Search PubMed⌕ Search

Biomedical subjects

H Matsubara

Publications and source records attributed to H Matsubara.

At least 217 records · Page 12Linked to original sources
PubMed1994

[Evaluation of the effects of vibro-acoustic stimulation test on the fetus during labor under epidural analgesia and evidence of its safety by way of auditory brainstem response].

This study was undertaken to elucidate the effects of epidural analgesia on vibro-acoustic stimulation test (VAST) during normal labor and auditory brainstem response (ABR) in fifty full term parturients without any complication. The safety of VAST in relation to the ABR of neonates was also studied. The results were as follows, 1) There were some changes in CTG monitoring in five cases (10%) in the 10 minutes after epidural analgesia. However, these changes were not ominous signs but all related to the coiling of the cord, as previously reported. 2) The ABR latency-intensity curve (audio-function of the newborn infants) revealed no hazardous change in VAST-loaded infants or in the controls. The response threshold was 10dB in this study. The application of VAST to the parturients under epidural analgesia was therefore not only harmless to the fetuses but also a useful way to evaluate fetal well-being.

Acoustic Stimulation↗
PubMed1993

Regulatory elements that mediate expression of the gene for the angiotensin II type 1a receptor for the rat.

To analyze the mechanism of the cell type-specific expression of rat angiotensin II type 1a receptor (AT1a-R) gene, we isolated the 5'-portion of the gene and identified multiple positive and negative regulatory sequences that regulate its transcription. Primer extension and S1 mapping identified a transcriptional initiation site at 33 (position +1) base pairs (bp) downstream of TATA sequence. The transcriptional activities of various 5'-deletion mutants of the AT1a-R gene upstream region, fused to the chloramphenicol acetyltransferase (CAT) gene, were examined using rat vascular smooth muscle cells (A10) and glial cells expressing AT1a-R mRNA predominantly or PC12 cells expressing AT2-R and very small amounts of AT1a-R mRNA. A 980-bp 5'-flanking sequence contained at least three positive elements, P1 (-560 to -489), P2 (-331 to -201), and P3 (-201 to -61). P1 and P3 were active in the tested three cells, and P2 was functional only in glial and PC12 cells. In addition to these positive elements, there was negative element, N1 (-489 to -331), which was active only in PC12 cells. These elements, when cotransfected with the AT1a-CAT fusion gene, had competitive effects against its promoter activity. The present study suggests the presence of multiple trans-acting factors that act on these positive and negative cis-acting elements and regulate the cell type-specific expression of the rat AT1a-R gene.

Animals↗
PubMed1993

Two novel mutations in the vasopressin V2 receptor gene in unrelated Japanese kindreds with nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is a rare X-linked disorder exhibiting renal resistance to the antidiuretic action of arginine vasopressin (AVP). Recent elucidation of the vasopressin V2 (renal type) receptor gene structure has enabled us to test the hypothesis that the genetic defect in the V2 receptor is the likely molecular basis of NDI. By using the polymerase chain reaction (PCR)-direct sequencing, we identified novel V2 receptor gene mutations in two unrelated Japanese kindreds with NDI. In the male patients of kindred A, a single codon deletion in one of two consecutive GTC triplets (nucleotide 832 to 837) was detected. This base change resulted in the loss of a valine residue in the 6th transmembrane domain. In the affected males of kindred B, a G to C substitution was found at nucleotide 428, altering codon 143 from arginine (CGT) to proline (CCT) in the second cytoplasmic domain. PCR-single strand conformation polymorphism (SSCP) analysis of family members demonstrated that the mutations cosegregated with clinically affected individuals and were absent in normal subjects. Our results suggest that different V2 receptor defects could be responsible for AVP resistance in individual NDI kindreds.

Adult↗
PubMed1993

The transcription of a mammalian voltage-gated potassium channel is regulated by cAMP in a cell-specific manner.

The transcript of Kv1.5, a Shaker-like delayed rectifier K+ channel cloned in our laboratory, is regulated in both tissue and developmentally specific manners. In this study we characterized the 5'-flanking region of the Kv1.5 gene. The gene lacks a canonical TATA box, has several transcription start sites, and the 5'-noncoding sequence is intronless. A cAMP response element (CRE) consensus signal was identified in the 5'-noncoding region. cAMP regulates the expression of Kv1.5 gene in a cell-specific manner. In primary cardiac cells, cAMP induces a 6-fold increase in the steady state levels of Kv1.5 transcript. However, in GH3 cells cAMP induces a 5-6-fold decrease in steady state levels of Kv1.5 transcript. The half-life of Kv1.5 transcript is 37 min and is not affected by cAMP. Nuclear run-on experiments show that in GH3 cells, cAMP reduces the transcription rate of Kv1.5 gene. Transient transfection assays using 5'-deletion mutations of Kv1.5 5'-flanking sequences revealed that the CRE located at +636 can confer the cAMP inducibility to Kv1.5 reporter gene constructs and binds to CRE-binding protein (CREB) and CRE modulator protein (CREM) in electromobility gel shift assays. Furthermore, KCl-induced depolarization can increase the steady state levels of Kv1.5 transcript in primary atrial cells and decrease it in GH3 cells. We conclude that cAMP and depolarization play an important role in regulating K+ channel expression and thus may induce long term effects on the pattern of electrical activity of excitable cells.

Animals↗
PubMed1993

Intramitochondrial protein sorting. Isolation and characterization of the yeast MSP1 gene which belongs to a novel family of putative ATPases.

Replacement of the presequence of yeast cytochrome c1 by the amino-terminal 61 residues of MAS70, a yeast mitochondrial outer membrane protein, resulted in exclusive localization of the fusion protein (termed the 61-mC1 protein) to the outer membrane. When a cytochrome c1-deficient yeast strain was transformed with a plasmid encoding the fusion protein, the cells could not grow on nonfermentable carbon sources such as glycerol. We isolated a novel yeast gene MSP1 (mitochondrial sorting of proteins) whose overexpression causes mislocalization of the 61mC1 fusion protein to the inner membrane, probably via the intermembrane space, and thereby allows the host cells to grow on glycerol. The predicted MSP1 protein (MSP1) is a hydrophilic 40-kDa polypeptide containing a putative membrane-spanning domain near the amino terminus. Further sequence analyses revealed that MSP1 is a member of a novel family of putative ATPases which share a highly conserved domain of about 185 amino acid residues, including a consensus motif for a nucleotide binding site. MSP1 was found to be an intrinsic mitochondrial outer membrane protein of an apparent molecular mass of 40 kDa with a large domain facing to the cytosol. The MSP1 gene is not essential for the cell growth either on fermentable or nonfermentable carbon sources.

Adenosine Triphosphatases↗
PubMed1993

Crystallization and preliminary X-ray diffraction studies of peroxidase from a fungus Arthromyces ramosus.

Peroxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P4(2)2(1)2, with unit cell dimensions of a = b = 74.5 A, c = 117.6 A. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 A resolution and are stable to X-rays.

Crystallization↗
PubMed1993

A comparison between ultrasonography and mammography, computed tomography and digital subtraction angiography for the detection of breast cancers.

Ultrasound (US) was compared with mammography (MMG), computed tomography (CT), and digital subtraction angiography (DSA) in its effectiveness to detect breast cancer masses and metastatic axillary nodes. Forty-seven breast cancer patients who all underwent MMG, US, CT, and DSA preoperatively in our institution between 1986 and 1990 were studied. US was able to detect tumors in all cases regardless of tumor size, whereas DSA detected T1-size tumors and MMG detected T2-size tumors in 40% and 64.7% of cases, respectively, being specifically inferior to US. It was found that MMG was least likely to detect papillotubular carcinoma, although microcalcification alone without a tumor mass on MMG improved detectability from 46.2% to 76.9%, according to the histological type. CT was found to be most sensitive to axillary node metastases (81.8%), followed by US (72.7%), but DSA was significantly unfavorable (42.9%). Thus, we concluded that US was superior to MMG, CT, and DSA for detecting breast cancer masses, but that CT was more advantageous than US, while DSA was of little value for evaluating axillary nodal status.

Adult↗
PubMed1993

Acidic regions of cytochrome c1 are essential for ubiquinol-cytochrome c reductase activity in yeast cells lacking the acidic QCR6 protein.

It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c1, a subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c1 more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c1 to complement the respiration deficiency of yeast cells lacking only cytochrome c1 or both cytochrome c1 and the QCR6 protein. The mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) was still functional in the cytochrome c1-deficient strain. Mutant cytochrome c1 with the deletion of the second acidic region (delta 168-179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant cytochrome c1 with altered charge distribution in the acidic regions (Asp170Asp171-->Asn170Asn171 or Asp170Asp171-->Asn170Lys171) made the cytochrome c1-deficient cells respiration-competent. On the other hand, mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) or altered charge distribution in the second region (Asp170Asp171-->Asn170Lys171) did not restore the respiration deficiency of the cells lacking not only cytochrome c1 but also the QCR6 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Rat angiotensin II (type 1A) receptor mRNA regulation and subtype expression in myocardial growth and hypertrophy.

Two subtypes of angiotensin II (Ang II) receptors (AT1 and AT2) are distinguished by using the respective specific antagonists. In the present study, we report the regulation of cardiac AT1 type A (AT1A) receptor mRNA levels and the expression pattern of AT1 and AT2 receptors in the growth of the heart and the development and regression of cardiac hypertrophy. The ventricular AT1A mRNA level and the density of Ang II receptors at the neonatal period were significantly increased (3.5-fold and 2.5-fold, respectively) and then downregulated with maturation. The cardiac hypertrophy established in spontaneously hypertensive rats or two-kidney one-clip renovascular hypertensive rats resulted in substantial increases in ventricular AT1A mRNA levels (threefold) and Ang II receptor densities (twofold) as compared with those in respective control rats, whereas the receptor affinity was similar. The proportion of AT1 and AT2 subtypes in the specific Ang II binding in ventricular membranes prepared from normal adult rats was nearly equal. This proportion did not change significantly in the development of myocardial hypertrophy. The regression of cardiac hypertrophy by the normalization of elevated blood pressure completely reversed the increased levels of AT1A mRNA and the receptor density to the control level. Thus, AT1 and AT2 receptors are present in rat ventricular myocardium, and their expression is developmentally regulated and upregulated in response to hypertrophic change. Ang II action exerted through the increased number of Ang II receptors may contribute to the growth of the heart and thus to the maintenance of established hypertrophy as one of the hormones involved in hypertrophy development.

Angiotensin II↗
PubMed1993

Shaker-related potassium channel, Kv1.4, mRNA regulation in cultured rat heart myocytes and differential expression of Kv1.4 and Kv1.5 genes in myocardial development and hypertrophy.

The multiple K+ channels are crucial for repolarization and configuration of the action potential in the neuronal and cardiac cells. In this study, we report the regulatory mechanisms of rapidly inactivating Shaker Kv1.4 channel transcript in the rat heart. Quantitative PCR analysis showed that stimulation with high concentration of KCl, BAY-K 8644, or 12-O-tetradecanoyl phorbol-13-acetate resulted in an immediate and substantial increase (two- to threefold) of Kv1.4 mRNA levels in spontaneously beating myocytes prepared from neonatal rat ventricles. The Kv1.4 mRNA in the ventricle remains at a steady state level after birth and gradually declines with maturation. These results suggest that the Kv1.4 mRNA level is not static and undergoes dynamic modulation by multiple factors that activate intracellular signals. In addition, the expression patterns of Kv1.4 as well as the delayed rectifier Shaker K+ channel Kv1.5 mRNAs were examined in hypertrophied ventricles in which a plateau phase of action potential is remarkably prolonged. The Kv1.5 mRNA level was dramatically repressed while the Kv1.4 mRNA level was remarkably increased. This differential regulation was completely reversed by the normalization of hypertrophy, suggesting that the pathological alterations of K+ channel gene regulation may be involved in the occurrence of ventricular arrhythmias in hypertrophic hearts.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Ca2+ sensitivity of contractile machinery and Ca2+ handling energy. Simulation.

Myocardial Ca2+ handling during excitation-contraction coupling has been modelled mathematically to gain a better insight into the expectation that Ca2+ sensitization of contractile machinery may save myocardial energy utilization for Ca2+ handling. The basic model of myocardial Ca2+ kinetics and mechanoenergetics involved the sarcoplasmic reticulum (SR), sarcoplasm, troponin C (Tn) and crossbridges (CB). The relations among the released Ca2+ ions from the SR, peak concentrations of sarcoplasmic free Ca2+ ([Ca2+]i) and Ca(2+)-bound troponin ([TnCa]) and peak contractile force were computed, based upon the assumptions that the released Ca2+ ions diffuse as free Ca2+ in sarcoplasm, bind kinetically with Tn with an association rate constant of k1, dissociate from TnCa with a dissociation rate constant of k2, and are sequestered into the SR with consumption of ATP. TnCa was associated with CB cycling to develop force with a set of given on and off rate constants. The association constant Ka (= k1/k2) of TnCa as an index of Ca2+ sensitivity of Tn was varied 32-fold from 0.25 to 8/microM. Results showed that Ca2+ sensitization from a lower Ka level could most sharply decrease the total Ca2+ release required to develop the same contractile force. Thus, it would reduce the total Ca2+ handling energy that the SR uses to maintain the same contractility.

Calcium↗
PubMed1993

Nucleotide sequence and genetic analysis of the region essential for functional expression of the gene for ferredoxin I, fdxN, in Rhodobacter capsulatus: sharing of one upstream activator sequence in opposite directions by two operons related to nitrogen fixation.

Nucleotide sequencing of the region upstream of two ferredoxin genes, fdxC and fdxN, of Rhodobacter capsulatus revealed the existence of one open reading frame (ORF), ORFU1, in the same orientation as these genes and two other ORFs, ORFU2 and ORFU3, in the opposite orientation. Two potential -24/-12 promoters were found in front of ORFU1 and ORFU2, respectively, and there was a putative upstream activator sequence (UAS) or NifA-binding site between them. The ORFs corresponded to no known nif genes. However, analysis of their putative products showed that the product of ORFU1 (M(r) 47,912) and that of ORFU3 (M(r) 19,090) had a flavodoxin-like domain and a 2[4Fe-4S] ferredoxin-like domain, respectively, and that the product of ORFU2 (M(r) 20,424) was a hydrophobic protein with six potential membrane-spanning portions. Results of interposon mutagenesis and complementation experiments indicated that ORFU2 but not ORFU1 is essential for nitrogen fixation and that additional gene(s) essential for nitrogen fixation must be present in the unsequenced region adjacent to ORFU3. Translational fusion analysis involving lacZYA and fdxN or ORFU3 provided evidence that the putative UAS is responsible for regulation of both ORFU1-fdxC-fdxN and ORFU2-ORFU3 operons in opposite orientations, and that the control of the latter is stricter than that of the former.

Amino Acid Sequence↗
PubMed1993

Identification of a nifDK-like gene (ORF467) involved in the biosynthesis of chlorophyll in the cyanobacterium Plectonema boryanum.

The frxC gene, which is found in chloroplast DNA (ctDNA) and in cyanobacteria, encodes a protein that is required for the light-independent reduction of protochlorophyllide (Pchlide) to chlorophyllide a (Chlide). A DNA fragment downstream of frxC in the filamentous cyanobacterium Plectonema boryanum was cloned and analyzed. Sequencing of the DNA fragment revealed an open reading frame (ORF) that encoded a protein of 467 amino acid residues (designated ORF467), which showed extensive homology to the proteins encoded by genes on ctDNAs (ORF465 in liverwort, gidA in pine and chlN in Chlamydomonas reinhardtii) and to ORF469 protein of the cyanobacterium Synechocystis sp. strain PCC 6803. We isolated a targeted mutant YFM6D-3 in which ORF467 was inactivated by the insertion of a kanamycin-resistance gene into the coding region. YFM6D-3 exhibited a phenotype similar to that of YFC1004, an frxC-disrupted mutant, which did not synthesize chlorophyll (Chl) and accumulated Pchlide, a precursor to Chl, in the dark. These phenotypic characteristics of YFM6D-3 indicate that the light-independent reduction of Pchlide requires not only the FrxC protein but also the ORF467 protein. The amino acid sequences of the homologues of ORF467 exhibit low but significant similarity to those of the alpha and beta subunits of nitrogenase MoFe-protein, suggesting a phylogenetic relationship between the light-independent Pchlide reductase and nitrogenase, as is observed between the FrxC protein and the Fe-protein of nitrogenase.

Amino Acid Sequence↗
PubMed1993

An improved technique for observing both membranous organelles and cytoskeleton in saponin-extracted cells.

Detergent-extraction of soluble cytoplasmic proteins under low osmotic pressure induces severe morphological damage, particularly on membranous organelles, although this processing is often required to observe cytoskeletal elements in the cells. In the present study, the osmotic adjustment of the rinsing solution and fixative, which were used after the detergent-extraction using saponin, has greatly improved the ultrastructure of membranous organelles. This improved method allowed us to observe both cytoskeletal elements and the fine structure of membranous organelles by electron microscopy.

Animals↗
PubMed1992

Molecular cloning and characterization of the gene encoding mouse melanoma antigen by cDNA library transfection.

We have isolated a cDNA (H52) of 2.8-kb-long encoding an 80-kDa mouse melanoma Ag that is defined by a syngeneic anti-B16 melanoma mAb with an ability to block anti-melanoma cytotoxic T cell responses. H52 transfectants were brightly stained with the antibody, and the 80-kDa molecule was immunoprecipitated from the transfectants. Northern blot analysis showed that this transcript was detected in mouse melanoma cells of C57BL/6 and DBA/2 origin, C1300 A/J neuroblastoma, L cell (C3H) and EL-4 T lymphoma (C57BL/6), faintly in BW5147 (AKR) T lymphoma, but not in other tumors, such as S913 fibrosarcoma (C57BL/10), NIH3T3, 70 Z/3 pre-B lymphoma, and P3U1 plasmacytoma (BALB/c). Since the transcripts were not found in normal C57BL/6 tissues of fetus, newborn, and adult origin, the H52 expression is associated with transforming phenotypes. However, no tissue- or cell type-specific expression was observed. Nucleotide sequence analysis has clearly demonstrated that H52 cDNA encodes the full length of the env gene and long terminal repeat region of endogenous ecotropic murine leukemia provirus of AKV-type, which is defective in C57BL/6. The H52 envelope protein has several amino acid changes compared to those of AKV, one of which is in the env 14 peptide region preferentially associated with MHC molecule, suggesting the possible reason for the difference of antibody reactivity even in H52-positive tumors. We also demonstrate that CTL against H52 transfectant kills B16 melanoma. Thus, the above results are direct evidence that even the endogenous self molecule, when constitutively expressed, does act as a tumor Ag.

Amino Acid Sequence↗
PubMed1992

Crystal structure of oxidized flavodoxin from a red alga Chondrus crispus refined at 1.8 A resolution. Description of the flavin mononucleotide binding site.

In order to describe the detailed conformation of the oxidized flavodoxin from a eukaryotic red alga, Chondrus crispus, the crystal structure has been refined by a restrained least-squares method. The crystallographic R factor is 0.168 for 13,899 reflections with F greater than 2 sigma F between 6.0 and 1.8 A resolution. The refined model includes 173 amino acid residues, flavin mononucleotide (FMN) and 110 water molecules. The root-mean-square deviation in bond lengths from ideal values is 0.015 A, and the mean co-ordinate error is estimated to be 0.2 A. The FMN is located at the periphery of the molecule. The orientation of the isoalloxazine ring is such that the C-7 and C-8 methyl groups are exposed to solvent and the pyrimidine moiety is buried in the protein. Three peptide segments, T8-T13, T55-T58 and D94-C103, are involved in FMN binding. The first segment of T8-T13 enfolds the phosphate group of the FMN. The three oxygen atoms in the phosphate group form extensive hydrogen bonds with amide groups of the main chain and the O gamma atoms of the side-chains in this segment. T55 O and W56 N epsilon 1 in the second segment form hydrogen bonds with O-2 in the ribityl moiety and one of the oxygen atoms in the phosphate group, respectively. The O gamma H of T58 forms a hydrogen bond with the N-5 atom in the isoalloxazine ring, which is expected to be protonated in the semiquinone form. The third segment is in contact with the isoalloxazine ring. It appears that the hydrogen bond acceptor of the NH of Asp94 in the third segment is O-2 rather than N-1 in the isoalloxazine ring. The isoalloxazine ring is flanked by the side-chains of Trp56 and Tyr98; it forms an angle of 38 degrees with the indole ring of Trp56 and is almost parallel to the benzene ring of Tyr98. The environment of the phosphate group is conserved as in other flavodoxins whereas that of the isoalloxazine ring differs. The relationship between the hydrogen bond to the N-5 in the ring and the redox potential for the oxidized/semiquinone couple is discussed.

Amino Acid Sequence↗