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A Nakazawa

Publications and source records attributed to A Nakazawa.

At least 55 records · Page 3Linked to original sources
PubMed1998

Isolation and characterization of mutated mitochondrial GTP:AMP phosphotransferase.

GTP:AMP phosphotransferase (adenylate kinase isozyme 3, AK3) mutants were obtained by using the ability of AK3 to complement a temperature-sensitive mutation of Escherichia coli adenylate kinase (AKe). Five mutants, P16L, G19S, G91D, G91S, and P93L, had mutation sites located at two loops that are involved in substrate binding of the enzyme. P16L and G19S bearing changes at the first loop showed reduced affinity for both GTP and AMP, the extent of reduction being slightly higher for GTP than AMP. In contrast, G91S and P93L having alterations at the second loop had lower affinities for AMP. Only the alterations at the second loop strongly influenced the Vmax value of the enzyme. Another mutant, D163N, had a substitution at the site forming a salt bridge in adenylate kinase isozyme 1 (AK1), which influenced the Vmax as well as the Km values for both substrates. The kinetic characteristics of these mutants were comparable to those of the corresponding AK1 or AKe mutants. Furthermore, from the results of mutations T201P and T201A that had alterations in all the kinetic parameters of AK3 and from a comparison with the structure and the kinetic parameters of AKe, we expect that a residue(s) around Thr201 is involved in recognition of the base of nucleoside triphosphate.

Adenylate Kinase↗
PubMed1998

Molecular cloning and characterization of the gene encoding human NeuroD.

NeuroD is a basic helix-loop-helix transcription factor that binds to an E-box sequence, CANNTG, in the target gene promoter region. The gene encoding NeuroD is expressed specifically in brain, intestine, and pancreatic cells and plays a pivotal role in tissue-specific differentiation. To investigate the regulation of human NeuroD gene expression, we isolated and sequenced a genomic DNA containing two exons and flanking regions of the NeuroD gene. The sizes of exons 1 and 2 were 168 and 2404 bp, respectively. CAT reporter analysis showed that the elements responsible for basal promoter activity lay in the proximal 408-bp region. Cotransfection of the CAT reporter plasmids with a NeuroD expression plasmid resulted in 5-fold enhancement of the CAT activity, indicating an autoregulatory mechanism is involved in human NeuroD gene expression.

Amino Acid Sequence↗
PubMed1998

cDNA cloning and tissue-specific expression of the gene encoding human adenylate kinase isozyme 2.

We isolated two kinds of cDNAs encoding human adenylate kinase (AK) isozyme 2 from a HeLa cell cDNA library using bovine AK2 cDNA as a probe. Nucleotide sequencing revealed that the cDNAs encoded 239- and 232-amino acid proteins with deduced molecular mass of 26.5 (AK2A) and 25.6kDa (AK2B), respectively. Northern blot analysis demonstrated that AK2 mRNA is strongly expressed in liver, heart, skeletal muscle and pancreas, and moderately in kidney, placenta and brain, and weakly in lung. However, Western blot analysis showed that AK2 protein was present in large amounts in liver, heart, kidney, and in a small amount in lung, and undetectable in brain and skeletal muscle. These results suggested the presence of the tissue-specific gene-expression including post-transcriptional regulation in expression of the AK2 gene.

Adenylate Kinase↗
PubMed1998

The rpoS gene regulates OP2, an operon for the lower pathway of xylene catabolism on the TOL plasmid, and the stress response in Pseudomonas putida mt-2.

Operon OP2 on the Pseudomonas putida TOL plasmid encodes enzymes for m-toluate catabolism; transcription of this operon is activated by XylS in the presence of m-toluate. Because transcriptional activation of OP2 specifically occurs in the stationary phase of growth both in P. putida and in Escherichia coli, we suspected that its transcription is dependent on RpoS (sigmaS). Therefore, we constructed a rpoS disruption strain of P. putida mt-2, and assayed OP2 expression and other phenotypes. OP2 transcription was dependent on rpoS, indicating that the stationary-phase specific activation of OP2 is due to positive regulation by RpoS in P. putida mt-2. The rpoS mutant exhibited reduced viability during the stationary phase and was sensitive to high salt concentrations and H2O2. P. putida mt-2 has two catalase isozymes, KatA and KatB. Expression of the katB gene was specific to the stationary phase and entirely dependent on the rpoS gene, while the katA gene expressed during log phase partially required rpoS. There were no significant changes in tolerance to high temperature or UV light in the rpoS mutant. No difference was observed between the E. coli rpoS mutants and their parents in their capacity to activate OP2, suggesting that the mechanism of OP2 activation in the stationary phase of growth differs between P. putida and E. coli.

Bacterial Proteins↗
PubMed1998

Increase of adenylate kinase isozyme 1 protein during neuronal differentiation in mouse embryonal carcinoma P19 cells and in rat brain primary cultured cells.

Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.

Adenylate Kinase↗
PubMed1998

Protein binding in vivo to OP2 promoter of the Pseudomonas putida TOL plasmid.

The transcription of OP2 encoding enzymes for m-toluate catabolism on the Pseudomonas putida TOL plasmid is activated by basal-level XylS protein in the presence of m-toluate or by overproduced XylS protein in the absence of m-toluate. In this study, in vivo dimethyl sulfate (DMS) footprinting was performed to understand the mechanism of transcriptional regulation of OP2 promoter by XylS. In the presence of overproduced XylS without m-toluate, several protected nucleotides were observed, indicating the binding of RNA polymerase to DNA. However, the protection was canceled upon addition of m-toluate. These results suggest that RNA polymerase is retained by XylS on the OP2 promoter in the absence of inducer, and is released by m-toluate binding to XylS, concomitant with transcription.

Bacterial Proteins↗
PubMed1998

Mechanism of mitochondrial import of adenylate kinase isozymes.

Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of the cellular adenine nucleotide composition. Three isozymes, AK1, AK2, and AK3, have so far been characterized in vertebrates. They are located in different tissues, while their primary and tertiary structures are similar. Among them, AK2 and AK3 are located in mitochondria, but unlike most mitochondrial proteins, both proteins lack a cleavable presequence. In this study, we first confirmed that AK2 is distributed in liver cells in both the cytosol and the intermembrane space of mitochondria, while AK3 is localized exclusively in the mitochondrial matrix. Next, we analyzed the process of import of AK2 and AK3 by incubating isolated rat mitochondria with proteins that were synthesized in a reticulocyte lysate translation system. The results indicated that both AK2 (an intermembrane-space-targeting protein) and AK3 (a matrix-targeting protein) require an inner membrane electrochemical potential for their import. This finding for AK2 is in contrast with those of other noncleavable intermembrane-space-targeting proteins such as cytochrome c and cytochrome c heme lyase, which do not require the membrane potential for their import. In the transport process, AK2 and AK3 competed with the adrenodoxin precursor, which is imported into the matrix through a mechanism common to other mitochondrial matrix proteins. Thus, AK2 and AK3 were thought to be translocated into mitochondria through the same pathway as that for most mitochondrial protein precursors. Neither AK2, that was previously synthesized in reticulocyte lysates, nor AK2, that was purified from an Escherichia coli overexpression system, was imported into mitochondria in a post-translational import manner. In contrast, AK3 was imported into mitochondria after completion of protein synthesis. Thus, the import of AK2 is likely to be co-translational, and the co-translational import mechanism might contribute to the bi-topological distribution of AK2 in both the cytosol and mitochondria.

Adenylate Kinase↗
PubMed1998

Immunohistochemical localization of peroxisomal enzymes in developing rat kidney tissues.

We studied the developmental changes in the localization of peroxisome-specific enzymes in rat kidney tissues from embryonic Day 16 to postnatal Week 10 by immunoblot analysis and immunohistochemistry, using antibodies for the peroxisomal enzymes catalase, d-amino acid oxidase, l-alpha-hydroxyacid oxidase (isozyme B), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein. Peroxisomal enzymes were detected in the neonatal kidney by immunoblot analysis and their amount increased with kidney development. By light microscopic immunohistochemistry, they were first localized in a few proximal tubules in the juxtamedullary cortex of 18-day embryos. The distribution of proximal tubules positive for them expanded towards the superficial cortex with development. The full thickness of the cortex became positive for the staining by 14 days after birth. Peroxisomes could be detected by electron microscopy in structurally immature proximal tubules in 18-day embryos. Their size increased and the ultrastructure of subcompartments became clear with continuing development of proximal tubules. These results show that peroxisomal enzymes appear in the immature proximal tubules in the kidney of embryos and that the ultrastructure of the peroxisomes and localization of the peroxisomal enzymes develop along with the maturation of proximal tubules and kidney tissues.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1998

[Antimicrobial activities of cefcapene against clinical isolates from respiratory tract infections of outpatients].

In order to evaluate antimicrobial activity of cefcapene (CFPN), minimum inhibitory concentrations (MICs) of CFPN and reference drugs were determined against clinical isolates from respiratory tract infection of out patients that were obtained in our laboratory from January to June of 1997. The results are summarized as follows; 1. The MIC90 of CFPN against penicillin (PC)-susceptible Streptococcus pneumoniae (PSSP) was equal to those of benzylpenicillin (PCG), ampicillin (ABPC) and cefditoren (CDTR), and was lower than those of cefaclor (CCL), cefdinir (CFDN) and erythromycin (EM). 2. The MIC90 of CFPN against PC-intermediate S. pneumoniae (PISP)/PC-resistant S. pneumoniae (PRSP) was equal to that of CDTR, and was lower than those of PCG, ABPC, CCL, CFDN and EM. CFPN showing strong antimicrobial activities against PISP. 3. CFPN showed strong antimicrobial activities against beta-lactamase producing and non-producing Haemophilus influenzae. The MIC90 of CFPN was stronger than those of ABPC, CCL, CFDN and EM, and was approximately equal to that of CDTR. CFPN also showed strong antimicrobial activities against strains which did not produce any beta-lactamase and were resistant to CCL with MIC of > or = 25 micrograms/ml. 4. Antimicrobial activities of CFPN against Moraxella subgenus Branhamella catarrhalis was stronger than that of ABPC and CCL, though the MIC90 of CFPN was rather high, 3.13 micrograms/ml. 5. CFPN showed strong antimicrobial activities against PISP and beta-lactamase producing H. influenzae, and also against the CCL-resistant H. influenzae indicative mutations of penicillin-binding proteins (PBPs). From those results, cefcapen-pivoxil was found to be clinically effective against community acquired respiratory tract infection.

Cephalosporins↗
PubMed1998

[Antimicrobial activity of ofloxacin against recent clinical isolates from otitis media and otitis externa].

In order to evaluate the annual changes of susceptibility, minimum inhibitory concentrations (MICs) of ofloxacin (OFLX) and 4 control drugs were determined against clinical isolates that were obtained from patients with otitis media and otitis externa during the periods between January and December 1993, and the periods between October 1996 and March 1997. The results are summarized as follows: 1. No annual changes were seen for MIC50 of OFLX, but MIC80 and MIC90 of that rose against methicillin-resistant Staphylococcus aureus (MRSA), coagulase-negative staphylococci (CNS) and Pseudomonas aeruginosa from 1993 to 1996. It appears that resistance to OFLX is increasing among these bacteria. Detection frequency of highly resistant strains to OFLX (MIC > 100 micrograms/ml) was lower than to other control drugs. 2. No annual changes were seen of MIC50, MIC80 and MIC90 of OFLX against methicillin-susceptible S. aureus (MSSA), Streptococcus spp., Proteus spp. and Haemophilus influenzae. OFLX showed strong antimicrobial activities against these bacteria. 3. Since there was no large annual changes in the antimicrobial activity of OFLX against clinical isolates that were obtained from patients with otitis media and otitis externa, OFLX otic solution was considered as one of the clinically useful drugs even now.

Anti-Infective Agents↗
PubMed1997

Differences in sarcoplasmic reticulum gene expression in myocardium from patients undergoing cardiac surgery. Quantification of steady-state levels of messenger RNA using the reverse transcription-polymerase chain reaction.

Little is known about any alterations in sarcoplasmic reticulum (SR) gene expression associated with cardiac diseases of varying degrees of severity. We assessed, using the reverse transcription-polymerase chain reaction (RT-PCR) technique, SR Ca2+ transport protein gene expression in small tissue samples from failing hearts in patients undergoing cardiac surgery. Total RNA was extracted from 30- to 50-mg samples from the hearts of 13 patients with coronary artery disease, congenital heart disease, or valvular heart disease. We used RT-PCR to synthesize and amplify cDNA encoding cardiac SR Ca(2+)-ATPase, ryanodine receptor (RYR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amount of each mRNA in the sample was expressed relative to the amount of GAPDH mRNA. The expression level of each mRNA was correlated with the cardiac functional index. The mRNA levels for Ca(2+)-ATPase and RYR varied between heart samples, but showed a positive correlation with left ventricular ejection fraction. Ca(2+)-ATPase mRNA levels showed in inverse relationship with plasma brain natriuretic peptide. In addition, we isolated partial cDNA encoding a human cardiac RYR. The cDNA consisted of 487 nucleotides, and the nucleotide and deduced amino acid sequences showed 93% and 99% homology, respectively, to those of rabbit cardiac RYR. These results suggest that decreased levels of mRNA for SR Ca2+ transport protein could be related to abnormal cardiac function, regardless of the etiology of the heart disease. RT-PCR provides a rapid and economical way of quantifying the expression of multiple genes in small specimens and may, therefore, aid understanding of the pathophysiology and treatment of heart disease.

Adolescent↗
PubMed1997

Wild-type p53 gene-induced morphological changes and growth suppression in hepatoma cells.

The human hepatocellular carcinoma (HCC) cell line, HLF, expresses only mutant-type p53 (mt-p53), which has an amino acid substitution at the 244th residue from glycine to alanine. HLF cells were transfected with wild-type p53 (wt-p53) cDNA construct pC53-SN3, mt-p53 cDNA construct pC53-SCX [which differs by a single nucleotide, resulting in alanine instead of valine at the 143rd residue in p53 (p53-143)], or pCMV-Neo-Bam, as a control, by a liposome method. After G418 selection, three wt-p53 stable transformants (WT), four mt-p53 transformants (MT), and three control vector transformants (VT) were obtained. We analyzed the cell growth and morphological changes of these transformants under different culture conditions [fetal calf serum (FCS), 10%, 1%, and 0%]. Whereas no difference from control in the growth rate and morphology was observed under the 10% FCS conditions, serum starvation induced remarkable phenotypical changes in all three WTs, but not in the other transformant. Corresponding to these phenotypical changes, the transcriptional activity of wt-p53 was increased more than nine fold. These results indicated that serum starvation would induce wt-p53 biological function, which is tightly linked to morphological changes and growth suppression. To induce these changes, the introduction of the wt-p53 gene itself was not sufficient, and additional triggering, i.e., serum starvation, was indispensable.

Carcinoma, Hepatocellular↗
PubMed1997

Targeting human breast tumour in xeno-grafted SCID mice with 99Tcm-glucarate.

99Tcm-glucarate accumulation in human mammary BT-20 tumours hosted in severe combined immune deficiency (SCID) mice was compared to 111In-monoclonal antibody 103D2-F(ab')2, 99Tcm-methoxyisobutyl isonitrile (99Tcm-MIBI) and 99Tcm-diethylenetriamine pentaacetate (99Tcm-DTPA). The intracellular tumour distribution of 99Tcm-glucarate was also determined. SCID mice injected with a mixture of 99Tcm-glucarate and 111In-103D2-F(ab')2 were imaged serially up until 24 h. Computer planimetered tumour-to-blood activity (in the heart) ratios (T/BH) to 8 h were significantly greater for 99Tcm-glucarate than 111In-103D2. The mean (+/-S.D.) tumour-to-blood ratio (T/B) from biodistribution was 1.21 +/- 0.31 and 0.35 +/- 0.06 (P < 0.0001) at 5 h, and 1.526 +/- 0.29 and 0.75 +/- 0.2 (P < 0.0001) at 8 h, for 99Tcm-glucarate and 111In-103D2 respectively. At 24 h, T/B for 111In-103D2 (1.76 +/- 0.22) exceeded that of 99Tcm-glucarate (1.44 +/- 0.2, P = 0.01). 99Tcm-glucarate uptake in the tumours at 5 h (1.133 +/- 0.25 %ID g-1) and 8 h (1.213 +/- 0.23 %ID g-1) was significantly greater than that of 99Tcm-MIBI (0.340 +/- 0.09, P = 0.0002; 0.220 +/- 0.04, P = 0.0001) and 99Tcm-DTPA (0.091 +/- 0.03, P < 0.0002; 0.016 +/- 0.01, P < 0.0001) respectively. Intracellular tumour distribution of 99Tcm-glucarate was 50.91 +/- 6.55% in the nuclear fraction, 34.34 +/- 2.88% in the cytoplasmic fraction and 14.75 +/- 7.66% in the mitochondrial fraction. Thus glucarate may provide a 99Tcm-based mammary tumour imaging modality for visualization of tumours very quickly after tracer administration with maximal targeting in the nuclei of the tumour cells.

Animals↗
PubMed1997

Increased blood plasminogen activator inhibitor-1 and intercellular adhesion molecule-1 as possible risk factors of atherosclerosis in Werner syndrome.

Werner syndrome is a rare premature aging syndrome accompanied by severe atherosclerosis. The etiology of atherosclerosis is suspected to be due to its complications, namely diabetes mellitus, hyperinsulinemia and hyperlipidemia. But from an autopsy case we found that some other risk factors may be involved in the mechanism of atherosclerosis in this syndrome. Previously we revealed that the plasminogen activator inhibitor-1 (PAI-1) gene was being overexpressed in skin fibroblasts from a patient with this syndrome. PAI-1 is a potent inhibitor of tissue plasminogen activator and a possible risk factor of atherosclerosis. This led us to assess the plasma concentration of PAI-1. Our working hypothesis was that the PAI-1 gene was upregulated or not fully suppressed in cells responsible for the production of PAI-1 in plasma as well as in fibroblasts. The results show a high concentration of plasma PAI-1. One of the well-known physiological substances that induce the PAI-1 gene is tumor necrosis factor-alpha, which also induces other possible risk factors of atherosclerosis, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1. We found the serum concentrations of ICAM-1 to be elevated in patients with this syndrome. We conclude that high concentrations of PAI-1 and ICAM-1 in blood may be one of the potent causes of severe atherosclerosis in Werner syndrome.

Adult↗
PubMed1997

[Antimicrobial activities of arbekacin against methicillin-resistant Staphylococcus aureus].

In order to evaluate antimicrobial activity of arbekacin (ABK), coagulase-type and minimum inhibitory concentrations (MICs) of ABK and other drugs were determined against 700 strains of methicillin-resistant Staphylococcus aureus (MRSA) that were obtained in our laboratory from 1990 to 1996, 7 years. The results are summarized as follows; 1. The MIC-distributions of ABK against 100 strains of MRSA obtained yearly did not show stochastically significant differences. 2. The coagulase-type distributions showed differences over the years. Coagulase-type II strains increased and type IV strains decreased, and MIC-distributions of ABK and other drugs were different according to coagulase-types. 3. The detection frequencies of ABK-resistant strains (MIC of ABK: > or = 12.5 micrograms/ml) were 2.0 approximately 8.0% through the years. The frequency reported in 1980 was equal to the frequency obtained in 1992 approximately 1993 in a nationwide survey. Coagulase-types II, IV and VII that were ABK-resistant strains were frequently obtained, and most of ABK-resistant strains were also highly resistant to gentamicin.

Aminoglycosides↗
PubMed1997

[Clinical and bacteriological effects of cefetamet pivoxil against community-acquired respiratory tract infections. Part II].

We investigated clinical and bacteriological effects of cefetamet pivoxil (CEMT-PI) in community-acquired respiratory tract infections and obtained the following findings. That method was approximately equal to that of investigation in 1994. 1. Of the 431 respiratory tract infection cases that were treated with CEMT-PI according to a same protocol at a total of 41 institutions in Tokyo, Kanagawa-ken, Saitama-ken and Chiba-ken from January to the beginning of March 1996. Outpatients accounted for 98.1% of the subjects. Regarding genders to patients, slightly more females (52.6%) than males were included. Diagnoses given to these patients included pharyngo-laryngitis (53.5%), tonsillitis (20.4%) and acute bronchitis (19.1%). 2. We investigated clinical efficacy rates (the ratio of those excellent + good) classified by diseases. The improvement rates of pharyngo-laryngitis, tonsillitis and acute bronchitis were more than 85.0%. Other cases were small in number. That of chronic bronchitis-acute increasing change for the worse was 66.7%, pneumonia was 50.0% and bronchiectasis infection was 16.7%. It was not studied that clinical efficacy rates among those who were treated with 1 CEMT-PI tablet twice and among those who were given 2 tablets twice were significant level. 3. For the bacteriological study, a written material describing the method of collecting specimens, storage and transport in detail was distributed to the above mentioned institutions. The isolation and identification of suspected causative bacteria, determination of minimum inhibitory concentrations (MICs) and investigation of beta-lactamase production were conducted all together at section of studies, Tokyo Clinical Research Center. Suspected causative bacteria were detected from 274 (63.6%) cases. They included 88 strains of Haemophilus influenzae, 47 strains of Streptococcus pneumoniae, 42 strains of Streptococcus pyogenes, 20 strains of Moraxella subgenus Branhamella catarrhalis and 17 strains of Klebsiella pneumoniae subsp. pneumoniae. Suspected causative bacteria classified by diseases were S. pyogenes (tonsillitis), S. pneumoniae (acute bronchitis and secondary infection of chronic respiratory infection) and H. influenzae (pharyngo-laryngitis), and the detection frequency of those was high. The clinical efficacies (the ratio of improvement) classified by suspected causative bacteria were 84.4% against organism that was indicating CEMT and were 69.2% against organism that was not indicating CEMT.

Adult↗