国立情報学研究所で電子化したコンテンツを使用している。 / 国立情報学研究所で電子化したコンテンツを使用している。

Nature is able to convert chemical energy into mechanical work under modest conditions, i.e., physiological pH and ambient temperature and pressure. One of the most interesting systems is muscle modeled as the "sliding filament" system. The sliding filament system is a combination of a thin actin filament and a thick myosin filament that slide over one another by breaking the "energy-rich" pyrophosphate bond of ATP. The energy from ATP hydrolysis is used for mechanical motion and the energy lost during this process is used to heat our body. In biology, the sliding filament system is taken as a fairly effective model. For engineering systems, the energy lost to heat needs to be reduced to build an efficient energy converter. In our research, we use a phosphate charged protein, casein, and react it with divinyl sulfone (DVS) through a Michael addition reaction to produce a cross-linked gel. The protein gel could be ephosphorylated at standard conditions using bovine phosphatase (bp) and re-phosporylated using casein kinase. When attached to the protein, the negatively charged phosphate groups cause the gel to expand from repulsion. When removed, the protein contracts. Therefore, work is realized without sliding friction, which is the origin of the large energy loss in muscle. FT-IR spectroscopy allows us to follow the two biochemical reactions. We also show a thermodynamic analysis of the work and offer an estimation of the most basic term. / Master of Science

The activity of vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.

Protein phosphorylation is an essential post-translational modification used in cells for regulating multiple biological processes in all organisms. Particularly, mitotic onset is regulated in all eukaryotes by an increase in cyclin-dependent kinase 1 (Cdk1) activity caused by the dephosphorylation of Cdk1 on a conserved tyrosine residue. PP2ARts1 is a phosphatase that participates in dephosphorylating the conserved tyrosine residue, tyrosine-19 (Y19). PP2ARts1 dephosphorylates phosphorylated serine and threonine residues. However, in vitro experiments suggest that in conjunction with the mammalian PP2A phosphatase activator (PTPA), PP2A gains phosphotyrosine specificity. My work indicates that Rrd1 and Rrd2 (the budding yeast homologs of PTPA) genetically interact with PP2ARts1 and the absence of these proteins cause a Swe1-dependent delay in mitosis. In parallel, utilizing a candidate approach to identify additional phosphatases specific to Cdk1-Y19, my work indicates that Ych1 and Arr2 act redundantly with Mih1 and Ptp1, and Ych1 may act downstream of PP2ARts1. In summation, my work provides the groundwork for how PP2ARts1 functions to dephosphorylate the conserved Y19 residue on Cdk1 and will lead to a better understanding of its role in regulating mitotic progression.

<p>Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation.</p><p>KEYWORDS</p><p>Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation</p>

<p>Viruses modulate biosynthetic machineries of the host cell for a rapid and efficient virus replication. One important way of modulating protein activity in eukaryotic cells is by reversible phosphorylation. In this thesis we have studied adenovirus and vaccinia virus, two DNA viruses with different replication stategies. Adenovirus replicates and assembles new virions in the nucleus, requiring the host cell transcription and splicing machinieries, whereas vaccinia virus replicates in the cytoplasm, only requiring the cellular translation machinery for its replication. </p><p>Adenovirus uses alternative RNA splicing to produce its proteins. We have shown that adenovirus takes over the cellular splicing machinery by modulating the activity of the essential cellular SR family of splicing factors. Vaccinia virus, that does not use RNA splicing, was shown to completely inactivate SR proteins as splicing regulatory factors. SR proteins are highly phosphorylated, a modification which is important for their activity as regulators of cellular pre-mRNA splicing. We have found that reversible phosphorylation of SR proteins is one mechanism to regulate alternative RNA splicing. We have demonstrated that adenovirus and vaccinia virus induce SR protein dephosphorylation, which inhibit their activity as splicing repressor and splicing activator proteins. We further showed that the adenovirus E4-ORF4 protein, which binds to the cellular protein phosphatase 2A, induced dephosphorylation of a specific SR protein, ASF/SF2, and that this mechanism was important for regulation of adenovirus alternative RNA splicing.</p><p>Inhibition of cellular pre-mRNA splicing results in a block in nuclear- to cytoplasmic transport of cellular mRNAs, ensuring free access of viral mRNAs to the translation machinery. We propose that SR protein dephosphorylation may be a general viral mechanism by which mammalian viruses take control over host cell gene expression.</p>

<p>Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing.</p><p>Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity <i>in vitro</i>.</p><p>Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns.</p><p>Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts.</p><p>Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in <i>E. Coli</i>.</p>

Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing. Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity in vitro. Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns. Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts. Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in E. Coli.

Viruses modulate biosynthetic machineries of the host cell for a rapid and efficient virus replication. One important way of modulating protein activity in eukaryotic cells is by reversible phosphorylation. In this thesis we have studied adenovirus and vaccinia virus, two DNA viruses with different replication stategies. Adenovirus replicates and assembles new virions in the nucleus, requiring the host cell transcription and splicing machinieries, whereas vaccinia virus replicates in the cytoplasm, only requiring the cellular translation machinery for its replication. Adenovirus uses alternative RNA splicing to produce its proteins. We have shown that adenovirus takes over the cellular splicing machinery by modulating the activity of the essential cellular SR family of splicing factors. Vaccinia virus, that does not use RNA splicing, was shown to completely inactivate SR proteins as splicing regulatory factors. SR proteins are highly phosphorylated, a modification which is important for their activity as regulators of cellular pre-mRNA splicing. We have found that reversible phosphorylation of SR proteins is one mechanism to regulate alternative RNA splicing. We have demonstrated that adenovirus and vaccinia virus induce SR protein dephosphorylation, which inhibit their activity as splicing repressor and splicing activator proteins. We further showed that the adenovirus E4-ORF4 protein, which binds to the cellular protein phosphatase 2A, induced dephosphorylation of a specific SR protein, ASF/SF2, and that this mechanism was important for regulation of adenovirus alternative RNA splicing. Inhibition of cellular pre-mRNA splicing results in a block in nuclear- to cytoplasmic transport of cellular mRNAs, ensuring free access of viral mRNAs to the translation machinery. We propose that SR protein dephosphorylation may be a general viral mechanism by which mammalian viruses take control over host cell gene expression.

Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation. KEYWORDS Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation