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Biomedical subjects

A Nakazawa

Publications and source records attributed to A Nakazawa.

At least 91 records · Page 5Linked to original sources
PubMed1994

Analysis of DNA bend structure of promoter regulatory regions of xylene-metabolizing genes on the Pseudomonas TOL plasmid.

The transcription of both the upper operon (OP1) coding for m-xylene-degrading enzymes and the positive regulatory gene xylS on the TOL plasmid depends on sigma 54-RNA polymerase and requires the activator protein XylR that binds to the cis-acting upstream regulatory sequence of each promoter. For transcription of OP1 in Escherichia coli, integration host factor (IHF) is also required. IHF binds to DNA between the upstream regulatory sequence and the promoter sequence of OP1. We showed that IHF induced a DNA bend in the promoter regulatory region of OP1 upon its binding, supporting the DNA-loop model for the activation of OP1 transcription. In contrast to OP1, the transcriptional activation of xylS does not require IHF. In the absence of IHF, the promoter regulatory region of xylS promoter was shown to have a weak but significant intrinsic DNA bend, which may be involved in stabilizing the DNA-loop structure for the activation of xylS transcription. When IHF was highly produced in E. coli, the xylS transcription was repressed. We found two weak binding sites for IHF, each overlapping with the promoter sequence and the upstream regulatory sequence. The IHF binding to these sites might result in repression of xylS expression by overproduced IHF. Evidence is presented that binding of IHF to these sites induces a DNA bend.

Bacterial Proteins↗
PubMed1993

Thiamin-triphosphate-synthesizing activity of mutant cytosolic adenylate kinases: significance of Arg-128 for substrate specificity.

The thiamin triphosphate (TTP)-synthesizing activity and the ATP-synthesizing activity of two mutant enzymes of chicken cytosolic adenylate kinase whose Arg-128 was substituted by Trp (cAK1(Trp)) or Ala (cAK1(Ala)) were compared to those of the wild-type enzyme. The TTP-synthesizing activity of both the mutant enzymes was higher due to higher affinity to thiamin diphosphate (cAK1(Trp)) or a larger Vmax (cAK1(Ala)). The optimal pH shifted to pH 9.0 from pH 10.5. The ATP-synthesizing activity of both the mutant enzymes was, on the other hand, markedly decreased with lower affinity for ADP and lower Vmax. These results suggest that Arg-128 plays an important role in the substrate specificity of the cytosolic adenylate kinase.

Adenylate Kinase↗
PubMed1993

Overproduction and purification of the colicin E1 immunity protein.

Colicin E1 immunity protein encoded by the plasmid colicin E1 was overproduced in Escherichia coli from an expression plasmid which was constructed by placing the immunity protein-encoding sequence downstream of the tac promoter and by properly positioning the ribosome binding site on a run-away replication vector. The immunity protein was solubilized with 0.5% Brij 58 from the membrane fraction and purified to homogeneity by a simple batch procedure with hydroxyapatite gel and reverse-phase chromatography. A 15-residue NH2-terminal amino acid sequence was determined to be the same as that deduced from the DNA sequence. The effect of the purified immunity protein on membranes was tested in vitro using solute-loaded liposomes. The immunity protein added to the liposomes showed a small but significant channel or lytic activity that is an indicator of its hydrophobic nature.

Base Sequence↗
PubMed1993

Tissue-specific and developmentally regulated expression of the genes encoding adenylate kinase isozymes.

Adenylate kinase (AK) is known to play an important role in homeostasis of adenine nucleotide metabolism. We isolated cDNAs for rat AK isozymes (AK1, AK2, and AK3), determined their mRNAs in rat tissues by Northern blot analysis, and measured the isozyme activities. Tissue-dependent activities of AK1 and AK2 paralleled the contents of mRNAs. Tissues with high AK1 levels showed low AK2 levels and vice versa, suggesting that tissue-specific expressions of the AK1 and AK2 genes are inversely regulated. AK3 mRNA was detected in most tissues examined, suggesting that AK3 gene expression is constitutive. We further examined developmental changes in mRNAs and enzyme activities of AK isozymes in rat skeletal muscle and liver. In the skeletal muscle, AK1 and AK3 activities started to increase at around the weaning period. AK1 mRNA accumulated at the prenatal stage and further increased during development, while AK3 mRNA was at high levels during the fetal stage and remained fairly constant during development. In the liver, AK2 and AK3 activities started to increase after birth and were further elevated during growth, whereas their mRNAs were present at relatively high levels throughout development. The physiological meanings of the tissue-specific expression of the AK isozyme genes are discussed.

Adenylate Kinase↗
PubMed1992

Analysis of an upstream regulatory sequence required for activation of the regulatory gene xylS in xylene metabolism directed by the TOL plasmid of Pseudomonas putida.

Transcription from the promoter of a positive regulatory gene, xylS, on the TOL plasmid of Pseudomonas putida is activated by another positive regulator, XylR, in the presence of m-xylene and is dependent on RNA polymerase containing the NtrA protein (sigma 54). Deletion analysis of the upstream region of the xylS gene revealed an upstream regulatory sequence (URS), located between 145 and 188 bp upstream from the transcription start site. The URS is active in either orientation and can be placed 3.9 kb further upstream without loss of activity. Dependence of activation on helical periodicity was observed in the region between the URS and the promoter of the xylS gene, suggesting DNA loop formation between these two sites, which are located about 100 bp apart. The expression of xylR was autogenously repressed by XylR protein. This autogenous repression is decreased in an NtrA- background, irrespective of the presence of the xylS promoter in cis, indicating that NtrA protein, or NtrA-containing RNA polymerase that is not bound to the xylS promoter, is involved in the binding of XylR protein to the URS.

Bacterial Proteins↗
PubMed1992

Detection of human papillomavirus types 16 and 18 in the exfoliated cervical cells using the polymerase chain reaction.

We applied the polymerase chain reaction (PCR) to detect HPV 16 and 18 in cytological samples obtained from the uterine cervices of Japanese women. HPV infection was detected in 17 (25%) of 67 with CIN and 11 (37%) of 30 with cervical carcinoma. It is notable that 11 (16%) of 69 women with normal cervices were infected with either HPV 16 or 18. The polymerase chain reaction is sensitive and useful for epidemiological studies.

Adolescent↗
PubMed1992

Sialyl-Tn, sialyl-Lewis Xi, CA 19-9, CA 125, carcinoembryonic antigen, and tissue polypeptide antigen in differentiating ovarian cancer from benign tumors.

Serum sialyl-Tn, sialyl-Lewis Xi, CA 19-9, CA 125, carcinoembryonic antigen (CEA), and tissue polypeptide antigen were measured in 65 women with early-stage ovarian cancer (45 stage I and 20 stage II cases) and 317 with benign pelvic masses. As a single assay, sialyl-Tn showed the best sensitivity and specificity, 46 and 92%, respectively. CA 19-9 detected the greatest number of cancer patients but had the lowest specificity. The combination of sialyl-Tn, CA 125, tissue polypeptide antigen, and CEA seemed to perform the best, with a sensitivity and specificity of 71 and 76%, respectively. The combination of sialyl-Tn, CA 125, and tissue polypeptide antigen gave similar results and may be more cost-effective. However, one-fifth of the patients with early-stage cancer still showed up as false negatives even with use of the six markers in combination. Approaches other than serum assay alone will be needed to detect all malignant pelvic masses at an early stage.

Antigens, Neoplasm↗
PubMed1992

Microchromosomal assignment of the chicken ovotransferrin and adenylate kinase genes.

Ovotransferrin, an egg-white protein implicated in the transfer of trace elements from the hen oviduct to the developing avian embryo, and cytosolic adenylate kinase, an essential enzyme involved in the interconversion of adenine nucleotides in energetically active tissues, have been mapped to two separate chicken microchromosomes by fluorescent in situ hybridization. Considering present and previous data, the possibility of loss of intron material resulting in the compactation of genes in chicken microchromosomes is briefly discussed.

Adenylate Kinase↗
PubMed1992

[Immunohistochemical study of proliferating cell nuclear antigen (PCNA) in gynecological tumors and their related lesions].

We have investigated whether monoclonal antibody (PC10) of proliferating cell nuclear antigen (PCNA) could be useful as a marker of proliferating cells within formalin-fixed, paraffin-embedded tissue sections of 140 gynecological tumors and their related lesions. There was a positive correlation (r = 0.76) between the labelling index for PCNA and that for Ki67. Immunohistochemical staining for PC10 was confined to the nucleus and showed a diffuse or granular pattern or a mixture of both. The distribution of PC10 staining in non-neoplastic tissues was localized to proliferating cell compartments. In malignant tissues, the localization of the distribution of PCNA-positive cells came to be lost and the proportion of positive cells varied from case to case as well as from field to field within the same tissue section. The cases in which more than 31% of cells were positive for PCNA were as follows: Cervical squamous dysplasia 2/3, squamous carcinoma in situ 2/5, microinvasive squamous carcinoma 2/2, invasive squamous carcinoma 9/13, adenocarcinoma in situ 4/4, microinvasive adenocarcinoma 3/3, invasive adenocarcinoma 6/7, endometrial adenocarcinoma 6/25, ovarian epithelial malignant tumors 11/17, sex cord stromal tumors 2/14, and germ cell tumors 3/22. It is concluded that immunohistochemical staining for PC10 may be useful as a marker for proliferating activity of the cells both in normal and tumor tissues rather than for malignancy.

Antigens, Neoplasm↗
PubMed1992

Progressive facial hemiatrophy with localized scleroderma.

A 46-year-old woman who presented with progressive facial hemiatrophy (PFH) following localized scleroderma is described. The patient had a markedly deformed and depressed plaque surrounded by erythema on the right cheek. She also showed linear scleroderma with hair loss on the occipital area and morphea lesions on the neck and shoulder. Histological findings of the facial and other atrophic lesions were consistent with localized scleroderma. Therefore, the PFH and localized scleroderma may have had the same pathological background in this case.

Facial Hemiatrophy↗
PubMed1991

Cloning and characterization of the gene encoding bovine mitochondrial adenylate kinase isozyme 3.

The structure of the gene (AK3) encoding bovine mitochondrial (mt) adenylate kinase isozyme 3 (AK3) has been determined. The gene spans about 46 kb and splits into five exons. The 5'-flanking region of the gene lacks typical transcriptional regulatory elements such as TATA and CAAT boxes. The G + C content of this region is high (71%), and seven GC boxes are recognized. Possible sequences responsive to transcription factors, AP-1, AP-2, Myf-6 and MyoD1, are present in this region. In the 5'-flanking region, there are sequences (mt sequence) which were found in the genes encoding other mt proteins. Transcriptional mapping analyses revealed one major mRNA start point. The promoter activity measurement in Chinese hamster ovary cells by transient expression of the cat gene connected with the 5'-flanking region, showed presence of a functional promoter.

Adenylate Kinase↗
PubMed1991

Synthesis and accumulation of thiamin triphosphate in Escherichia coli cells expressing chicken cytosolic adenylate kinase.

To examine whether cytosolic adenylate kinase (AK1, EC 2.7.4.3) catalyzes synthesis of thiamin triphosphate (TTP) in vivo, chicken AK1 was expressed in Escherichia coli, and cellular AK1 activity and TTP content were determined. E. coli harboring the vector plasmid was used as a control. Chicken AK1 was expressed in the producer strain at a high level (83 U/mg protein) even without inducers, and this expression was doubled (153 U/mg protein) by beta-D-isopropylthiogalactopyranoside (IPTG). TTP was accumulated in the producer cells at a high level (5.7 nmol/g dry weight) without IPTG and this was also doubled (10.2 nmol/g dry weight) by IPTG. TTP content in the control strain was very low (0.2-0.9 nmol/g dry weight) and was unaffected by IPTG. Neither bacterial growth curve nor cellular content of AMP, ADP, ATP, thiamin diphosphate or total thiamin (sum of thiamin and its phosphate esters) was different between the producer and the control strains. These results indicate that chicken AK1 expressed in E. coli catalyzed the synthesis and accumulation of TTP within the bacterial cells.

Adenosine Diphosphate↗
PubMed1991

Structural analyses of a channel-forming fragment of colicin E1 incorporated into lipid vesicles. Fourier-transform infrared and tryptophan fluorescence studies.

Structural changes upon binding to the membrane of a COOH-terminal channel-forming thermolytic fragment of colicin E1 have been studied by means of a variety of spectroscopic techniques. Circular dichroism measurements show that the thermolytic fragment predominantly takes a helical structure in aqueous and detergent solutions. Fourier transform infrared spectroscopic measurements indicate that the content of the beta-structure is significantly increased when the thermolytic fragment is bound to vesicles. On the basis of the result of tryptophan fluorescence measurements, we have concluded that each of the three tryptophan residues of the thermolytic fragment exists in different environments, i.e. one is buried in the lipid bilayer, one exists on the cis side of the vesicles, and one exists near the surface of the lipid bilayer. The Fourier transform infrared and fluorescence data have been used along with the crystal structure of colicin A, which is highly homologous to colicin E1 in structure and function, to propose a model of the thermolytic fragment bound to the lipid vesicles.

Amino Acid Sequence↗
PubMed1991

Characterization of a processed pseudogene derived from the gene encoding bovine adenylate kinase isozyme 2.

We have isolated an intronless processed pseudogene for an isoform of bovine adenylate kinase isozyme 2 (AK2B). The pseudogene which was flanked by 11-nucleotide (nt) direct repeats contained an open reading frame of 519 bp which encodes 173 amino acids (aa), 61 aa shorter than AK2B. In the entire pseudogene sequence, substitutions as well as deletions and insertions were noted in 78 nt positions as compared with the AK2B cDNA (95.1% homology). Northern-blot analysis showed that this pseudogene was not expressed in bovine tissues.

Adenylate Kinase↗