Bombesin-like peptides stimulate phosphatidylinositol turnover in rat brain slices

The ability of bombesin (BN)-like peptides to stimulate phosphatidylinositol turnover in rat brain slices was investigated. BN (1 μM) significantly stimulated inositol-1-phosphate (IP₁) but not inositol-4,5-biphosphate (IP₂) or inositol-1,4,5-trisphosphate (IP₃) production using frontal cortex slices in the presence of LiCl (7.5 mM); BN had no effect on cAMP or cGMP levels. BN and the structurally-related gastrin releasing peptide (GRP) elevated IP₁ levels in a dose-dependent manner. Similarly, nanomolar concentrations of the GRP fragment (Ac-GRP^20–27) significantly elevated IP₁ levels, whereas micromolar concentrations of the inactive GRP^1–16 did not. BN significantly elevated IP₁ levels in those brain regions enriched in BN receptors such as the olfactory bulb, hippocampus, striatum, thalamus and frontal cortex, whereas IP₁ levels were not significantly increased in areas which have a low density of BN receptors such as the cerebellum, medulla/pons and midbrain. These data suggest that CNS BN receptors may utilize phosphatidylinositol as a second messenger.     

Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli

The global pool of all metabolites in a cell, or metabolome, is a reflection of all the metabolic functions of an organism under any particular growth condition. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. In the past, a variety of cell lysis methods were adopted for assays of individual metabolites or groups of intermediates in pathways. In this study, metabolites were extracted from Escherichia coli using six different commonly used procedures including acid or alkaline treatments, permeabilization by freezing with methanol, high-temperature extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol. Metabolites were extracted by the six methods from cells grown under identical conditions and labeled with [14C]glucose. The metabolomes were compared after 2-dimensional thin-layer chromatography of labeled compounds. For global analysis, extraction with cold (-40 degrees C) methanol showed the greatest promise, allowing simultaneous resolution of more than 95 metabolite spots. In contrast, 80 or less spots were obtained with other extraction methods. Extraction also influenced quantitative analysis of particular compounds. Metabolites such as adenosine exhibited up to 20-fold higher abundance after cold methanol extraction than after extraction with acid, alkali, or chloroform. The simplicity, rapidity, and universality of cold methanol extraction offer great promise if a single method of lysis is to be adopted in metabolome analysis.     

nodD alleles of Sinorhizobium fredii USDA191 differentially influence soybean nodulation, nodC expression, and production of exopolysaccharides

All Rhizobium strains examined to date have one or multiple alleles of nodD. At least one copy of nodD and the presence of flavonoid exudates are required for nod gene induction and nodulation. Sinorhizobium fredii USDA191 has two copies of nodD. In this study, we demonstrate that inactivation of either copy of nodD caused a reduction in basal levels of expression of nodC. Extra copies of nodD1 had no effect on the expression of nodC when compared with the wild type, but extra copies of nodD2 abolished the inducer requirement, thereby rendering nodC constitutive. A nodD1 mutant was unable to nodulate soybean cultivars 'Peking' and 'McCall'. Inactivation of nodD2 or addition of extra copies of nodD1 or nodD2 caused delayed nodulation on Peking, and reduced the number of nodules on McCall. Both nodD alleles of S. fredii USDA191 appear to be involved in regulation of exopolysaccharide production; however, nodD2 appears to be more important in this respect than nodD1.     

Genistein: A Potent Anti-Breast Cancer Agent

Genistein is an isoflavonoid present in high quantities in soybeans. Possessing a wide range of bioactives, it is being studied extensively for its tumoricidal effects. Investigations into mechanisms of the anti-cancer activity have revealed many pathways including induction of cell proliferation, suppression of tyrosine kinases, regulation of Hedgehog-Gli1 signaling, modulation of epigenetic activities, seizing of cell cycle and Akt and MEK signaling pathways, among others via which the cancer cell proliferation can be controlled. Notwithstanding, the observed activities have been time- and dose-dependent. In addition, genistein has also shown varying results in women depending on the physiological parameters, such as the early or post-menopausal states.     

The molecular basis of erectile dysfunction: from bench to bedside

Erectile dysfunction is a common problem affecting many men across all age groups. Its etiology is multifactorial. Hormonal, vascular, neurogenic, lifestyle, and psychological entities have all been implicated as causative agents. The molecular basis underlying its etiology and progression is complex and still challenges researchers in the field. Nonetheless, newly discovered common pathways and targets of its pathogenesis have opened a new era for both prevention and active treatment of the disease. This review describes some of the known molecular mechanisms contributing to erectile dysfunction and discusses the future of gene therapy for the disease.     

Role of pH on dimeric interactions for DENV envelope protein: an insight from molecular dynamics study

The entry of dengue viruses is mediated by pH triggering in the host cells. Here we have studied the DENV E protein stability and binding of its units at low and normal pH using MD and MM-PB/SA method for the first time. To investigate the role of pH in dissociation of dimeric protein, we have performed a concise study of hydrogen bonding and other interactions between units of dimer at low and normal pH. The Generalized Born calculation connotes that dimeric unit was relatively less stable and less proned for dimerisation at low pH. Our results provide a theoretical verification for previous assumptions of pH triggering mechanism of dengue envelope protein. During the pH alteration, we found a large decrement in salt bridges which were observed at normal pH. We also compared the flexibility of each unit and found that they exhibit different fluctuations during molecular dynamics simulations.     

Prechemistry versus preorganization in DNA replication fidelity

The molecular origin of nucleotide insertion catalysis and fidelity of DNA polymerases is explored by means of computational simulations. Special attention is paid to the examination of the validity of proposals that invoke prechemistry effects, checkpoints concepts, and dynamical effects. The simulations reproduce the observed fidelity in Pol β, starting with the relevant observed X-ray structures of the complex with the right (R) and wrong (W) nucleotides. The generation of free energy surfaces for the R and W systems also allowed us to analyze different proposals about the origin of the fidelity and to reach several important conclusions. It is found that the potential of mean force (PMF) obtained by proper sampling does not support QM/MM-based proposals of a large barrier before the prechemistry state. Furthermore, examination of dynamical proposals by the renormalization approach indicates that the motions from open to close configurations do not contribute to catalysis or fidelity. Finally we discuss and analyze the induced fit concept and show that, despite its importance, it does not explain fidelity. That is, the fidelity is apparently due to the change in the preorganization of the chemical site, as a result of the relaxation of the binding site upon binding of the incorrect nucleotide. Finally and importantly, since the issue is the barrier associated with the enzyme-substrate (ES)/DNA complex at the chemical transition state and not the path to this complex formation (unless this path involves rate determining steps), it is also not useful to invoke checkpoints while discussing fidelity.     

Use of synthetic gramicidins in the determination of channel structure and mechanism

The syntheses of (1-13C) Trp9 gramicidin A (GA), (1-13C) Trp11 GA, (1-13C) Trp13 GA, (1-13C) Trp15 GA, and D . Leu2 GA are verified by means of high performance liquid chromatography, carbon-13 nuclear magnetic resonance, circular dichroism and characterization of transport properties. The use of these and other synthetic gramicidins is discussed in terms of determining ion binding sites within the channel, helix sense of the channel, the basis of monovalent vs divalent cation selectivity, and means of modulating channel conductance.