Search PubMed⌕ Search

Biomedical subjects

A Nakazawa

Publications and source records attributed to A Nakazawa.

At least 109 records · Page 6Linked to original sources
PubMed1991

Detection of human papillomavirus type 16 in sexual partners of patients having cervical cancer by polymerase chain reaction.

The polymerase chain reaction (PCR) was employed to detect human papillomavirus (HPV) 16 and 18 in cytological samples from the uterine cervix and in urine samples from the male consorts. HPV 16 was detected in 2 (25%) of 8 males whose wives were positive for HPV 16, while it was detected in only one (7%) of 14 consorts whose wives were negative for HPV 16 and 18. This is the first report of the detection of HPV 16 in urine. Viral detection in urine samples by the PCR method is a non-invasive, convenient and useful tool for large-scale epidemiologic studies and investigations of the mechanism of virus transmission.

Adenocarcinoma↗
PubMed1991

[Detection of HPV DNA in the uterine cervical lesions by polymerase chain reaction and in situ hybridization].

Human papillomavirus (HPV) is found in close association with carcinogenesis of the uterine cervix. We applied a new in vitro gene amplification technology, the polymerase chain reaction (PCR) to detect HPV 16 and 18 in cervical exfoliated cells. HPV infections were detected in 5 (16%) of 31 women with no pathological lesions of the uterine cervix (normal), 16 (24%) of 67 with cervical intraepithelial neoplasia (CIN) and 6 (38%) of 16 with invasive cervical cancer. Moreover, 10% formalin-fixed and paraffin-embedded tissue sections were prepared from the uterine cervix of these 27 women with PCR-proven HPV infection and were examined for the histological localization of HPV-DNA by in situ hybridization with biotin-labeled DNA probes of HPV types 6/11, 16/18 and 31/33/35. HPV-DNA type 16/18 was detected in 3 of 5 normal women, 2 of 4 CINs I, 2 of 3 CINs II, 6 of 9 CINs III and 6 of 6 invasive cervical cancers. HPV-DNA type 6/11 was detected in 6 of 6 condylomas. Viral DNA sequence was detected in the superficial cells of CIN I and II, and it was distributed through entire thickness layer of undifferentiated cells derived from CIN III and squamous cell carcinoma. In addition, the staining intensity became weak as the lesion progressed. These differences between lesions might be due to the difference in the viral form in the nuclei, ie whether an episomal or integrated form. Thus, an in situ hybridization technique with a biotin-labeled DNA probe as well as the PCR method is useful for the detection of HPV in clinical samples.

Base Sequence↗
PubMed1991

[Alcohol-induced variant angina and considerations of its mechanism: a case report].

A 64-year-old man had episodes of angina pectoris several hours after ingestion of alcohol. Otherwise, anginal attacks never occurred. He was diagnosed as having variant angina based on the typical ST elevation in leads II, III and aVF during the anginal attacks. We performed an alcohol challenge test on his 4th admission day. He was given 540 ml of "sake" at 6:00 p.m. and anginal attacks with ST elevations occurred 9.5 hours after its ingestion. The peak value of plasma ethanol was 136 mg/dl at 9:00 p.m. and it returned to 0 when angina occurred. By alcohol ingestion, urinary excretion of Mg increased in association with a slight decrease in serum Mg. The ratio of serum Ca to Mg was increased from 4.0 at the control state before taking alcohol to 4.5 at the occurrence of anginal attack. Mg content in red blood cells and in plasma catecholamines did not differ between before and after ingesting alcohol. We concluded that the change in the extracellular Ca-Mg equilibrium may contribute to the mechanism of alcohol-induced variant angina.

Aged↗
PubMed1990

Mechanism of adenylate kinase. Critical evaluation of the X-ray model and assignment of the AMP site.

The substrate binding sites of adenylate kinase (AK) proposed by X-ray crystallographic studies [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45, and subsequent revisions] were evaluated by site-specific mutagenesis in conjunction with structural analysis by NMR. The residues examined in this report include two near an adenosine site (threonine-39 and arginine-44) and two in the phosphate binding region (arginine-128 and arginine-149). The results and conclusions are summarized as follows: (a) Although Thr-39 is very close to an adenine site [Egner, U., Tomasselli, A. G., & Schulz, G. E. (1987) J. Mol. Biol. 195, 649-658], it is nonessential either structurally or functionally. (b) The R44M mutant enzyme showed significant increases in the Michaelis and dissociation constants of adenosine 5'-monophosphate (AMP) (36- and 22-fold, respectively) while all other kinetic parameters were relatively unperturbed. The proton NMR property of this mutant was unchanged in the free enzyme and only slightly perturbed in the binary complexes with AMP and with MgATP (adenosine 5'-triphosphate), and in the ternary complex with MgAP5A [P1,P5-bis(5'-adenosyl) pentaphosphate]. These results indicate that Arg-44 interacts specifically with AMP starting at the binary complex, and suggest that the MgATP site proposed by Pai et al. (1977) is likely to be the AMP site. (c) The kinetic parameters of R149M were dramatically perturbed: kcat decreased by a factor of 1540, Km increased to 130-fold, and kcat/Km decreased by a factor of 2 X 10(5).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1990

Upstream regulatory sequence for transcriptional activator XylR in the first operon of xylene metabolism on the TOL plasmid.

Transcription of the first operon coding for m-xylene-degrading enzymes on the TOL plasmid of Pseudomonas putida is activated by the xylR gene product in the presence of m-xylene. The operon has the consensus sequence of the ntr/nif promoters at -24 and -12 regions, and the transcription is dependent on an RNA polymerase containing a sigma factor NtrA (RpoN or sigma 54). Deletion derivatives of the upstream sequence of the operon promoter were made in vitro and connected with the xylE gene on a plasmid. Their promoter activities were analyzed in Escherichia coli by monitoring catechol 2,3-dioxygenase activity, the xylE gene product. A cis-acting DNA element was identified, which is required for activation of the operon promoter by XylR protein in the presence of the inducer. This regulatory sequence of about 40 base-pairs in length was located 150 base-pairs upstream from the transcription start site. Analysis of the mutants containing insertions between the upstream regulatory sequence and the promoter sequence demonstrated strong dependence of the activation upon helical periodicity of DNA. The regulatory sequence functioned in the inverse orientation or at a distance of more than 1 x 10(3) base-pairs upstream from the promoter though less efficient. These results indicated that this upstream regulatory sequence might be the binding site for XylR protein. DNA-loop formation through protein-protein interaction between XylR protein attached to the upstream sequence and the NtrA-containing RNA polymerase bound by the promoter sequence was suggested for activation of the operon transcription. A sequence similar to the regulatory sequence of the first operon of xylene metabolism was found in the upstream region of the xylS gene, which is also activated by XylR protein in the presence of m-xylene.

Base Sequence↗
PubMed1990

Isolation and characterization of the gene encoding bovine adenylate kinase isozyme 2.

Mitochondrial adenylate kinase isozyme 2 (AK2) exists in two isoforms, AK2A and AK2B, which have the same amino-acid sequence except for the C-terminal portion. We have isolated the gene encoding AK2 from a bovine genomic library. The gene covers about 25 kb and consists of seven exons and six introns. The nucleotide sequences from exon 1 to the 5' half of exon 6 encode the portion common to AK2A and AK2B, while the sequences of the 3' half of exon 6 and exon 7 direct the unique portions of AK2A and AK2B, respectively. Therefore, an alternative splicing mechanism is suggested in generating two types of mRNA encoding AK2A and AK2B. The 5'-flanking region of the gene lacks a TATA box, but contains three CAAT boxes. The G + C content of this region is high and eight copies of GC box are found. These features of the promoter region resemble those of 'housekeeping' genes. S1 mapping and primer extension analyses revealed multiple transcription start points. The 581-bp region just upstream from the start codon functions as a promoter in the expression of the cat gene in Chinese hamster ovary cells.

Adenylate Kinase↗
PubMed1990

Mechanism of adenylate kinase. Are the essential lysines essential?

Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli. The two residues were chosen since according to the nuclear magnetic resonance (NMR) model [Mildvan, A. S., & Fry, D. C. (1987) Adv. Enzymol. 58, 241-313], they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex. In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins. The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK. For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK. Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183. The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR. Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Kinase↗
PubMed1990

Gene structures of three vertebrate adenylate kinase isozymes.

Adenylate kinase is an ubiquitous enzyme which contributes to homeostasis of adenine nucleotide composition in the cell. In vertebrates, three isozymes (AK1, AK2, and AK3) are characterized which have distinct distribution in tissues as well as subcellular compartments. The genetic backgrounds of these adenylate kinase isozymes were analyzed. cDNA clones for AK1 were isolated from poly(A)+RNA of chicken skeletal muscle. The results of mRNA analysis in various tissues using the AK1 cDNA indicated that the AK1 gene expression is regulated both tissue-specifically and developmentally at the transcriptional level. The AK1 gene was cloned from chicken and human DNA and characterized. Both genes were split into seven exons. The intron positions in both genes coincided. cDNA clones for AK2 isolated from bovine liver poly(A)+RNA contained two types. One type (AK2A) encoded the same amino acid sequence as that reported for bovine heart AK2. The other type (AK2B) encoded the same sequence as AK2 except for the COOH terminus. The mRNA species corresponding to the two cDNA clones were identified in bovine liver and heart. Both the cDNA sequences were found to direct the active adenylate kinase synthesis in E. coli. The AK2 gene was cloned and characterized. It consisted of seven exons and six introns. From genomic structure analysis, the two cDNA species were shown to be derived from a single gene by the alternative splicing mechanism. Three types of cDNA clones for AK3 were isolated from bovine liver poly(A)+RNA, which contained the common AK3-coding region and different 3' portions. No NH2-terminal presequence of mitochondrial targeting was identified in AK3 from the sequencing and expression analyses of the cDNA. Upon expression of the cDNA sequence in E. coli, AK3 protein was recovered in the periplasmic space of the bacteria, indicating that AK3 without presequence was exported through the inner bacterial membrane as it is imported through the mitochondrial membranes. Internal targeting signals may be responsible for the translocation process. The AK3 gene was cloned and partially characterized. It is split into at least five exons. The comparisons of amino acid sequences and genomic structure of three isozymes revealed that a segment corresponding to either exon 5 of the AK2 gene or a part of exon 3 of the AK3 gene is missing in the AK1 gene. Phylogenetic analysis suggested that AK1, a shorter molecule, would have been separated from a longer molecule very early in evolution of adenylate kinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylate Kinase↗
PubMed1990

[Inhibition of vasospastic angina by alcohol ingestion].

A 66-year-old man having a long history of angina on effort has started to show frequent episodes of angina at rest since 6 months ago. He noticed that chest pain was uncommon after taking alcohol. A variant form of angina pectoris (variant angina) was diagnosed by documentation of typical ST elevation during anginal attack and also by inducing coronary arterial spasm with intracoronary administration of ergonovine maleate. Ambulatory ECG monitoring revealed frequent ST elevation during sleep. Since the history suggested that alcohol ingestion could be effective for preventing variant angina, this effect was examined by giving 540 ml of "sake" in the evening. Variant angina was inhibited, while plasma ethanol was detected. The plasma ethanol reached its peak value as 152 mg/dl at 10 o'clock pm and returned to zero after 12 hours. When ethanol disappeared in the plasma, variant angina recurred again. Although the precise mechanism for inhibition of variant angina by alcohol ingestion is not clear, alcohol or its metabolite such as acetaldehyde seems to be able to inhibit coronary arterial spasm.

Aged↗
PubMed1990

[Intermittent administration of the combination chemotherapy with cis-platin, adriamycin and cyclophosphamide (cyclic-PAC chemotherapy) for the ovarian cancers].

Despite cis-platin-based combination chemotherapy with initially high response rates in advanced ovarian cancer, the overall survival rate remains unsatisfactory. This prompted us to design a new systematic approach using a combination chemotherapy consisting of cis-platin, adriamycin and cyclophosphamide (PAC), namely cyclic-PAC chemotherapy: Three-step chemotherapies consisting of 3 courses of the PAC regimen in each step were administered for 18 months to patients with Stage Ic-IV, after cytoreductive surgery and chemotherapy. In the present study, the survival rate with the cyclic-PAC chemotherapy was compared to that with short course-PAC and FAM (5-Fu, Alkylating agent and Mitomycin C) chemotherapies. The cyclic-PAC, PAC and FAM groups included 24 cases, 31 cases and 30 cases, respectively. Treatment of Stage Ic-IV diseases by cyclic-PAC improved the outcome (66% 3-year survival rate) compared to PAC and FAM groups (PAC 35% and FAM 30% 3-year survival rates), while no difference was observed between PAC and FAM. The outcome for patients with Stage III. IV was also superior for the cyclic-PAC group compared to the PAC and FAM groups (cyclic-PAC 54%, PAC 18% and FAM 13% 3-year survival rates). Cyclic-PAC chemotherapy is capable of dramatically improving the long-term survival rate of ovarian cancer patients.

Adult↗
PubMed1989

Cloning and sequence analysis of the ntrA (rpoN) gene of Pseudomonas putida.

The gene encoding a sigma factor NtrA (RpoN) was cloned from Pseudomonas putida by cross-hybridization with a probe containing a part of the corresponding Escherichia coli gene. The cloned gene complemented an ntrA mutation of E. coli in activation of xyl genes on the TOL plasmid. The predicted amino acid (aa) sequence of P. putida NtrA (497 aa; Mr 56,215) is highly homologous to NtrA proteins from Azotobacter vinelandii (81.7%), Klebsiella pneumoniae (52.6%), and Rhizobium meliloti (36.1%). There are two other open reading frames (ORF1, ORF3) upstream and downstream from the ntrA gene, respectively. These ntrA-flanking ORFs are also highly homologous to the corresponding ORFs of K. pneumoniae, A. vinelandii, and R. meliloti.

Amino Acid Sequence↗
PubMed1989

Cloning and characterization of cDNA for mitochondrial GTP:AMP phosphotransferase of bovine liver.

Three different types of cDNA clones for mitochondrial GTP:AMP phosphotransferase (AK3) were isolated from a cDNA library of bovine liver poly(A)+ RNA. Nucleotide sequencing revealed that each of these clones consisted of a common 5'-untranslated region, a common AK3-coding sequence and a 3'-untranslated region with different sizes. By Northern blot analysis, three species of AK3 mRNA apparently corresponding to the isolated cDNA clones were detected, which would be a result of varying terminations and polyadenylations of the primary transcript. From comparison of the size of the product synthesized in vitro from the message directed by the isolated cDNA with that of the purified AK3 protein, AK3 appeared to have no cleavable NH2-terminal sequence as found in other mitochondrial proteins. The AK3 cDNA was expressed in Escherichia coli, which resulted in complementation of an adenylate kinase mutation of E. coli. The AK3 product was exported to the periplasmic space through the bacterial inner membrane. The possible involvement of the NH2-terminal sequence of the protein in targeting to the mitochondrial matrix was discussed.

Amino Acid Sequence↗
PubMed1989

Properties of a novel D-stereospecific aminopeptidase from Ochrobactrum anthropi.

A novel aminopeptidase active toward D-amino acid-containing peptides, D-amino acid amides, and D-amino acid esters has been purified 2,800-fold to homogeneity from a bacterium Ochrobactrum anthropi SCRC C1-38, which had been isolated from soil. The enzyme has a molecular weight of about 122,000 and is composed of two identical subunits (Mr = 59,000). The optimal pH for activity was 8.0. It showed strict D-stereospecificity toward substrates including low molecular weight D-amino acid amides such as D-alanine amide, D-alpha-aminobutyric acid amides, and D-serine amide; D-alanine N-alkylamides such as D-alanine-p-nitroanilide, D-alanine benzylamide, and D-alanine n-butylamide; and peptides with a D-alanine at the NH2 terminus such as D-alanylglycine, D-alanylglycylglycine, D-alanyl-L-alanyl-L-alanine, and D-alanine oligomers. Generally, the enzyme did not act on substrates composed of L-amino acid at the NH2 terminus, although it showed low stereospecificity only toward substrates such as the methyl esters of L-alanine, L-serine, and L-alanine-p-nitroanilide. Comparing the Km and Vmax values for the major substrates, it is clear that the enzyme prefers peptides to amino acid arylamides or amino acid amides. The enzyme was tentatively named as "D-aminopeptidase." EDTA and divalent cations have no effect on the enzyme activity. The enzyme appears to be a thiol peptidase.

Alcaligenes↗
PubMed1989

A new D-stereospecific amino acid amidase from Ochrobactrum anthropi.

A new D-stereospecific amino acid amidase has been partially purified from Ochrobactrum anthropi SCRC SV3, which had been isolated and selected from soil. The Mr of the enzyme was estimated to be about 38,000, and its isoelectric point was 5.3. The enzyme catalyzes the stereospecific hydrolysis of D-amino acid amide to yield D-amino acid and ammonia. The major substrates included D-phenylalanine amide, D-tyrosine amide, D-tryptophan amide, D-leucine amide, and D-alanine amide.

Alcaligenes↗
PubMed1989

Human adenylate kinase deficiency associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of the cytosolic adenylate kinase.

Adenylate kinase deficiency in the erythrocyte is a rare genetic disorder associated with hemolytic anemia. To determine the molecular basis of this disorder, we first cloned the normal gene encoding human cytosolic adenylate kinase (AK1) and determined the structure. The gene was 12 kilobase pairs long and was split into 7 exons. The structures of 5'- and 3'-flanking regions were determined by primer extension and RNA blot analysis. The results showed that two species of mRNA with 0.9 and 2.5 kilobases, which differed at the 3'-end portion, were generated by the AK1 gene. Alu sequences were found in the largest intron (intron 5) and in the noncoding region of exon 7. Next, both alleles of the AK1 gene were cloned from DNA of a patient bearing the adenylate kinase deficiency and their nucleotide sequences determined. A transition (C----T) was found in exon 6 on an allele, which resulted in an Arg to Trp (CGG----TGG) substitution at the 128th residue of AK1. Since chicken AK1 is highly homologous to human AK1 with respect to the amino acid sequence, we introduced an Arg to Trp substitution to chicken AK1 at the same position by oligodeoxynucleotide-directed mutagenesis. The mutant chicken AK1 expressed in Escherichia coli showed a reduced catalytic activity as well as a decreased solubility and a change in affinity to phosphocellulose. Thus it was considered that the observed C----T transition was a cause of the decreased AK1 activity of the patient's erythrocyte. Analysis on phosphocellulose chromatography of erythrocyte AK1 of the patient and parents revealed that the patient's mutant allele was derived from the mother.

Adenylate Kinase↗
PubMed1989

Decrease of anion selectivity caused by mutation of Thr501 and Gly502 to Glu in the hydrophobic domain of the colicin E1 channel.

Structure-function relations of the colicin E1 ion channel were studied through the effects of mutations in the 35-residue hydrophobic region of the channel polypeptide and neighboring residues in the channel domain. Mutation of neutral residues threonine 501 and glycine 502 to a more polar or charged glutamic acid generated a protein whose channel conductance properties in each case had a decreased selectivity for anions. There was no significant effect on ion selectivity caused by mutations that changed residue charge outside the hydrophobic domain at the neighboring aspartic acid 509 or at glycine 439. The Thr501----Glu and Gly502----Glu mutants possessed lower cytotoxic and in vitro activity. An altered thermolysin cleavage pattern and a greater binding to membrane vesicles at pH greater than 4.5 of the Gly502----Glu mutant indicated greater exposure of its COOH-terminal hydrophobic domain in solution. It is concluded that the hydrophobic nature of threonine 501 and glycine 502 is important in the structure of the channel lumen and the soluble colicin. Altering proline 462, a residue conserved in five sequenced channel-forming colicins, had no significant effect on channel properties. These conclusions are discussed in the context of sequence-structure-function concepts for channel proteins.

Amino Acid Sequence↗