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Biomedical subjects

K Ohyama

Publications and source records attributed to K Ohyama.

At least 19 recordsLinked to original sources
PubMed1992

Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis.

To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor.

Angiotensin II↗
PubMed1992

Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis.

To determine the specific mechanism of ligand binding to angiotensin (Ang II) receptor AT1, mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to peptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys199 to Gln in the intramembrane domain strongly reduced the affinity to both [125I] Ang II and [125I]-1Sar, 8Ile-Ang II whereas mutation of two other Lys had little effect, indicating involvement of Lys199 in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity, indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in N-glycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies.

Angiotensin II↗
PubMed1992

Transfer RNA genes in the mitochondrial genome from a liverwort, Marchantia polymorpha: the absence of chloroplast-like tRNAs.

Twenty-nine genes for 27 species of tRNAs were deduced from the complete nucleotide sequence of the mitochondrial genome from a liverwort, Marchantia polymorpha. One to three species of tRNA genes corresponded to each of 20 amino acids including three species for leucine and arginine, two species for serine and glycine, and one for the rest of the amino acids. Interestingly, all tRNA genes were located in the semicircle of the liverwort mitochondrial genome except for the trnY and trnR genes. The region containing these tRNA genes was originally duplicated, and two trnR genes have diverged from each other. On the other hand, trnY and trnfM are present as two identical copies. The G:U and U:N wobbling between the first nucleotide of the anticodon and the third nucleotide of the codon permit the 27 tRNA identified species to translate almost all codons. However, at least two additional tRNA genes, trnl-GAU for AUY codon and trnT-UGU for ACR codon, are required to read all codons used in the liverwort mitochondrial genome. All of the identified tRNA genes are 'native' in liverwort mitochondria, not 'chloroplast-like' tRNAs as are found in the mitochondria of higher plants. This result implies that the tRNA gene transfer from chloroplast to mitochondrial genome in higher plants has occurred after the divergence from bryophytes.

Base Sequence↗
PubMed1992

Gene clusters for ribosomal proteins in the mitochondrial genome of a liverwort, Marchantia polymorpha.

We detected 16 genes for ribosomal proteins in the complete sequence of the mitochondrial DNA from a liverwort, Marchantia polymorpha. The genes formed two major clusters, rps12-rps7 and rps10-rpl2-rps19-rps3-rpl16-rpl5- rps14-rps8- rpl6-rps13-rps11-rps1, very similar in organization to Escherichia coli ribosomal protein operons (str and S10-spc-alpha operons, respectively). In contrast, rps2 and rps4 genes were located separately in the liverwort mitochondrial genome (the latter was part of the alpha operon in E. coli). Furthermore, several ribosomal proteins encoded by the liverwort mitochondrial genome differed substantially in size from their counterparts in E. coli and liverwort chloroplast.

Amino Acid Sequence↗
PubMed1992

Mechanism of interferon gamma-induced protection of human gliosarcoma cells from lymphokine-activated killer lysis: division of lymphokine-activated killer cells into natural killer- and T-like cells.

The mechanism by which interferon gamma (IFN-gamma) decreases the susceptibility of the established cultured gliosarcoma line Gl-1 to lymphokine-activated killer (LAK) lysis was analyzed. The results of monolayer depletion and lectin-dependent cellular cytotoxicity assays by LAK cells revealed that the resistance to LAK lysis of IFN-gamma-treated Gl-1 cells is manifested at the stage of LAK cell target recognition alone. We have also divided LAK cells into populations of phenotypically natural killer (NK)- and T-like cells with monoclonal antibodies and complement, respectively. We have used these cells to examine the mechanism of IFN-gamma-induced protection of Gl-1 cells from LAK lysis in cold target inhibition, monolayer depletion, and direct binding assays. The results revealed that NK-like cells do not recognize IFN-gamma-treated Gl-1 cells as efficiently as they do untreated targets, whereas T-like cells show the opposite tendency. In conclusion, we have demonstrated that the IFN-gamma induced protection of tumor cells from LAK lysis is predominantly regulated by the target recognition of NK-like cells. On the other hand, IFN-gamma-treated tumor cells may bind to T-like cells but fail to trigger them to initiate further stages for lysis as effectively as NK-like cells.

Cell Division↗
PubMed1992

Mitochondrial DNA of Marchantia polymorpha as a single circular form with no incorporation of foreign DNA.

A cosmid library and physical maps of mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, were constructed using the cosmid clones. Electrophoresis profile and the physical maps indicated that the liverwort mtDNA was approximately 183 kb long, the smallest among plant mtDNAs, and that it consisted of a single circular molecule. Southern hybridization analysis showed that genes typical to the mitochondrial genome existed in a single copy, and also that there was no incorporation of chloroplast DNA fragments into the mitochondrial genome.

Base Sequence↗
PubMed1992

Cloning and nucleotide sequence of a frxC-ORF469 gene cluster of Synechocystis PCC6803: conservation with liverwort chloroplast frxC-ORF465 and nif operon.

A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase. Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known. Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469-ORF248) was isolated from the cyanobacterium Synechocystis PCC6803. In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively. Synechocystis frxC showed 31% amino acid sequence identity with nifH1 from Clostridium pasteurianum. Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C. pasteurianum nifK product, which is the beta subunit of a molybdenum-iron protein of a nitrogenase complex. Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.

Amino Acid Sequence↗
PubMed1991

Presence of influenza virus-reactive glycophorins other than glycophorin A in human erythrocyte membranes.

The reactivities against seven hemagglutinins including influenza A and B viruses of six sialoglycoprotein (glycophorin) fractions, M-Frs.1-3 and N-Frs.1-3, separated from human OMs and ONs erythrocyte membranes by a combination of the LIS-phenol method and gel filtration were studied. The serological results show that M-Fr.1 and N-Fr.1 were glycophorins AM and AN, respectively, the reactivities against influenza A and B viruses of both M-Fr.2 and N-Fr.2 which contained glycophorins B and C were remarkably higher than that of glycophorin A and the reactivities against influenza viruses of both M-Fr.3 and N-Fr.3 which contained glycophorins B and D were considerably lower than that of glycophorin A.

Amino Acids↗
PubMed1991

Involvement of the Chox-4 chicken homeobox genes in determination of anteroposterior axial polarity during limb development.

We have isolated and identified four chicken homeobox genes in the upstream region of the Chox-4 complex. The Chox-4g and -4f genes, at the 5' extremity of the complex, were expressed locally in the vicinity of the zone of polarizing activity (ZPA) at early stages of limb development, substantiating the involvement of the genes in anteroposterior axis determination. To confirm their function, we implanted a bead containing retinoic acid, or the ZPA itself, in the anterior margin of the limb bud, leading to formation of mirror-image duplicated digits, and observed the resultant change in gene expression. Expression of the Chox-4g and -4f genes was induced in the new digit-forming region. Those results suggest that positional information assigned by a ZPA morphogen is imprinted on cellular memory by expression of the Chox-4 genes to maintain positional signaling along the anteroposterior axis in the limb field.

Amino Acid Sequence↗
PubMed1991

Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud.

Retinoic acid is a putative morphogen in limb formation in the chick and other vertebrates. In chick limb formation, it is thought that retinoic acid is released from the zone of polarizing activity (ZPA) and the concentration gradient of retinoic acid formed from the posterior to the anterior provides positional cues for digit formation. Implantation of a bead containing retinoic acid at the anterior margin of the limb bud induces a mirror-image symmetrical duplication of the digit pattern similar to that observed when the ZPA is grafted into the anterior margin of the host limb bud. Also, the level of endogenous retinoic acid (25 nM on average) is higher in the posterior one third of the limb bud. We found that when the bead containing either retinoic acid or an analogue but not the ZPA, was implanted in the anterior margin of the chick limb bud, expression of the retinoic acid receptor type-beta gene was induced around the bead within 4 h. These results indicate that exogenous retinoic acid is not identical with the ZPA morphogen. As the anterior tissue exposed to retinoic acid has polarizing activity, we conclude that the primary function of exogenous retinoic acid is to induce polarizing activity in the limb bud.

Amino Acid Sequence↗
PubMed1991

Radial communication between the perilymphatic scalae of the cochlea. I: Estimation by tracer perfusion.

The degree to which radial exchange between scala tympani (ST) and scala vestibuli (SV) can occur has not previously been quantified. We have measured the amount of cross-communication in the third turn of the guinea pig cochlea using an ionic tracer, trimethylphenylammonium (TMPA). TMPA was perfused through one scala while TMPA concentrations were measured simultaneously in the perfused and non-perfused scalae. On the basis of the time course of TMPA increase recorded in the non-perfused scala, we were able to calculate rate constants for cross-leak and clearance. Cross-leak in the third turn occurred remarkably rapidly with a rate constant from ST to SV of 0.049 min-1 and from SV to ST of 0.031 min-1. These correspond to transfer half times of 14.2 and 22.4 min respectively. This result demonstrates that ST and SV in the third turn cannot be regarded as independent compartments.

Animals↗
PubMed1991

Radial communication between the perilymphatic scalae of the cochlea. II: Estimation by bolus injection of tracer into the sealed cochlea.

Radial communication between ST and SV was measured in the sealed cochlea by monitoring the dispersal of an ionic tracer, trimethylphenylammonium (TMPA) injected in the form of a minute bolus. Tracer movements were recorded by a pair of ion-selective electrodes sealed into the injected and non-injected scalae close to the injection site. Measurements were made in the basal or third turn of the guinea pig cochlea. In the third turn, radial communication occurred rapidly with a ST half time from ST to SV of 25 min and from SV to ST of 26 min. In the basal turn the communication was markedly slower, with a ST half time from ST to SV of 170 min and from SV to ST of 240 min. However, the difference between the basal and third turns can be shown to arise almost totally from differences in cross-sectional area of the perilymphatic scalae. When normalized with respect to scala cross-section, the process of tracer movement across the spiral ligament is similar in the basal and third turns. These results demonstrate that radial communication between scala tympani and scala vestibuli is an important route which must be considered in studies involving perilymph.

Animals↗
PubMed1991

Spontaneous otoacoustic emissions in the guinea pig.

Measurement of spontaneous otoacoustic emission (SOAE) in a large number of animals was conducted in the guinea pig. A remarkably high incidence of SOAE (51 ears out of 248 ears; 20.6%) was observed in the animals that were raised under unexceptional conditions. The frequencies of SOAE were distributed between 0.7 kHz and 2.3 kHz (average: 1.16 kHz). The emission signals disappeared with hypoxia and with furosemide injection, and recovered after certain periods with some frequency fluctuations. Their electrical correlates that behaved in exactly the same manner as SOAE were clearly distinguished by FFT analysis of the AC potential from the cochlear surface. The signals could be divided into 4 types: (1) emission with pure and stable frequency spectrum and steady level, (2) emission with stable frequency and fluctuating level, (3) emission alternating between two closely situated frequency points, (4) emission having an irregular, wide spectrum band. Light microscopy revealed no abnormalities in the organ of Corti that have been widely accepted to be SOAE-related, i.e., patchy lesions of the organ of Corti and/or irregularities in hair cell arrangement. This suggests that the spontaneous oscillation within the guinea pig cochlea can be generated by some minor, functional, and probably reversible disturbances of the active cochlear transduction mechanism, without major structural derangement of the organ of Corti.

Acoustics↗
PubMed1991

Mechanisms of stimulation of collagen synthesis during chick embryonic skin development.

To study how collagen synthesis is regulated in developing chick embryonic skin, hydroxyproline synthesis, incorporation of proline, and translational activity and content of collagen mRNA in 12-, 15-, and 18-day skins were determined and compared with each other. Hydroxyproline synthesis in the 18-day skins was markedly increased over that in the 12-day skins, whereas proline incorporation was moderately increased. The increase in collagen synthesis from day 15 to 18 was accompanied by increases in both the translational activity and the content of type I procollagen mRNA, with a selective increase in the lower-molecular-weight species of pro alpha 1 (I) collagen mRNA. In contrast, the stimulation of collagen synthesis from day 12 to day 15 did not parallel the levels of type I procollagen mRNA. These results suggest that the stimulation of collagen synthesis is regulated by collagen mRNA levels only in the later stage of development (from day 15 to day 18). Both the collagen synthesis and type I procollagen mRNA levels in the fibroblasts isolated on each corresponding day were constant. The difference in collagen synthesis under two different culture conditions suggests that cell-matrix interaction and/or some serum factors, including several growth factors, are essential for the marked stimulation of collagen synthesis observed in 12- to 18-day skin.

Actins↗
PubMed1991

Secretion of a unique collagen by spontaneously transformed murine keratinocytes (PAM cells) in vitro.

A spontaneously transformed murine keratinocyte cell line (PAM cell) was found to secrete two nondisulfide-linked collagenous polypeptides with apparent molecular weight (MW) 190-kd and 120-kd. Pulse-chase experiments indicated that the 190-kd polypeptide was secreted into the culture medium in 2 h and processed to the 120-kd collagen component within 4 h. This process was inhibited by EDTA. The 120-kd polypeptide was sensitive to pepsin, and a 50-kd fragment was produced by a mild pepsin treatment at 4 degrees C. A cyanogen bromide peptide map of the 120-kd polypeptide was distinct from that of types I, II, III, IV, and V collagens. These properties indicate similarities to the type VIII-related collagen produced by human astrocytoma cells. The secretion of the collagen rapidly reached a maximum level on the first day of culture and subsequently declined with cell proliferation. An accelerated processing to the 120-kd polypeptide was observed under culture conditions of high cell density. Similar collagens were also found to be produced by normal human keratinocytes. These results indicate that the 120-kd polypeptide is a potentially functional protein that may participate in the formation of the extracellular matrix of keratinocytes.

Animals↗
PubMed1991

Effect of cyproterone acetate on active and inactive renin secretion in patients with precocious puberty and genetic short stature.

To evaluate the effect of cyproterone acetate (CA) on the renin-angiotensin-aldosterone axis, we measured the plasma active, inactive and total renin concentrations (PARC, PIRC and PTRC) during and after CA treatment in patients with precocious puberty and genetic short stature. CA was administered at a daily dose of 150-170 mg/m2 in all subjects. PARC and PTRC were measured by immunoradiometric assays. During CA treatment, PARC, PIRC, PTRC and the PARC/PTRC ratio were significantly decreased. The plasma renin activity, measured by enzymatic assay, and the plasma aldosterone concentration were also decreased. After CA discontinuation, all of these were increased immediately along normal ranges. PARC closely correlated with plasma renin activity. These results suggest that CA produces mineralocorticoid action and suppresses the production and activation of renin.

Androgen Antagonists↗
PubMed1991

Gene encoding a putative zinc finger protein in Synechocystis PCC6803.

A 5.5-kb HindIII fragment of Synechocystis PCC6803 containing a liverwort (ORF316) homolog encoding a putative zinc finger protein was cloned. Nucleotide sequence analysis showed that the homology of the amino acid sequence deduced from the ORF326 of Synechocystis PCC6803 with the counterparts of a liverwort and tobacco was 50% and 46%, respectively. Synechocystis ORF326 also showed 38% homology with the dedB gene in Escherichia coli. The gene organization of the region in these species of organisms was quite different. This suggests that the Synechocystis ORF326 and liverwort ORF316 genes may be related to a common regulatory gene, but not photosynthetic gene characteristic to chloroplasts.

Amino Acid Sequence↗