Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.     

Dissecting amelogenin protein nanospheres: characterization of metastable oligomers

Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp(161), Trp(45), and Trp(25)) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (R(H)) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg · ml(-1). We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp(161)) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp(25) and Trp(45) is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.     

Mutagenesis of varicella-zoster virus glycoprotein I (gI) identifies a cysteine residue critical for gE/gI heterodimer formation, gI structure, and virulence in skin cells

Varicella-zoster virus (VZV) is the alphaherpesvirus that causes chicken pox (varicella) and shingles (zoster). The two VZV glycoproteins gE and gI form a heterodimer that mediates efficient cell-to-cell spread. Deletion of gI yields a small-plaque-phenotype virus, ΔgI virus, which is avirulent in human skin using the xenograft model of VZV pathogenesis. In the present study, 10 mutant viruses were generated to determine which residues were required for the typical function of gI. Three phosphorylation sites in the cytoplasmic domain of gI were not required for VZV virulence in vivo. Two deletion mutants mapped a gE binding region in gI to residues 105 to 125. A glycosylation site, N116, in this region did not affect virulence. Substitution of four cysteine residues highly conserved in the Alphaherpesvirinae established that C95 is required for gE/gI heterodimer formation. The C95A and Δ105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ΔgI virus. The Δ105-125 virus was avirulent for human skin in vivo. In contrast, the C95A mutant replicated in vivo but with significantly reduced kinetics compared to those of the wild-type virus. In addition to abolished gE/gI heterodimer formation, gI from the C95A or the Δ105-125 mutant was not recognized by monoclonal antibodies that detect the canonical conformation of gI, demonstrating structural disruption of gI in these viruses. This alteration prevented gI incorporation into virus particles. Thus, residues C95 and 105 to 125 are critical for gI structure required for gE/gI heterodimer formation, virion incorporation, and ultimately, effective viral spread in human skin.     

A database for the predicted pharmacophoric features of medicinal compounds

Pharmacophore feature is defined by a set of chemical structure patterns having the active site of drug like molecule. Pharmacophore can be used to assist in building hypothesis about desirable chemical properties in drug molecule and hence it can be used to refine and modify drug candidates. We predicted the pharmacophoric features of 150 medicinal compounds from plants for anti-cancer, anti-carcinogenic, anti-diabetic, anti-microbial, and anti-oxidant. Estimation of pharmacophoric feature is necessary to ensure the optimal supramolecular interaction with a biological target and to trigger or block its biological response. We subsequently make this data available to open access using a database at the URL: http://www.hccbif.info/index.htm AVAILABILITY: The database is available for free at http://www.hccbif.info/index.htm.     

The prolyl hydroxylase oxygen-sensing pathway is cytoprotective and allows maintenance of mitochondrial membrane potential during metabolic inhibition

The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. The addition of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential (_mito) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, _mito is partially maintained during metabolic inhibition and recovers completely on washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse _mito in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of _mito in PHI-treated myocytes, whereas a role for the "reverse mode" operation of the F₁F₀-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only 15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of preinsult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult. fumarate; hibernation; cardioprotection; anaplerotic     

Isolation and characterization of dihydromonacolin-MV from <Emphasis Type="Italic">Monascus purpureus</Emphasis> for antioxidant properties

The methanolic extract of Monascus purpureus cultivated by solid-state fermentation on rice showed strong 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and better yield as compared to other polarity based extracted fractions. It was selected for further purification of the antioxidant. The activity-guided repeated fractionation of methanolic extract on a silica gel column chromatography yielded a compound that exhibited strong antioxidant activity. Based on the spectroscopic analysis by UV, IR, ¹H NMR, ¹³C NMR, 2D-HSQCT NMR, and MS, the antioxidant isolated was elucidated as a derivative of dihydromonacolin-K, where the ester group is 2-methyl propionate, designated as dihydromonacolin-MV. The DPPH radical was significantly scavenged by the dihydromonacolin-MV (IC₅₀ 20±1 μg ml⁻¹). The dihydromonacolin-MV showed strong inhibition of lipid peroxidation in a liposome model with an IC₅₀ value of 5.71±0.38 μg ml⁻¹ and superoxide radical scavenging activity with an IC₅₀ value of 163.97±2.68 μg ml⁻¹.     

Role of pectin from cucumber (Cucumis sativus) in modulation of protein kinase C activity and regulation of glycogen metabolism in rats

The regulatory role of protein kinase C (PKC) in glycogen metabolism in pectin fed rats was investigated. Administration of pectin (5 g/kg body wt/day) from cucumber (Cucumis sativius L.) led to inhibitory effects on PKC activity in the liver of rats. In the brain and pancreas, PKC activity was significantly higher in pectin-treated rats as compared to the control group. Level of blood glucose was significantly lowered and the level of glycogen in the liver was significantly increased in pectin-administered rats. Glycogen synthase activity was enhanced, while glycogen phosphorylase enzyme showed inhibition in pectin-treated rats. Results indicated that pectin administration might have caused an increase in the secretion of the insulin, which, in turn, had a stimulatory effect on the PKC activity in the pancreas. The decreased PKC activity in the liver and increased PKC activity in the brain and pancreas on pectin administration indicated enhanced glycogenesis and reduced glycogenolysis.     

A LuxP-FRET-based reporter for the detection and quantification of AI-2 bacterial quorum-sensing signal compounds

Various bacterial species produce and monitor low-molecular weight signaling molecules that regulate specific sets of genes in a population density-dependent manner. This process is known as quorum sensing (QS). To date, the detection of QS signaling molecules from Gram-negative bacteria has relied primarily on bacterial reporter strains. These bioassays are subject to substantial interference by compounds that affect the growth and metabolism of the reporter strains. In addition, the sensitivity of reporter strains to QS signaling molecules is population density-dependent. Here, we describe the development of an in vitro assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer 2 (AI-2), QS molecules. The sensor is based on ligand binding-induced changes in fluorescence resonance energy transfer (FRET) between a cyan and yellow variant of GFP fused to the termini of the BAI-2 receptor, LuxP. Unexpectedly, the addition of synthetic BAI-2 to the purified biosensor induces a decrease in the level of FRET between the terminal fluorophores. Several lines of evidence, including mutation of the ligand binding sites, indicate that the observed FRET changes are BAI-2-dependent. The FRET-based BAI-2 biosensor responded to the addition of culture filtrates from wild-type Vibrio harveyi but exhibited no response to culture filtrates from V. harveyi mutants defective in BAI-2 synthesis. The sensitivity of the biosensor to BAI-2 (apparent Kd = 270 nM) was similar to that of BAI-2 bioassay systems. The limitations of microbial bioassay systems and the advantages and potential applications for the FRET-based BAI-2 biosensor are discussed.     

Noninvasive Evaluation of Heavy Metal Uptake and Storage in Micoralgae Using a Fluorescence Resonance Energy Transfer-Based Heavy Metal Biosensor

We have developed a fluorescence resonance energy transfer (FRET)-based heavy metal biosensor for the quantification of bioavailable free heavy metals in the cytoplasm of the microalga Chlamydomonas reinhardtii. The biosensor is composed of an end-to-end fusion of cyan fluorescent protein (CFP), chicken metallothionein II (MT-II), and yellow fluorescent protein (YFP). In vitro measurements of YFP/CFP fluorescence emission ratios indicated that the addition of metals to the purified biosensor enhanced FRET between CFP and YFP, consistent with heavy metal-induced folding of MT-II. A maximum YFP/CFP FRET ratio of 2.8 was observed in the presence of saturating concentrations of heavy metals. The sensitivity of the biosensor was greatest for Hg²⁺ followed by Cd²⁺ ≈ Pb²⁺ > Zn²⁺ > Cu²⁺. The heavy metal biosensor was unresponsive to metals that do not bind to MT-II (Na⁺ and Mg²⁺). When expressed in C. reinhardtii, we observed a differential metal-dependent response to saturating external concentrations (1.6 mm) of heavy metals (Pb²⁺ > Cd²⁺) that was unlike that observed for the isolated biosensor (in vitro). Significantly, analysis of metal uptake kinetics indicated that equilibration of the cytoplasm with externally applied heavy metals occurred within seconds. Our results also indicated that algae have substantial buffering capacity for free heavy metals in their cytosol, even at high external metal concentrations.Many proteins utilize metals to stabilize their structures or as cofactors to catalyze redox and other chemical reactions. Metals such as zinc, copper, iron, magnesium, cobalt, and manganese are required by most living organisms for their normal cellular functions. Essential metals are often present at low concentrations in the environment, however, and must be imported into cells, often at the expense of energy (Hanikenne et al., 2005; Merchant et al., 2006). In contrast to essential metals, toxic metals such as cadmium, lead, and mercury can disrupt cellular functions by competing with essential metals for their metal-binding sites and/or by altering the redox state of cells. Exposure of organisms to high concentrations of toxic metals can impair their cellular functions, growth, and reproduction. To prevent metal-induced cellular anomalies, organisms have evolved a variety of strategies to reduce the toxicity of heavy metals. One such strategy involves the selective binding of toxic metals in the cytoplasm by metal-binding proteins and other small molecules. As discussed below, both enzymatically and ribosomally synthesized Cys-rich peptides, including phytochelatins and metallothioneins (MTs), are utilized by a variety of organisms to sequester toxic heavy metals, including cadmium, mercury, lead, silver, and gold. The peptides may also serve as storage reserves for essential metals such as copper and zinc (Cobbett and Goldsbrough, 2002).Phytochelatins are enzymatically synthesized polypeptides containing repeating units of (γ-Glu-Cys)_n-Gly, where n = 2 to 11 (Rauser, 1990), whereas MTs are genetically encoded, ribosomally synthesized polypeptides (Cobbett and Goldsbrough, 2002). MTs have molecular mass values ranging from 6 to 7 kD and contain approximately 20 conserved Cys residues (Cobbett and Goldsbrough, 2002; Romero-Isart and Vasák, 2002). Metals are characteristically bound to MT via the thiolate sulfur ligands of Cys residues (Kägi, 1991). It is estimated that the metal-saturated MT contains about 10% thiolate sulfur and bound metals by mass (Romero-Isart and Vasák, 2002). Structural analyses of metal-free and metal-complexed MTs demonstrated that MTs undergo a structural transition from a metal-free random-coil structure to a metal-bound compact dumbbell-shaped structure having metal saturated α- and β-domains (Pearce et al., 2000; Romero-Isart and Vasak, 2002; Hong and Maret, 2003). The N-terminal β-domain binds three metal ion equivalents, and the C-terminal α-domain binds four metal ion equivalents (Romero-Isart and Vasák, 2002; Vasák, 2005). Furthermore, several decades of work on MTs have provided a great deal of information regarding their metal-binding affinity, specificity, and domain selectivity for select metals (Cobbett and Goldsbrough, 2002; Romero-Isart and Vasák, 2002; Vasák, 2005).Fluorescence resonance energy transfer (FRET) involves the nonradioactive transfer of energy between the excited state of a luminescent or fluorescent donor molecule and a nearby acceptor molecule that has overlapping excited state transitions. Proteins that are modified to have efficient energy donor and acceptor domains and that undergo structural changes upon binding a specific ligand are good candidates for FRET-based sensors. For ligand-specific FRET-based biosensors, the distance and/or the orientation between the energy donor and acceptor molecules is changed upon ligand binding in a concentration-dependent manner (Selvin, 1995; Weiss, 2000; Hong and Maret, 2003; Looger et al., 2005). Relevant to this discussion, a FRET-based biosensor with GFP variants fused to MT was previously shown to be an effective means to monitor metal release during nitric oxide-induced signaling in endothelial cells (Pearce et al., 2000).Unicellular algae such as Chlamydomonas species are often found in areas that might be contaminated by toxic heavy metals (Merchant et al., 2006). Chlamydomonas species have also been shown to sequester toxic metals (e.g. cadmium and mercury) and have potential use for bioremediation of these metals (Cai et al., 1999; Adhiya et al., 2002; Siripornadulsil et al., 2002; He et al., 2011; Priyadarshani et al., 2011). To determine the kinetics and selectivity of exogenous heavy metal uptake as well as free heavy metal concentration in the cytoplasm of Chlamydomonas species, we developed an MT, FRET-based metal-binding sensor and expressed this in the cytoplasm of the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that heavy metal uptake is rapid in C. reinhardtii and that cytoplasmic free heavy metal concentrations are substantially lower than exogenous free heavy metal concentrations, implying that heavy metals are rapidly sequestered by various biological molecules in the cell.