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Biomedical subjects

M Muramatsu

Publications and source records attributed to M Muramatsu.

At least 127 records · Page 7Linked to original sources
PubMed1999

Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain.

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.

Amino Acid Sequence↗
PubMed1999

Carboxy-terminally truncated form of a coactivator UTF1 stimulates transcription from a variety of gene promoters through the TATA Box.

We have recently isolated a novel transcriptional coactivator, UTF1, which is expressed mainly in pluripotent embryonic stem cells (Okuda, A., Fukushima, A., Nishimoto, M., Orimo, A., Yamagishi, T., Nabeshima, Y., Kuro-o, M., Nabeshima, Y., Boon, K., Keaveney, M., Stunnenberg, H. G., and Muramatsu, M. EMBO J. 17, 2019-2032, 1998). The UTF1 does not activate transcription nonspecifically, but boosts the level of transcription strictly in a specific upstream factor, ATF-2, dependent manner in mammalian cells. However, when expressed in yeast cells, the UTF1 displays a distinct activity, being able to augment the activity of minimal promoter bearing only the TATA element. Thus, these results indicate that certain domains of UTF1 render the factor inactive in terms of stimulating transcription through the basal transcription machinery in the absence of promoter-bound ATF-2 in mammalian cells. Here we report that the region bearing the leucine zipper motif is responsible for such biochemical properties of the UTF1. Indeed, UTF1 lacking functional leucine zipper is able to rather promiscuously stimulate transcription from a number of basal gene promoters such as those of hsp70 and E1B genes in mammalian cells. We have also shown that this activation is executed through TATA box by the experiments using a TBP allele with an altered TATA-binding specificity. Moreover, we have found that Dr1-mediated repression of transcription can be overcome by expression of this mutant UTF1, indicating that the observed stimulation of transcription is at least in part due to its action as an anti-repressor.

Adenovirus E1B Proteins↗
PubMed1999

Isolation and characterization of a novel human gene (HFB30) which encodes a protein with a RING finger motif.

A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.

Amino Acid Sequence↗
PubMed1999

Cytotoxic effects of NSL-1406, a new thienopyrimidine derivative, on leukocytes and osteoclasts.

We synthesized a series of thienopyrimidine derivatives and examined their cytotoxic effects on several cell lines. One of the derivatives, NSL-1406, was shown to exert potent cytotoxic effects on leukemia cell line including P388 cells and J774 cells. It was also inhibitory on mouse osteoclasts and suppressed the in vitro bone resorption by osteoclasts at nanomolar concentrations.

Animals↗
PubMed1999

Characterization of the carp myosin heavy chain multigene family.

We isolated partial coding sequences for 29 carp myosin heavy chain genes (MyoHCs) and determined the nucleotide sequences around the region encoding the loop 2 of the myosin molecule. The predicted amino acid sequences from the isolated genes all showed very high similarity to those of skeletal and cardiac muscles from higher vertebrates, but not to those of smooth and non-muscle counterparts. Among all clones isolated, carp MyoHC10, MyoHCI-1-3 and MyoHC30 showed exon-nucleotide sequences identical to those of cDNAs encoding the loop 2 region of the 10 degrees C-, intermediate- and 30 degrees C-type fast skeletal isoforms [Hirayama and Watabe, Euro. J. Biochem. 246 (1997) 380-387]. The loop 2 of 28 types of carp MyoHCs was encoded by two exons separated by an intron corresponding to that of the 16th in higher vertebrate MyoHCs, whilst this intron was not found in carp MyoHC30. Although carp MyoHC30 had a gene organization different from those of higher vertebrates and other carp MyoHCs, its predicted amino acid sequence for loop 2 showed the highest homology to those of higher vertebrates among carp MyoHCs. In the 28 carp MyoHCs containing the intron, a combination of different nucleotide sequences for the two resulted in 14 distinct series for the combined coding sequence. These different nucleotide sequences encoded nine distinct amino acid sequences. Phylogenetic analysis for the present loop 2 and light meromyosin previously reported for carp MyoHCs [Imai et al., J. Exp. Biol. 200 (1997) 27-34] revealed that carp MyoHCs have recently diverged and are more closely related to each other than to MyoHCs from other species.

Amino Acid Sequence↗
PubMed1999

Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.

We have developed a method for high-efficiency window separation of cDNA display by increasing the specificity of priming in reverse transcription. In the conventional method, two-base anchored oligo(dT) primers (5'dT16VN3', where N is any base and V is G, A or C) are used to make windows for the display of transcripts. However, reverse transcriptase often extends misprimed oligonucleotides. To avoid mispriming from dT16VN primers, we have developed two new technologies. One is higher temperature priming with reverse transcriptase thermoactivated by the disaccharide trehalose. The other is the use of competitive oligonucleotide blockers that hybridize to the non-selectively primed mRNAs, preventing the mispriming from the VN site. These methods were combined to improve restriction landmark cDNA scanning (RLCS), resulting in the elimination of the redundant signals that appear in different windows. This was achieved by the increased specificity of initiation of reverse trans-cription from the beginning of poly(A) sites. This method paves the way for the precise visualization of transcripts to allow expression profiles in individual tissues and at each developmental stage to be understood.

Animals↗
PubMed1999

NMDA receptor type 2D gene as target for estrogen receptor in the brain.

Although it is well known that estrogen exerts its effect in the brain, the direct target genes transcriptionally regulated by estrogen or rather estrogen receptor (ER) are almost unknown. During the search for estrogen receptor-binding sites from human CpG island library, we found one genomic DNA fragment corresponding to the putative 3'-untranslated region of human NMDA receptor subunit 2D (NR2D) gene. It contained at least four half palindromic estrogen responsive elements (hEREs) within two hundred nucleotides, which was conserved also in the rat. Interestingly, the NR2D mRNA is co-localized with ERalpha and/or ERbeta mRNA in a number of regions of rat brain. We have also demonstrated that NR2D mRNA is up-regulated in rat hypothalamus by estrogen possibly via hEREs identified here. Thus, we suggest that NR2D is one of the direct targets of estrogen receptors which are involved in reproductive as well as non-reproductive actions in the brain.

Animals↗
PubMed1999

Fractalkine and macrophage-derived chemokine: T cell-attracting chemokines expressed in T cell area dendritic cells.

Dendritic cells (DC) are a system of antigen-presenting cells specialized in interaction with T cells. Recently it has been reported that DC can produce CC (beta) chemokines that attract T cells. In this study we isolated mouse fractalkine and macrophage-derived chemokine (MDC) belonging to CX3C (delta) and CC chemokine families, respectively, from bone marrow-derived mature DC. While expression of fractalkine, which has so far been only examined in the brain and in vitro endothelial cells so far, was rather ubiquitous, MDC, which has been reported to be synthesized by macrophages and DC, was expressed specifically in the thymus and lymph node. This is the first report that indicates fractalkine expression by DC. Expression of fractalkine and MDC mRNA increased with maturation of DC during in vitro culture of bone marrow cells. Spleen- and epidermis-derived mature DC in culture also expressed these chemokines. Furthermore, their expression was detected selectively by Northern hybridization in CD11c+ B220- DC freshly purified from lymph nodes, and in large stellate cells in the lymph node T cell areas by in situ hybridization. Conditioned media of 293T cells transfected with these chemokine cDNA were chemotactic to Con A-activated splenic T cells as well as the mouse T cell line EL4. In conclusion, while fractalkine and MDC belong to different families of chemokines, both may be involved in recruitment of T cells for interaction with mature DC in the immune response.

Animals↗
PubMed1999

Isolation and characterization of the fission yeast gene rpa42+, which encodes a subunit shared by RNA polymerases I and III.

Eukaryotic RNA polymerases I and III share two distinct alpha-related subunits that show limited homology to the alpha subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of alpha-related subunits in eukaryotes, we have previously identified the alpha-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42+, which encodes the alpha-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42+ encodes a polypeptide with an apparent molecular mass of 42 kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42+ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct alpha-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.

Amino Acid Sequence↗
PubMed1999

The fission yeast rpa17+ gene encodes a functional homolog of AC19, a subunit of RNA polymerases I and III of Saccharomyces cerevisiae.

Eukaryotic RNA polymerases I and III consist of multiple subunits. Each of these enzymes includes two distinct and evolutionarily conserved subunits called alpha-related subunits which are shared only by polymerases I and III. The alpha-related subunits show limited homology with the alpha-subunit of prokaryotic RNA polymerase. To gain further insight into the structure and function of alpha-related subunits, we cloned and characterized a gene from Schizosaccharomyces pombe that encodes a protein of 17 kDa which can functionally replace AC19 - an alpha-related subunit of RNA polymerases I and III of Saccharomyces cerevisiae - and was thus named rpa17+. RPA17 has 125 amino acids and shows 63% identity to AC19 over a 108-residue stretch, whereas the N-terminal regions of the two proteins are highly divergent. Disruption of rpa17+ shows that the gene is essential for cell growth. Sequence comparison with other alpha-related subunits from different species showed that RPA17 contains an 81-amino acid block that is evolutionarily conserved. Deletion analysis of the N- and C-terminal regions of RPA17 and AC19 confirms that the 81-amino acid block is important for the function of the alpha-related subunits.

Amino Acid Sequence↗
PubMed1999

Isolation and chromosomal assignment of human genes encoding cofactor of LIM homeodomain proteins, CLIM1 and CLIM2.

Cofactors of LIM homeodomain proteins (CLIM) are transcriptional activators that associate with the LIM homeoproteins and coordinate transcription. LIM homeoproteins and CLIMs are involved in a variety of developmental processes. Two CLIMs, CLIM1 and CLIM2, have been identified in the mouse. Here we report the isolation of human CLIM1 and CLIM2 cDNAs and the determination of their chromosome locations by using a human-rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. The proteins deduced from human CLIM1 and CLIM2 cDNAs were composed of 373 and 375 amino acids, respectively, and had 97.3% and 98.7% amino acid identity, respectively, to their mouse counterparts. Human CLIM1 and CLIM2 proteins were 75.5% identical. Human CLIM1 and CLIM2 genes were mapped to the chromosome on 4p15.3 and 10q24-q25 regions, respectively. Mapping of a pair of developmentally important genes may provide new clues to the understanding of genetic disorders caused by these chromosome regions.

Amino Acid Sequence↗
PubMed1999

A novel human gene whose product shares significant homology with the bovine brain-specific protein p25 on chromosome 5p15.3.

Here, we report on the sequence features and chromosomal location of a novel human gene which shares significant homology with the bovine brain-specific protein p25. Based on polymerase chain reaction analysis with a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped on to p15.3 region of chromosome 5.

Amino Acid Sequence↗
PubMed1999

Isolation and chromosomal assignment of a human gene encoding protein inhibitor of activated STAT3 (PIAS3).

Mouse PIAS3 (protein inhibitor of activated STAT3) is a specific inhibitor of STAT3 that downregulates its signaling pathway. Here we report the isolation and chromosome mapping of the human PIAS3 gene. Human PIAS3 cDNA encoded a predicted protein of 619 aa which has 83% overall amino acid identity to the mouse counterpart. Based on polymerase chain reaction assisted analysis of a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel, the human PIAS3 gene was mapped to the chromosome 1q21 region. Mapping of a crucial gene in modulating the STAT3 signaling pathway may provide new clues to the understanding of malignancies or genetic disorders caused by this chromosome region.

Amino Acid Sequence↗
PubMed1999

Isolation, tissue expression, and chromosomal assignment of a human LIM protein gene, showing homology to rat enigma homologue (ENH).

Rat ENH (Enigma homolog) is a LIM domain protein that associates with protein kinase C in an isoform-specific manner. We have identified a human cDNA which shares a significant sequence homology with rat ENH. The isolated cDNA clone, designated human ENH (hENH), was 3287 bp in length and encoded a predicted protein of 596 amino acids which had 88% overall identity to rat ENH protein. Northern blot analysis revealed that 1.9 kb of the hENH messenger RNA was predominantly expressed in heart and skeletal muscle, while 5.6 kb of the hENH messenger RNA was ubiquitously expressed in various human tissues. The chromosomal location of the gene was determined on chromosome 4q22 region, between markers WI-2900 and WI-3273, by polymerase chain reaction (PCR)-based analyses using both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

Adaptor Proteins, Signal Transducing↗
PubMed1999

A thermophilic cyanobacterium Synechococcus elongatus has three different Class I prenyltransferase genes.

Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C5) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences. Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus. Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C20) diphosphate synthase, another encodes a farnesyl (C15) diphosphate synthase whose optimal reaction temperature is 60 degrees C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 degrees C. The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C25), hexaprenyl (C30), and heptaprenyl (C35) diphosphates from dimethylallyl (C5) diphosphate, geranyl (C10) diphosphate, or farnesyl diphosphate as the allylic substrates. The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates. The situations of these three S. elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al. (1996).

Amino Acid Sequence↗
PubMed1999

Multiple mammalian proteasomal ATPases, but not proteasome itself, are associated with TATA-binding protein and a novel transcriptional activator, TIP120.

BACKGROUND: SUG1 belongs to proteasomal ATPase. Previous studies have demonstrated that SUG1 is associated with TBP. It is assumed to be involved in transcriptional regulation in addition to proteolysis. In this study, we investigated the association of mammalian SUG1 with TBP in more detail. RESULTS: Pull-down experiments with TBP revealed multiple TBP-interacting proteins (TIPs) that were recovered dependent upon the presence of C-terminal conserved domain of TBP. By 2-D electrophoresis, we identified SUG1 in TIPs. By using far-Western analysis, we identified two proteins that could directly bind to TBP: SUG1 and another proteasomal ATPase (S4). Protein microsequencing and Western blotting identified all the remaining proteasomal ATPases (MSS1, TBP1, TBP7, and SUG2) in the TIP preparations. We present evidence that TBP and at least SUG1, MSS1, and S4 form a complex in the cell. However, no evidence of association of TBP with the 26S proteasome or its 19S regulatory unit was obtained. The molecular mass of the TBP/ATPases-complex, which also included a novel transcription regulatory factor, TIP120, was estimated to be approximately 800 kDa. CONCLUSION: These results suggest that there is a novel multisubunit complex containing TBP and proteasomal ATPases. Based on our findings, we hypothesize that proteasomal ATPases are involved in transcriptional regulation in addition to proteolysis.

Adenosine Triphosphatases↗
PubMed1999

Interaction of the new histamine H2-receptor antagonist pibutidine hydrochloride with canine cloned H2-receptor expressed cells.

The effect of a new histamine H2-receptor antagonist, pibutidine hydrochloride ((Z)-3-amino-4-{4-{4-[(piperidinomethyl)pyrid-2-yl]oxy} but-2-enylamino}cyclobut-3-ene-1,2-dione monohydrochloride, CAS 126463-66-9, IT-066 displaced the binding of [3H]tiotidine to cells transfected with the wild-type H2-receptor in a concentration-dependent manner. Compared with the 50% inhibitory concentration values (IC50) among five H2-receptor antagonists tested, the value of IT-066 was lowest. The inhibitory effect of IT-066 was enhanced by preincubation of IT-066 with the cells, and preincubation for 60 min increased potency about 2-fold. Famotidine also has such effects. When H2-receptor transfected cells were treated with IT-066 for 30 min, the inhibitory effect of IT-066 on the [3H]tiotidine binding still remained even after the cells were extensively washed. Scatchard plot analysis supported the non-competitive and irreversible action of IT-066. When the canine mutated H2-receptor-expressed cells were used, which have substituted amino acid of 190Alanine (Ala; position: 190th amino acid) for 190Threonine (Thr) in the fifth transmembrane domain, IT-066 also inhibited [3H]tiotidine binding to the cells concentration-dependently. However, in the case of the 190Ala mutated cells, the inhibitory effect of IT-066 attenuated with a decrease in maximal response after washing of the cells. These results show that IT-066 could tightly bind H2-receptor in a time-dependent manner and suggest the possibility that the irreversible inhibition of IT-066 is important in the interaction with 190Thr in the fifth transmembrane domain of H2-receptor.

Alanine↗