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Research was conducted to examine the DNA sequencing, gene cloning and the expression of thermostable beta-mannanase from Bacillus stearothermophilus. The detection of recombinant beta mannanase activity was done based on the clearing of halos surrounding the colonies of Escherichia coli grown on a dye-labelled substrate. The high level of expression of recombinant mannanase in addition to its thermostability indicate its possible commercial use in biobleaching.

Aims During pregnancy, there is a significant increase in heart rate (HR) potentially associated with an increased risk of arrhythmias or exacerbation of pre-existing cardiac conditions endangering both mother and foetus. Calcium homeostasis plays an important role in regulating automaticity of the sinoatrial node (SAN); however, its contribution to the accelerated HR during pregnancy remains unknown. Methods and results Using murine SAN cells, we showed that pregnancy increased L-type Ca2+ current (I CaL) and CaV1.3 mRNA expression, whereas T-type Ca2+ current (I CaT) and its underlying channel were unchanged. Analysis of SAN intra-cellular Ca2+ oscillations showed that the rate of spontaneous Ca2+ transients was significantly higher in pregnant mice along with a higher mRNA expression of ryanodine receptor. Assessment of supra-ventricular arrhythmias using programmed electrical stimulation protocols on anaesthetized mice revealed higher susceptibility in pregnancy. Of note, the modifications associated with pregnancy were reversible following delivery. Furthermore, chronic administration of 17β-estradiol (E2) to nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), control mice, oestrogen-receptor-β knockout (ERKOβ) but not ERKOα mice, accelerated cardiac automaticity, recapitulating the pregnancy phenotype in both mouse and human SAN cell models. Conclusion Together, these results indicate that pregnancy considerably alters intra-cellular Ca2+ homeostasis sustaining faster HR during pregnancy. Importantly, these changes were dependent on an oestrogen receptor α (ERα) mechanism that resulted in increased I CaL and spontaneous Ca2+ release from the sarcoplasmic reticulum, highlighting a novel role for oestrogen in regulating HR. [ABSTRACT FROM AUTHOR]

In the current model of γ-aminobutyric acid (GABA) B receptor function, there is a requirement for GABA-B1/2 heterodimerisation for targetting to the cell surface. However, different lines of evidence suggest that the GABA-B1 subunit can form a functional receptor in the absence of GABA-B2. We observed coupling of endogenous GABA-B1 receptors in the DI-TNC1 glial cell line to the ERK pathway in response to baclofen even though these cells do not express GABA-B2. GABA-B1A receptors were also able to mediate a rapid, transient, and dose-dependent activation of the ERK1/2 MAP kinase pathway when transfected alone into HEK 293 cells. The response was abolished by Gi/o and MEK inhibition, potentiated by inhibitors of phospholipase C and protein kinase C and did not involve PI-3-kinase activity. Finally, using bioluminescence resonance energy transfer and co-immunoprecipitation, we show the existence of homodimeric GABA-B1A receptors in transfected HEK293 cells. Altogether, our observations show that GABA-B1A receptors are able to activate the ERK1/2 pathway despite the absence of surface targetting partner GABA-B2 in both HEK 293 cells and the DI-TNC1 cell line. [ABSTRACT FROM AUTHOR]

Abstract: Objective: We sought to determine if different β-adrenergic receptor (βAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. Methods: Employing enriched nuclear preparations, we assayed the specific presence of βAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. Results: Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of β1AR and β3AR but, surprisingly, not the β2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear βAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the β3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another β3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a β1AR-selective agonist, xamoterol, nor a β2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear βAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. Conclusion: These results highlight differential subcellular localization for βAR subtypes and indicate that βAR may have specific roles in regulating nuclear function in cardiomyocytes. [Copyright &y& Elsevier]

Investigates on the genomic library constructed from Bacillus stearothermophilus, a gram positive, facultative thermophilic aerobe that secretes a thermostable...-mannnanase. Methodologies in deoxyribonucleic acid cloning; Analysis on nucleotide sequencing; Similarities of amino acid sequence from the Bacillus aerobe.

Heterotrimeric G proteins play a central role in intracellular communication mediated by extracellular signals, and both Galpha and Gbetagamma subunits regulate effectors downstream of activated receptors. The particular constituents of the G protein heterotrimer affect both specificity and efficiency of signal transduction. However, little is known about mechanistic aspects of G protein assembly in the cell that would certainly contribute to formation of heterotrimers of specific composition. It was recently shown that phosducin-like protein (PhLP) modulated both Gbetagamma expression and subsequent signaling by chaperoning nascent Gbeta and facilitating heterodimer formation with Ggamma subunits (Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E., and Willardson, B. M. (2005) EMBO J. 24, 1965-1975; Humrich, J., Bermel, C., Bunemann, M., Harmark, L., Frost, R., Quitterer, U., and Lohse, M. J. (2005) J. Biol. Chem. 280, 20042-20050). Here we demonstrate using a variety of techniques that DRiP78, an endoplasmic reticulum resident protein known to regulate the trafficking of several seven transmembrane receptors, interacts specifically with the Ggamma subunit but not Gbeta or Galpha subunits. Furthermore, we demonstrate that DRiP78 and the Gbeta subunit can compete for the Ggamma subunit. DRiP78 also protects Ggamma from degradation until a stable partner such as Gbeta is provided. Furthermore, DRiP78 interaction may represent a mechanism for assembly of specific Gbetagamma heterodimers, as selectivity was observed among Ggamma isoforms for interaction with DRiP78 depending on the presence of particular Gbeta subunits. Interestingly, we could detect an interaction between DRiP78 and PhLP, suggesting a role of DRiP78 in the assembly of Gbetagamma by linking Ggamma to PhLP.Gbeta complexes. Our results, therefore, suggest a role of DRiP78 as a chaperone in the assembly of Gbetagamma subunits of the G protein.

We have previously demonstrated that adenylyl cyclase II (ACII) interacts with beta2-adrenergic receptors and heterotrimeric G proteins as part of a pre-assembled signalling complex. In this study, we further show that AC interacts with these proteins before it is targetted to the cell surface. Using a combination of approaches including bioluminescence resonance energy transfer (BRET) in concert with subcellular fractionation, we show that ACII and beta2AR initially interact in the ER. Further, dominant-negative Rab1 and Sar1 GTPases which block anterograde trafficking out of the ER have no effect on either ACII/receptor or ACII/Gbetagamma protein interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Galpha subunits in ACII signalling complexes suggesting it assembles into the complex at a slightly later stage. Thus, like Kir3.1 inwardly rectifying potassium channels, signalosomes containing ACII are formed during their biosynthesis and not in response to agonist at the cell surface.

Objective: We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes., Methods: Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays., Results: Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels., Conclusion: These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.