions. Association of caveolin-1 with VE-cadherin/catenin complexes is essential for the barrier function decrease in response to the pro-inflammatory mediator thrombin, which causes a reorganization of the complex in a rope ladder-like pattern accompanied by a loss of junction-associated actin filaments. Mechanistically, we show that in response to thrombin stimulation the protease-activated receptor 1 (PAR-1) causes phosphorylation of caveolin-1, which increasingly associates with β- and γ-catenin. Consequently, the association of β- and γ-catenin with VE-cadherin is weakened, thus allowing junction reorganization and a decrease in barrier function. Thrombin-induced opening of cell junctions is lost in caveolin-1-knockout endothelial cells and after expression of a Y/F-caveolin-1 mutant but is completely reconstituted after expression of wild-type caveolin-1.¦CONCLUSION: Our results highlight the pivotal role of caveolin-1 in VE-cadherin-mediated cell adhesion via catenins and, in turn, in barrier function regulation.

The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.

The effect of exercise, alcohol or both combined on health and physical performance.

Alcohol (ethanol) is consumed on a daily basis by a large fraction of the population, and in many countries, light-to-moderate alcohol consumption is considered as an integral part of the diet. Although the relationship between alcohol intake and obesity is controversial, regular consumption of alcohol, through its effects in suppressing fat oxidation, is regarded as a risk factor for weight gain, increased abdominal obesity and hypertriglyceridemia. Indeed, alcohol taken with a meal leads to an increase in postprandial lipemia-an effect on postprandial metabolism that is opposite to that observed with exercise. Furthermore, although regular exercise training and/or a preprandial exercise session reduce postprandial lipemia independently of alcohol ingestion, the exercise-induced reduction in postprandial lipemia is nonetheless less pronounced when alcohol is also consumed with the meal. Whether or not alcohol influences exercise and sport performance remains contradictory. It is believed that alcohol has deleterious effects on the performance, although it may contribute to reduce pain and anxiety. The alcohol effects on sports performance depend on the type and dosage of alcohol, acute vs chronic administration, the alcohol elimination rate as well as the type of exercise.

Beta-lactams against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) have developed resistance to virtually all non-experimental antibiotics. They are intrinsically resistant to beta-lactams by virtue of newly acquired low-affinity penicillin-binding protein 2A (PBP2A). Because PBP2A can build the wall when other PBPs are blocked by beta-lactams, designing beta-lactams capable of blocking this additional target should help solve the issue. Older molecules including penicillin G, amoxicillin and ampicillin had relatively good PBP2A affinities, and successfully treated experimental endocarditis caused by MRSA, provided that the bacterial penicillinase could be inhibited. Newer anti-PBP2A beta-lactams with over 10-fold greater PBP2A affinities and low minimal inhibitory concentrations were developed, primarily in the cephem and carbapenem classes. They are also very resistant to penicillinase. Most have demonstrated anti-MRSA activity in animal models of infection, and two--the carbapenem CS-023 and the cephalosporin ceftopibrole medocaril--have proceeded to Phase II and Phase III clinical evaluation. Thus, clinically useful anti-MRSA beta-lactams are imminent.

Decreased blood pressure response in mice deficient of the alpha1b-adrenergic receptor.

To investigate the functional role of different alpha1-adrenergic receptor (alpha1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the alpha1b-AR (alpha1b-/-). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the alpha1-AR subtypes in various tissues of alpha1b +/+ and -/- mice. Total alpha1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the alpha1b -/- as compared with +/+ mice. Because of the large decrease of alpha1-AR in the heart and the loss of the alpha1b-AR mRNA in the aorta of the alpha1b-/- mice, the in vivo blood pressure and in vitro aorta contractile responses to alpha1-agonists were investigated in alpha1b +/+ and -/- mice. Our findings provide strong evidence that the alpha1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by alpha1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in alpha1b -/- as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in alpha1b-/- mice. The alpha1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different alpha1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.