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Biomedical subjects

H Aoki

Publications and source records attributed to H Aoki.

At least 37 records · Page 2Linked to original sources
PubMed1997

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis.

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.

Chromosomes, Bacterial↗
PubMed1997

Interlamellar waters in dimyristoylphosphatidylethanolamine-water system as studied by calorimetry and X-ray diffraction.

The number of water molecules incorporated into the interlamellar region in a gel phase of dimyristoylphosphatidylethanolamine (DMPE)-water system containing up to about 40 g% water was estimated by techniques of calorimetry and X-ray diffraction. The calorimetric estimation based upon enthalpy changes of deconvoluted ice-melting peaks revealed that bulk water existing outside lipid bilayers begins to appear although the gel phase is not fully hydrated. The gel phase showed a linear depression of its transition temperature proportional to the amount of freezable waters interposed between bilayers. For a fully hydrated gel phase, the numbers of non-freezable and freezable interlamellar waters estimated by calorimetric analysis were about 2.3 and 3.7 molecules per lipid, respectively. The limiting, total number of interlamellar waters, 6 H2O/lipid, agreed with that estimated from both the X-ray diffraction data and the absolute specific volume for a DMPE molecule. Furthermore, the analysis for the lamellar intensity data is also consistent with the result of calorimetric analysis.

Calorimetry, Differential Scanning↗
PubMed1997

Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload.

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the alpha-skeletal actin promoter 1.7-fold whereas the alpha-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1. 6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.

Animals↗
PubMed1997

Molecular cloning and heterologous expression of the isopullulanase gene from Aspergillus niger A.T.C.C. 9642.

Isopullulanase (IPU) from Aspergillus niger A.T.C.C. (American Type Culture Collection) 9642 hydrolyses pullulan to isopanose. IPU is important for the production of isopanose and is used in the structural analysis of oligosaccharides with alpha-1,4 and alpha-1,6 glucosidic linkages. We have isolated the ipuA gene encoding IPU from the filamentous fungi A. niger A.T.C.C. 9642. The ipuA gene encodes an open reading frame of 1695 bp (564 amino acids). IPU contained a signal sequence of 19 amino acids, and the molecular mass of the mature form was calculated to be 59 kDa. IPU has no amino-acid-sequence similarity with the other pullulan-hydrolysing enzymes, which are pullulanase, neopullulanase and glucoamylase. However, IPU showed a high amino-acid-sequence similarity with dextranases from Penicillium minioluteum (61%) and Arthrobacter sp. (56%). When the ipuA gene was expressed in Aspergillus oryzae, the expressed protein (recombinant IPU) had IPU activity and was immunologically reactive with antibodies raised against native IPU. The substrate specificity, thermostability and pH profile of recombinant IPU were identical with those of the native enzyme, but recombinant IPU (90 kDa) was larger than the native enzyme (69-71 kDa). After deglycosylation with peptide-N-glycosidase F, the deglycosylated recombinant IPU had the same molecular mass as deglycosylated native enzyme (59 kDa). This result suggests that the carbohydrate chain of recombinant IPU differed from that of the native enzyme.

Amino Acid Sequence↗
PubMed1997

Identification of two closely related genes, inducible and noninducible carbonyl reductases in the rat ovary.

Two closely related cDNAs encoding carbonyl reductase (CR) were isolated from the rat ovary and testis. One (inducible CR, iCR) was rapidly and strongly induced in the ovary by pregnant mare serum gonadotropin (PMSG), whereas the other (non-inducible CR, nCR) was constitutively expressed in the ovary and was not induced by PMSG. Both genes were also expressed in the rat testis. The cDNAs of rat iCR and nCR encode highly homologous proteins (86% identity in amino acid) with 277 and 276 amino acid residues, respectively. However, the 5'-upstream regions flanking respective genes are completely unrelated to each other except for a short (38 bp) overlap. These results indicate the presence of two closely related but differently regulated CR genes in rat gonadal tissues. Strong induction of iCR by PMSG suggests the importance of the gene in the ovarian follicular development.

Alcohol Oxidoreductases↗
PubMed1997

Low serum cholesterol in suicide attempters.

Previous studies have shown an association between low serum cholesterol concentration and suicide; however, conflicting results have also been reported. To examine this potential association, cholesterol levels in 99 patients admitted to an emergency ward following an attempted suicide were compared with those in 74 nonsuicidal psychiatric inpatients, and those in 39 psychiatrically normal individuals with accidental injuries. Cholesterol concentrations in suicide attempters were found to be significantly lower compared with both psychiatric and normal controls, when sex, age, psychiatric diagnosis, and physical conditions (serum total protein and red blood cell count) were adjusted for. This significant relationship was observed in mood disorders and personality or neurotic disorders, but not in schizophrenia spectrum disorders. These results support the previous claim that lower cholesterol level is associated with an increased risk of suicidal behavior.

Adult↗
PubMed1997

Transient expression of M-CSF is important for osteoclast-like cell differentiation in a monocytic leukemia cell line.

Cells of U937, a human monocytic leukemia cell line, differentiate into macrophages by treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA), whereas cells treated with 1 alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3] continue to grow without undergoing differentiation. When U937 cells were successively treated with TPA and 1,25-(OH)2D3, tartrate-resistant acid phosphatase-positive multinucleated cells appeared at 5 days after the treatment. These osteoclast-like cells released a soluble form of 45Ca from 45Ca-labeled bone particles. These cells were not formed when the order of treatment with TPA and 1,25-(OH)2D3 was reversed. Use of either dexamethasone or interferon-gamma (IFN-gamma) was effective in inhibiting the formation of these osteoclast-like cells. The expression of c-src, c-fms, and macrophage colony stimulating factor (M-CSF) was induced by TPA treatment; however, TPA-induced M-CSF gene transcription was attenuated by the subsequent addition of 1,25-(OH)2D3. Furthermore, both dexamethasone and IFN-gamma impaired the attenuation of M-CSF expression, suggesting that the transient expression of M-CSF may be important for the formation of osteoclast-like cells.

Acid Phosphatase↗
PubMed1997

Synovial sarcoma of the hand.

We report on a 23-year-old woman with a poorly differentiated synovial sarcoma on the palm of her hand which presented as a painless nodule. The MRI findings are presented along with the clinical and pathologic features. Because of similar morphological features, these tumors can be confused with benign lesions such as aggressive fibromatosis or ganglion cysts, especially when very small. The possibility of a synovial sarcoma mimicking a benign lesion needs to be considered when the mass does not have an unequivocal benign diagnosis on MR imaging. Following wide resection and reconstruction, our patient has been disease free with good function for 28 months.

Adult↗
PubMed1997

Reproduction of excursive tooth contact in an articulator with computerized axiography data.

PURPOSE: This study assessed the reproduction of excursive tooth contacts with a SAM2 "P" articulator set up with the aid of computerized axiography. MATERIAL AND METHODS: Articulator excursive tooth contacts were compared with intraoral excursive contacts identified with occlusogram wax. The maxillary casts were mounted with a kinematic face-bow, and the mandibular cast was oriented to the intercuspal position. The articulator was set using information obtained from computerized axiography. Protrusion and laterotrusion to the left and right sides were examined. The first 4 mm of excursive tooth contacts in the occlusogram were compared with the 4 mm of excursive tooth contacts in the articulator. RESULTS: The articulator reproduced 82% of the teeth with protrusive tooth contacts and 90% of the teeth with laterotrusive tooth contacts. The exact locations of excursive tooth contacts were reproduced in 66% of protrusive contacts and 81% of laterotrusive contacts. The articulator also created additional eccentric dental contacts that were not originally present in the occlusogram. Twenty percent of the subjects showed these additional contacts during protrusion; in lateral movement of the articulator these additional contacts were present in 27% to the left side and 20% to the right. Clinically, these findings suggest that there are limits to the ability of the articulator to reproduce excursive tooth contacts. These limitations should be kept in mind when an articulator is used for diagnostic and restorative dental procedures.

Adolescent↗
PubMed1997

Molecular characterization of the prokaryotic efp gene product involved in a peptidyltransferase reaction.

The translation factor EF-P is required for efficient prokaryotic peptide bond synthesis on 70S ribosomes from fMet-tRNAfMet. This protein has been purified from Escherichia coli cells and the gene, efp, encoding it has been cloned and sequenced. We have isolated recombinant clones which overexpress a protein that co-migrates with purified EF-P upon SDS-PAGE analysis. Using these clones, we report the purification, crystallization and initial characterization of the efp gene product. The mechanism by which EF-P stimulates peptide-bond synthesis was studied using several antibiotics that inhibit translocation, peptide-bond synthesis and decoding. The stimulation of peptidyltransferase by EF-P was not inhibited by antibiotics that affect translocation and occupation of the A site (in the elongation state), ie thiostrepton, viomycin, neomycin and fusidic acid but was inhibited by streptomycin as well as by inhibitors of peptidyltransferase, chloramphenicol and lincomycin. This observation and the requirement for L16 but not for the L7/L12 nor L6 or L11 r-proteins suggest that the binding site for EF-P may overlap the peptidyltransferase center of the ribosome.

Anti-Bacterial Agents↗
PubMed1997

Characterization of an interleukin 1 receptor antagonist protein released from ultraviolet-irradiated epidermal sheet.

An interleukin 1 (IL-1) inhibitor was detected in the culture supernatant of mouse epidermal sheet which had been irradiated with middle-wavelength ultraviolet (UVB) either in vitro or in vivo. Properties of the IL-1 inhibitor were consistent with those of IL-1 receptor antagonist (IL-1ra), i.e., approximate molecular size of 17 kDa by gel filtration, specific inhibition of IL-1-induced thymocyte proliferation, inhibition of binding of IL-1 to IL-1 receptor on the T-cell surface, and reactivity to anti-mouse IL-1ra antibody shown by immunoblotting and enzyme-linked immunosorbent assay (ELISA). These results indicate that this IL-1 inhibitor, which appears to be identical with IL-1ra, was released from the epidermis upon UVB irradiation, and that the inhibitor may participate in the regulation of inflammation mediated by IL-1, as well as in the pathogenesis of ultraviolet (UV)-induced immunosuppression.

Animals↗
PubMed1997

The promoter of an androgen dependent gene in the hamster flank organ.

Hamster flank organs are useful for studying androgen-dependent growth of hair follicles and sebaceous glands. A cDNA clone (FAR-17a) was isolated from the hamster flank organ, whose expression was highly sensitive to androgen. The mRNA level of this gene was reduced after castration but reappeared after testosterone treatment. To elucidate the mechanism of expression of this gene regulated by androgen we isolated a genomic clone, from a hamster genomic library, that includes the promoter and upsteam region. The promoter region was used to drive a luciferase reporter gene in Cos 7 cells. This construct was activated five to six times higher over a control plasmid lacking the promoter region. We tested the effects of testosterone by transfection of the reporter plasmid into androgen dependent SC-3 cells. The results showed up to fivefold stimulation after the addition of androgen. The sequence of this promoter region was analyzed and the transcription factor binding sites were predicted. Since no obvious androgen responsive elements were included in the promoter region, we suggest that the stimulation of the reporter construct has to be mediated indirectly by androgen-dependent transcription factor(s).

Amino Acid Sequence↗
PubMed1997

Analyses of human gliomas by restriction landmark genomic scanning.

The 16 primary gliomas were examined for changes in genomic DNA using a recently developed 2-dimensional gel electrophoresis method called restriction landmark genomic scanning (RLGS). This approach allows detection of DNA amplification, deletion, methylation and potentially other genetic rearrangements represented as decreases and increases in spot/fragment intensity on an autoradiogram. Approximately 2,000 landmark sites in tumor DNA were compared with those of DNA isolated from normal brain tissues. Seven spots showing intensified signal were consistently detected in at least 50% of tumors, implying activation of corresponding DNA sequences, and 8 additional spots having reduced signal were observed, again in more than 50% of all tumors, suggesting inactivation by the loss of 1 allele or homozygous deletion. Decreased signal may also infer relative CpG island methylation state. Of those spots consistently identified in tumors, 2 amplified and 4 reduced spots were found to be characteristic of low- and high-grade tumors, while the remaining 5 amplified and 4 reduced spots were associated with high-grade gliomas only, suggesting a link of specific mutations to degree of malignancy. A separate subset of glioblastomas evaluated, however, showed no alterations in these 'hot spots' which were detected in even low grade astrocytomas. The results demonstrate the genetic heterogeneity of glioblastoma and implicate the progression of neoplasia via differing genetic pathways.

Astrocytoma↗
PubMed1997

Elevated plasma levels of interleukin-1 receptor antagonist and interleukin-10 in patients with acute myocardial infarction.

The study was undertaken to measure plasma interleukin-1 (IL-1) receptor antagonist and IL-10 concentrations in patients with acute myocardial infarction and to analyze their relationship to the hemodynamics, severity, and prognosis of myocardial infarction in its acute stages. We attempted to define the kinetics of IL-1 receptor antagonist and IL-10 in patients with acute myocardial infarction (n = 34, age 42-91 years, mean 68 years). Plasma IL-1 receptor antagonist and IL-10 levels were measured by enzyme-linked immunosorbent assay. Patients in group A (n = 17) had uncomplicated acute myocardial infarction (Killip class I). Patients in group B (n = 17) had severe acute myocardial infarction (Killip class II, III, or IV). Peak Il-1 receptor antagonist and IL-10 levels in group B were significantly higher (p < 0.05) than those of group A. In group B, the peak IL-1 receptor antagonist levels were significantly correlated with white blood cell counts (r = 0.63, p = 0.006), pulmonary capillary wedge pressure (r = 0.78, p = 0.0002), and cardiac index (r = -0.51, p = 0.04). Peak IL-10 levels were significantly correlated with white blood cell counts (r = 0.60, p = 0.01), the pulmonary wedge pressure (r = 0.73, p = 0.0008), and cardiac index (r = -0.50, p = 0.04). Moreover, a significant correlation was found between the peak IL-1 receptor antagonist and IL-10 levels (r = 0.91, p < 0.0001). The peak IL-1 receptor antagonist levels in nonsurvivors (n = 13) were significantly higher (p < 0.01) than those in survivors (n = 21). The plasma IL-1 receptor antagonist and IL-10 levels were closely correlated with the severity of hemodynamics in acute myocardial infarction and with the clinical status of patients with severe acute myocardial infarction. Results suggest that plasma IL-1 receptor antagonist and IL-10 can serve as prognostic indicators in cases of sever acute myocardial infarction.

Adult↗
PubMed1997

Platelet function is inhibited by nitric oxide liberation during nitroglycerin-induced hypotension anaesthesia.

The inhibitory effects of nitroglycerin (NTG) on platelet function and the mechanisms of inhibition have been studied in vitro, but not in vivo. Therefore, we have investigated the effects of NTG on platelet function in eight patients undergoing orthopaedic surgery. Simultaneous measurements of platelet aggregation and change in intracellular calcium concentrations were performed in Fura-2 loaded platelets using thrombin as a stimulator. Intraplatelet concentrations of cyclic 3',5'-guanine monophosphate (cGMP) were measured by radioimmunoassay, and the concentration of nitrite ion was also measured. Continuous i.v. infusion of NTG 4-8 micrograms kg-1 min-1 significantly inhibited platelet aggregation and the increase in intracellular Ca2+ concentration (first phase, mean 439.9 (SEM 68.7) vs 210.6 (38.7) nmol litre-1; second phase, 154.4 (19.8) vs 106.7 (18.0) nmol litre-1). The concentration of cGMP (from 0.633 (0.098) to 1.764 (0.578) pmol/10(9) platelets) and the concentration of nitrite ion (from 532.6 (17.6) to 724.4 (34.8) nmol litre-1) also increased significantly after infusion of NTG. The NTG concentration in plasma was of the order of 10(-8) mol litre-1. We have demonstrated that in vivo, NTG increased intraplatelet cGMP concentrations and inhibited platelet function; one mechanisms of this effect is likely to be related to nitric oxide liberation from NTG bioconversion.

Adult↗
PubMed1997

Angiotensin II and serum differentially regulate expression of cyclins, activity of cyclin-dependent kinases, and phosphorylation of retinoblastoma gene product in neonatal cardiac myocytes.

The hypertrophic response in cardiac myocytes and the mitogenic response in other cell types share various early cellular responses. However, how the subsequent cell growth response, such as cell cycle machinery, is regulated in cardiac hypertrophy is not understood. Using cultured neonatal rat cardiac myocytes, we examined the effect of angiotensin II (Ang II), a hypertrophic stimulus, on mRNA and protein expression of cyclins and cyclin-dependent protein kinases (cdks), activity of cdks, and phosphorylation of retinoblastoma gene product (pRb). The effect of FCS, a stimulus that was previously reported to initiate both protein and DNA synthesis in cardiac myocytes, was also examined for comparison. Ang II activated cdk4 and caused phosphorylation of pRb, peaking at 12 hours, but subsequently downregulated cyclin D1, D3, and A expression and cdk2 activity. FCS increased the expression of G1-S cyclins, caused activation of cdk4, cdk2, and cdc2, and strongly phosphorylated pRb but failed to significantly stimulate DNA synthesis in neonatal cardiac myocytes. These results suggest that Ang II transiently activates but subsequently downregulates cell cycle regulators. Induction of G1 and G1-S cyclins and activation of cdks by FCS are not sufficient to drive cardiac myocytes into S phase. The functional role of pRb phosphorylation by Ang II and serum stimulation and, by inference, the subsequent liberation of E2F in terminally differentiated myocytes remain to be elucidated.

Angiotensin II↗