Biomedical subjects

T Yamazaki

Publications and source records attributed to T Yamazaki.

At least 433 records · Page 24Linked to original sources

PubMed1995

Solution structure of the activator contact domain of the RNA polymerase alpha subunit.

The structure of the carboxyl-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alpha CTD), which is regarded as the contact site for transcription activator proteins and for the promoter UP element, was determined by nuclear magnetic resonance spectroscopy. Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins. The UP element binding site was found on the surface comprising helix 1, the amino-terminal end of helix 4, and the preceding loop. Mutation experiments indicated that the contact sites for transcription activator proteins are also on the same surface.

Base Sequence↗

PubMed1995

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup.

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920-1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.

Animals↗

PubMed1995

Nitrite reductase from the magnetotactic bacterium Magnetospirillum magnetotacticum. A novel cytochrome cd1 with Fe(II):nitrite oxidoreductase activity.

Cytochrome cd1 nitrite reductase was isolated from magnetite-containing cells of the magnetotactic bacterium Magnetospirillum (formerly Aquaspirillum) magnetotacticum, which was microaerobically cultivated under denitrifying conditions. The enzyme showed absorption maxima at 643 nm and 409 nm in the oxidized form, and at 663, 551, 522, and 418 nm in the reduced form. A distinctive split absorption band did not occur at about 550 nm. The pyridine ferrohemochrome spectra suggested the presence of heme c and heme d1 in the molecule. The enzyme was composed of two identical subunits each with a molecular mass of 54 kDa; each subunit contained one c-type and one d-type heme. The isoelectric point was 9.2. The redox potentials of heme c and heme d1 were estimated to be +191 mV and +180 mV, respectively. Although the enzyme showed cyanide-sensitive N,N,N',N'-tetramethyl-p-phenylenediamine-O2 oxidoreductase activity and N,N,N',N'-tetramethyl-p-phenylenediamine-nitrite oxidoreductase activity, the enzyme did not oxidize M. magnetotacticum ferrocytochrome c-550 and Pseudomonas aeruginosa ferrocytochrome c-551 in the presence of nitrite. Furthermore, sodium succinate did not cause the reduction of cytochrome cd1 in the crude cell-free extract prepared from the magnetite-containing bacterial cells. However, M. magnetotacticum cytochrome cd1 showed a novel Fe(II):nitrite oxidoreductase activity whereas P. aeruginosa cytochromes cd1 had no Fe(II):nitrite oxidoreductase activity. These results suggest that M. magnetotacticum cytochrome cd1 may function as a Fe(II)-oxidizing enzyme under microaerobic conditions using nitrite as electron acceptor.

Cytochrome c Group↗

PubMed1995

Structural and dynamic characterization of the phosphotyrosine binding region of a Src homology 2 domain--phosphopeptide complex by NMR relaxation, proton exchange, and chemical shift approaches.

Arginine side chains are often involved in protein--protein and protein--nucleic acid interactions. Due to a number of factors, resonance assignment and detection of NOEs involving the arginine side chains via standard NMR techniques can be difficult. We present here an approach to characterization of the interaction between a phosphopeptide (pY1021) and four arginine residues that line the phosphotyrosine-binding pocket of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2). Previously published [Pascal, S. M., et al. (1994) Cell 77, 461] NOE data provide a partial description of this interaction, including contacts between the aliphatic region of Arg 59 and the phosphotyrosine (pTyr) aromatic ring. Further characterization has now been accomplished by using 15N and 13C NMR relaxation studies of arginine N episilon and C zeta spins, respectively, and proton exchange rates of arginine H episilon nuclei. Differences between the chemical shifts of the arginine guanidino groups of the free SH2 domain in imidazole and phosphate buffers or in complex with pY1021 have provided insight into specific interactions with the phosphate and the aromatic ring of the pTyr. The resulting data are consistent with the most stable hydrogen bonds to phosphate donated by the Arg 39 epsilon-NH and the two Arg 37 eta-NH2 groups and with pTyr aromatic ring interactions involving the Arg 39 and possibly the Arg 18 guanidino groups.

Amino Acid Sequence↗

PubMed1995

Routine high-performance liquid chromatographic determination of myocardial interstitial norepinephrine.

The present study describes a high-performance liquid chromatographic-electrochemical detection (HPLC-ED) system for routine measurement of the low levels of norepinephrine (NE) found in the myocardial interstitial space. In this system, an in vivo detection limit of 100 fg in a 50-microliters injection was achieved for NE. Using cardiac dialysis technique, 20-microliters dialysates were sampled from the myocardial interstitial space at 2-min intervals. The basal dialysate NE concentration was 16.6 +/- 4.0 pg/ml. This low detection limit allowed the dialysate NE concentration to be monitored for dysfunction of the cardiac sympathetic nerve terminal. This system offers a new possibility for routine analysis of myocardial interstitial NE levels.

Animals↗

PubMed1995

Initial spectroscopic characterization of the ciliate photoreceptor stentorin.

Stentorin serves as the primary photosensor in the single cell ciliate, Stentor coeruleus, for its photophobic and phototactic response to light of visible wavelengths. We separated two subunits, stentorin-2A and -2B, from the previous stentorin complex ('stentorin-2') of greater than half a million molecular mass isolated from the photoreceptor organelle (pigment granule). Stentorin-2B bears the chromophore covalently linked to an approx. 50 kDa apoprotein, as determined by SDS-urea-PAGE. Partial amino acid sequences were obtained from this 50 kDa subunit. Its visible and CD spectra were found to be similar to those of stentorin-2. The steady-state and time-resolved fluorescence spectra of stentorin-2B, in H2O and D2O buffers, were also similar to those of stentorin-2. This suggests that the 50 kDa subunit retains the spectral integrity and primary photoreactivity of the stentorin-complex. The picosecond time-resolved fluorescence study revealed that the short picosecond emission component (tau F approximately equal to 8-10 ps) was the predominant emitting species in stentorin-2B and -2, followed by longer decaying species. No deuterium solvent effect was seen in this fast-decaying species. The possible mechanism for the primary photoreaction appears to involve electron transfer coupled with proton transfer.

Amino Acids↗

PubMed1995

Incorporation of bovine adrenal 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase into phospholipid vesicles.

3 beta-Hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD/I) and cytochrome P-450C21 were co-purified from bovine adrenocortical microsomes by an improved method. The 3 beta-HSD/I was successfully incorporated into liposomal membranes in which the enzyme activity was greatly stabilized. Enzymatic activities and kinetic parameters of the 3 beta-HSD/I proteoliposomes were almost the same as those of the solubilized form.

17-alpha-Hydroxypregnenolone↗

PubMed1995

Induction of G1 arrest by down-regulation of cyclin D3 in T cell hybridomas.

The relationship between activation-induced growth inhibition and regulation of the cell cycle progression was investigated in T cell hybridomas by studying the function of the cell cycle-regulating genes such as G1 cyclins and their associated kinases. Activation of T cell hybridomas by anti-T cell receptor antibody induces growth arrest at G1 phase of the cell cycle and subsequently results in activation-driven cell death. Rapid reduction of both messenger RNA and protein level of the cyclin D3 is accompanied by growth arrest upon activation. Although the residual cyclin D3 protein forms a complex with cdk4 protein, cyclin D3-dependent kinase activity is severely impaired. Stable transfectants engineered to express cyclin D3 override the growth arrest upon activation. These results imply that the activation signal through T cell receptor induces the down-regulation of cyclin D3 expression and cyclin D3-dependent kinase activity, leading to growth arrest in G1 phase of the cell cycle in T cells.

Animals↗

PubMed1995

Stretching mesangial cells stimulates tyrosine phosphorylation of focal adhesion kinase pp125FAK.

Mechanical loading of cultured mesangial cells has been shown to induce both increased production of matrix proteins and cell proliferation. This in vitro phenomenon has been implicated in the progression of glomerular sclerosis observed in a variety of glomerular diseases. However, it is not yet known how cells sense mechanical stress and transfer this sense into biochemical and biological events. In this study, we show that mesangial stretching rapidly stimulates tyrosine phosphorylation of non-receptor tyrosine kinase pp125FAK. Since pp125FAK is a major constituent of focal adhesions, our findings suggest that the focal adhesion may be a site where mechanical forces are translated into biochemical events, and that pp125FAK may play an important role in this signaling cascade.

Animals↗

PubMed1995

Avian neurosteroids. I. Pregnenolone biosynthesis in the quail brain.

The present study was carried out to determine steroid biosynthesis from cholesterol in the brain of adult male Japanese quails. As an initial step of the experiments, the concentrations of pregnenolone, dehydroepiandrosterone and their sulfate esters in the brain and plasma extracts were measured by specific radioimmunoassays (RIAs). To exclude the possibility that these steroids in the brain are derived from peripheral steroidogenic glands, hypophysectomized and sham-operated birds were used in this experiment. The pregnenolone concentration was much larger in the brain than in the plasma in these two groups. Hypophysectomy led to decreases in the plasma and brain pregnenolone concentrations, but the change in the brain was less pronounced than that in the plasma. Although pregnenolone sulfate ester, dehydroepiandrosterone and its sulfate ester were also detected in brain extracts, those levels were low in both hypophysectomized and sham-operated birds. The biochemical demonstration of cholesterol metabolism was further conducted in intact mitochondria. When mitochondrial fractions obtained from the whole brain were incubated with cholesterol at 37 degrees C, the pregnenolone level in mitochondria increased as a function of incubation time. Finally, Western immunoblot analysis using a purified IgG with polyclonal antibodies against the purified bovine adrenal cytochrome P450scc was performed after SDS-gel electrophoresis of homogenates of the hypothalamus and preoptic area. A protein recognized the antibody as a band of electrophoretic mobility in the proximity of reference bovine P450scc. These results suggest that the brain produces pregnenolone from cholesterol in adult male Japanese quails. Most accumulation of pregnenolone in the brain may be independent of the peripheral endocrine gland system.

Animals↗

PubMed1995

Avian neurosteroids. II. Localization of a cytochrome P450scc-like substance in the quail brain.

We have recently demonstrated that the avian brain as well as the peripheral steroidogenic glands produces pregnenolone, the main precursor of steroid hormones, on the basis of biochemical studies. Therefore, the immunohistochemical and Western immunoblotting analyses with a polyclonal antibody directed against the purified bovine adrenal cytochrome P450scc, which is involved in pregnenolone formation, was undertaken to investigate the localization of a cytochrome P450scc-like substance in the brain of adult male Japanese quails. P450scc-like immunoreactive cells were distributed in several telencephalic, diencephalic and mesencephalic regions. An intense immunoreaction was observed in somata of Purkinje cells and in the dendrites extending through the cerebellar molecular layer. Clusters of immunoreactive cell bodies were also detected in the hyperstriatum accessorium (HA), the ventral portions of both the archistriatum and the corticoid area, the preoptic area (POA), the anterior hypothalamus (AHy) and the dorsolateral thalamus (DL). Preadsorbing the antibody with cytochrome P450scc resulted in a complete absence of P450scc-like immunoreactivity in all of positively stained cells. On Western immunoblotting with the P450scc antibody, a P450scc-like substance was present in homogenates of the several brain regions that contain immunohistochemically stained cells. Immunohistochemical experiments using both antibodies against P450scc and the glial fibrillary acidic protein (GFA), a specific marker of glial cells, indicated that many HA cells contained both P450scc-like and GFA-like immunoreactivities. However, no immunoreactivity for GFA was observed in Purkinje cells and the cells localized in the ventral portion of the corticoid area, despite the presence of P450scc-like immunoreactivity. These results confirm our previous findings of pregnenolone biosynthesis and suggest the presence of a P450scc-like substance in both neuronal and glial cells of the quail brain.

Animals↗

PubMed1995

The molecular analysis of brown eye color mutations isolated from geographically discrete populations of Drosophila melanogaster.

A large proportion of spontaneous mutations in Drosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessive brown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caught Drosophila melanogaster females, bw mutations were isolated from seven separate geographic sites distributed among Japan, California. Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in the bw gene. The remainder are inseparable from wild-type bw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneous bw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.

Alleles↗

PubMed1995

P450scc-dependent cholesterol metabolism in rat adrenal mitochondria is inhibited by low concentrations of matrix Ca2+.

Pregnenolone formation at P450scc in isolated rat adrenal mitochondria is determined equally by the amount of ACTH-stimulated reactive inner membrane cholesterol and by the matrix generation of NADPH. Evidence is presented here that both are sensitive to an increase in matrix Ca2+ produced by low levels of external Ca2+ (0.4-4 microM). Cholesterol availability to P450scc and intramitochondrial NADPH are shown to be highly interdependent. The proportion of mitochondrial cholesterol which is readily available to P450scc in the intact mitochondria increases as conditions become more favorable for NADPH generation. Preincubation of mitochondria without reductant causes substantial decreases in cholesterol metabolism when weaker reducing conditions are used without effect on NADPH generation as evidenced by unchanged metabolism of 20 alpha-hydroxycholesterol at P450scc or DOC at P450(11) beta. Increased Ca2+free not only increases this sensitivity to preincubation but also inhibits reductant transfer from succinate and isocitrate to both P450scc and P450(11) beta. Succinate activity was inhibited much more than that of isocitrate. These actions of Ca2+, which quantitatively explain the stimulatory characteristics of EGTA in standard media, were reversible providing exposure was short (< or = 3 min). Ruthenium red (inhibits Ca2+ uptake antiporter) is as effective as EGTA in preventing all these effects of Ca2+ while cyclosporin A (prevents opening of Ca(2+)-activated inner membrane channels) is partially effective. Ca2+ entry into the matrix is, therefore a necessary step prior to inhibition of cholesterol metabolism by several mechanisms, including the consequences of changes in inner membrane permeability.

Adenosine Diphosphate↗

PubMed1995

Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein.

Many signal transduction processes are mediated by the binding of Scr-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins. Although most SH2-pTyr interactions occur between two different types of molecules, some appear to involve only a single molecular type. It has been proposed that the enzymatic activity and substrate recognition of the Src-family kinases, and the protein-binding and transforming activity of Crk-family adaptor proteins, are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding activity of Stat transcription factors seems to be regulated by SH2-mediated homodimerization. Here we examine the phosphorylated and non-phosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively) using a combination of physical techniques. The Crk protein contains a single SH2 domain and two SH3 domains in the order SH2-SH3-SH3. There is a tyrosine-phosphorylation site between the two SH3 domains at residue 221 which is phosphorylated in vivo by the Abl tyrosine kinase. Using NMR spectroscopic analysis, we show here that the SH2 domain of purified p-mCrk is bound to pTyr, and by hydrodynamic measurements that the phosphorylated protein is monomeric. These results provide direct demonstration of an intramolecular SH2-pTyr interaction in a signalling molecule.

Amino Acid Sequence↗

PubMed1995

Molecular characterization of galactosemia (type 1) mutations in Japanese.

We characterized two novel mutations of the galactose-1-phosphate uridyltransferase (GALT) gene in two Japanese patients with GALT deficiency and identified N314D and R333W mutations, previously found in Caucasians. One novel missense mutation was an G-to-A transition in exon 8, resulting in the substitution of arginine by histidine at the codon 231 (R231H). GALT activity of the R231H mutant construct was reduced to 15% of normal controls in a COS cell expression system. The other was a splicing mutation, an A-to-G transition at the 38th nucleotide in exon 3 (318A-->G), resulting in a 38-bp deletion in the GALT cDNA by activating a cryptic splice acceptor site. In seven Japanese families (14 alleles for classic form and one allele for Duarte variant) with GALT deficiency, the R231H and 318A-->G mutations were found only on both alleles of the proband. The N314D and R333W mutations were found on one allele each. The Q188R was prevalent in the United States but not in Japanese patients. The N314D mutation was associated with the Duarte variant in Japanese persons, as well as in the United States. We speculate that classic galactosemia mutations appear to differ between Japanese and Caucasian patients. Our limited data set on galactosemia mutations in Japanese suggests that the N314D GALT mutation encoding the Duarte variant arose before Asian and Caucasian people diverged and that classic galactosemia mutations arose and/or accumulated after the divergence of Asian and Caucasian populations.

Amino Acid Sequence↗

PubMed1995

Evolution of the response patterns to dietary carbohydrates and the developmental differentiation of gene expression of alpha-amylase in Drosophila.

Intraspecific variation of alpha-amylase activity in D. melanogaster and D. immigrans, which is distantly related to D. melanogaster, and interspecific variation of alpha-amylase activity in 18 Drosophila species were examined. The amount of intraspecific variation of alpha-amylase activities measured in terms of coefficient of variation in D. melanogaster and D. immigrans was one-half and one-tenth or less, respectively, of the interspecific variation in 18 Drosophila species. We also surveyed the response patterns of alpha-amylase activity to dietary carbohydrates at the larval and adult stages. The levels of alpha-amylase activity depended on both repression by dietary glucose (glucose repression) and induction by dietary starch (starch induction). In general, our data suggest that glucose repression was conserved among species at both stages while starch induction was mainly observed in larvae, although the degree of the response depended on species. In D. lebanonensis lebanonensis and D. serrata, larvae expressed electrophoretically different alpha-amylase variants (isozymes) from those of adult flies. These results may suggest that the regulatory systems responsible both for the response to environment and developmental expression are different among species in Drosophila.

Animals↗

PubMed1995

MR findings of avulsive cortical irregularity of the distal femur.

Avulsive cortical irregularity, a benign condition occurring only among children and adolescents, has been known to simulate malignancy not only radiologically but also microscopically. Therefore, in addition to plain radiographs, further studies including by magnetic resonance (MR) imaging may occasionally be required. MR images of seven cases of avulsive cortical irregularity of the femur were reviewed. In all cases, the lesion appeared hypointense on T1-weighted images and hyperintense on T2-weighted images, with a dark rim on both sequences at or near the sites of the bony attachment of the medial head of the gastrocnemius muscle. In all cases, bilateral involvement was demonstrated by plain radiography, computed tomography, and/or MR imaging. The authors suggest that avulsive cortical irregularity involves both femora much more frequently than has been reported previously.

Adolescent↗

PubMed1995

Metabolism of exogenous cholesterol by rat adrenal mitochondria is stimulated equally by physiological levels of free Ca2+ and by GTP.

Adrenal mitochondria metabolize cholesterol at inner membrane (IM) cytochrome P450scc. Exogenous and outer membrane (OM) cholesterol are metabolized more slowly due to a limiting transfer of cholesterol from OM to IM. This process is stimulated by in vivo ACTH treatment and inhibited by cycloheximide (CX)-induced depletion of labile regulatory proteins. In isolated rat adrenal mitochondria, GTP enhances the metabolism of exogenous cholesterol, consistent with enhanced intermembrane cholesterol transfer (Xu et al. (1989) J. Biol Chem. 264, 17674), but metabolism of 20 alpha-hydroxycholesterol, which readily traverses mitochondrial membranes, is not affected. The non-hydrolyzable analog, GTP gamma S, completely inhibits the activation of cholesterol metabolism by GTP, suggesting a requirement for GTP hydrolysis. Low concentrations of Ca2+ (0.4-4 microM) stimulate two independent cholesterol transport processes. For exogenous cholesterol, a Ca(2+)-mediated process can replace GTP since each produces comparable stimulation and the combination produces little additional activity. This Ca2+ stimulation is insensitive to GTP gamma S and also to Ruthenium Red (RR), which prevents Ca2+ entry into the matrix. Ca2+ also enhances availability to P450 scc of endogenous OM cholesterol, which accumulates during in vivo CX-inhibition. This stimulation is, however, distinguished by insensitivity to GTP and complete inhibition by RR. Ca2+, therefore, enhances intermembrane transfer of exogenous cholesterol from OM without entry into the matrix through a process which is independently stimulated by GTP. Ca2+ induces transfer of endogenous OM cholesterol through a completely different mechanism involving RR-inhibited matrix changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗