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Biomedical subjects

K Mizobuchi

Publications and source records attributed to K Mizobuchi.

At least 55 records · Page 3Linked to original sources
PubMed1995

[Circadian rhythms in peak expiratory flow rate of asthmatic patients before and after treatment with beclomethasone dipropionate].

Asthmatic patients have a circadian rhythm in peak expiratory flow rate (PEFR). The present study was done to measure the effect of inhaled beclomethasone dipropionate (BDP) on the circadian rhythm of PEFR in asthmatic patients. After two weeks of observation, BDP (400 micrograms) was given by metered dose inhaler to nine asthmatic patients. The dose of inhaled BDP (800-1200 micrograms) was increased every two weeks until PEFR varied by no more than 20% each day. PEFR was measured four times daily: on waking, around noon, in the evening, and at bedtime. Nine asthmatic patients had a significant (p < 0.05) rhythm detectable by single cosinor analysis, both during the observation period and during treatment. Analysis by the group mean-cosinor method showed that the mean mesor was 397.3 +/- 6.8 l/min, the mean amplitude was 54.3 +/- 7.1 l/min, and the mean acrophase was at 16:31 +/- 0.27 before treatment. After treatment, the mean mesor was 543.8 +/- 4.4 l/min, the mean amplitude was 30.5 +/- 4.9 l/minm, and the mean acrophase was at 16:25 +/- 0.31. The mean mesor had increased significantly (p < 0.05), and the mean amplitude had decreased significantly (p < 0.05) after treatment. The mean acrophase did not change. These data indicate that inhaled BDP increases PEFR at a constant acrophase in asthmatic patients.

Adolescent↗
PubMed1995

Conformational changes of purine repressor DNA-binding domain upon complexation with DNA.

The purine repressor (PurR) consists of two functional domains: an N-terminal DNA-binding domain and a C-terminal corepressor-binding domain. Recently, the structure of PurR-corepressor-operator ternary complex was determined by X-ray crystallography. In the complex the DNA-binding domain, consisting of 56 amino acids, was composed of four helices. Here, we have determined the solution structure of the DNA-binding domain in its DNA free state by NMR. It consists of three helices and the fourth helix (the hinge helix) region is diordered. The architecture of the first three helices of its DNA free state is very similar to that of its DNA-bound form. The hinge helix is induced by the specific DNA binding and by the dimerization of PurR which is provided by the corepressor-binding domain.

Bacterial Proteins↗
PubMed1994

Evolution of the replication regions of IncI alpha and IncFII plasmids by exchanging their replication control systems.

The basic replicons of bacterial plasmids consist of two sets of genetic systems, the replication-structural system and the replication control system. Comparison of nucleotide sequences suggested that the basic replicons of plasmids P307 (IncFI) and pMU2200 (IncZ) were generated by reciprocal recombination between ancestors of R100 (IncFII) and ColIb-P9 (IncI alpha), or vice versa. The plasmids of each pair, P307/pMU2200 and R100/ColIb-P9, are structurally unrelated to each other. Based on this information, we constructed in vitro and analyzed P307-like chimeric replicons from ColIb-P9 and R100. When the replication-structural region of ColIb-P9 was combined with the whole replication control region of R100, the resultant replicon replicated stably as R100 did. These results revealed that the basic replicons of the plasmids diverged by exchanging their replication control systems. Thus, we propose that the replication control systems of plasmids, in some cases, evolved independently of their structural systems, although these two systems work together to maintain the replication functions. We also showed that the reciprocal recombination was specified by the unique secondary structures of RNA involved in the control of expression of the genes encoding the replication initiator proteins.

Amino Acid Sequence↗
PubMed1994

Malignant peripheral nerve sheath tumor with prominent intracytoplasmic vacuolation: report of a case.

A case of malignant peripheral nerve sheath tumor with uncommon features is reported. A mass was noted in the left thigh of a 16 year old man. Histologically, most areas of the tumor exhibited the typical appearance of malignant peripheral nerve sheath tumor, but some tumor cells had rounded nuclei and cytoplasm, resembling an epithelioid pattern. It was noted that some rounded tumor cells showed prominent intracytoplasmic vacuolation. Immunohistochemically, almost all of the tumor cells, including the rounded and vacuolated ones, were positive for S-100 protein and vimentin. Electron microscopic study revealed well-developed cytoplasmic processes, intracytoplasmic intermediate-sized filaments, basement lamina formation and extracellular long-spacing collagens. These findings were compatible with those of Schwann cell differentiation. Moreover, ultrastructurally, the vacuolated spaces contained a few granular materials and were derived from the dilatation of the rough endoplasmic reticulum. It is speculated that intracytoplasmic vacuolation in malignant peripheral nerve sheath tumor would be caused by degeneration of the tumor cells.

Adolescent↗
PubMed1994

Cloning, sequencing, and expression of bacteriophage BF23 late genes 24 and 25 encoding tail proteins.

Two bacteriophage BF23 late genes, genes 24 and 25, were isolated on a 7.4-kb PstI fragment from the phage DNA, and their nucleotide sequences were determined. Gene 24 encodes a minor tail protein with the expected M(r) of 34,309, and gene 25 located 4 bp upstream of gene 24 encodes a major tail protein with the expected M(r) of 50,329. When total cellular RNA isolated from either phage-infected cells or cells bearing the cloned genes was analyzed by the primer extension method using the primers specific to either gene 25 or gene 24, we identified a possible late gene promoter, designated P25, in the 5'-flanking region of gene 25. This promoter was similar in structure to Escherichia coli promoters for sigma 70. Studies of the translational gene 25- and gene 24-lacZ fusions in the cloned gene system revealed that the promoter P25 was responsible for the expression of both genes 25 and 24 even in the absence of the regulatory genes which were absolutely required for late gene expression in the normal phage-infected cells. These results indicate that the two genes constitute an operon under the control of P25 and that the regulatory gene products of BF23 do not participate directly in specifying the late gene promoter.

Amino Acid Sequence↗
PubMed1994

[Dynamics of pleural liquid in hydrothorax].

The basics of pleural liquid dynamics are summarized. The normal pleural cavity contains a small amount of pleural liquid (0.1-0.3 ml/kg). Its protein concentration is about 1.0 g/dl and its pH is 7.6. The normal flow of pleural liquid is gravity dependent, and pleural liquid flows from the costal to the mediastinal region. In experimental hydrothorax, the pleural liquid was removed mainly via lymphatics. The turnover of the pleural liquid is rapid, and it depends on the area of contact between pleural liquid and pleural and on the blood flow to the pleura.

Animals↗
PubMed1994

[Detection of intratumoral DNA heterogeneity in primary lung cancer using a multiple sampling method].

The nuclear DNA contents of 18 freshly resected specimens of primary lung cancer tissue were determined by flow cytometry with a multiple sampling method. A DNA aneuploidy pattern was observed in all cases. In three cases, a diploid DNA pattern was observed along with abnormal DNA stem lines. Twelve cases (66%) had two or more abnormal DNA stem lines, and were classified as DNA multiploidy. The frequencies of DNA aneuploidy and DNA multiploidy were higher than in previously reported studies. Intratumoral DNA heterogeneity was observed in 10 cases (55%). Three of these were a mixture of DNA diploidy and DNA aneuploidy, and the other seven were a mixture of DNA aneuploidy and DNA multiploidy. If multiple sampling had not been performed in these cases, their ploidy patterns would have been misinterpreted. We conclude that multiple sampling is important in determining the precise DNA ploidy patterns of primary lung cancers.

Adenocarcinoma↗
PubMed1993

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12.

We isolated a transposon Tn10 insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.

Amino Acid Sequence↗
PubMed1992

Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.

Bacterial Proteins↗
PubMed1992

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells.

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100 beta protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.

Adult↗
PubMed1992

15N and 13C labeling of Escherichia coli tRNAs toward the NMR analysis.

Escherichia coli tRNAs were labeled with stable isotope 15N in vivo. Three species of tRNA, tRNA(Glu), tRNA(Lys) and tRNA(Ile), were purified by an HPLC system and their NMR spectra were observed. In heteronuclear 1H-15N multiple or single quantum coherence (HMQC or HSQC) spectra, the crosspeaks corresponding to NH3 of U and NH1 of G can be distinguished clearly since their 15N chemical shifts are significantly different from each other. Thus, this combination of 15N-labeling and the proton detected heteronuclear experiments are useful for the signal assignment and the conformational analysis of tRNAs. Furthermore, C1'- selective 13C-labeling of nucleotides was examined in vivo in order to resolve the H1' signals of tRNAs. By using a newly constructed E. coli mutant strain, the isotopic enrichments of more than 90% at C1' and of less than 10% for other ribose carbons were achieved.

Carbon Isotopes↗
PubMed1991

An induced mRNA secondary structure enhances repZ translation in plasmid ColIb-P9.

Translation of the repZ gene encoding a DNA replication initiation protein of plasmid ColIb-P9 depends on not only the translation of a transcribed leader sequence (repY) but also the specific intergenic base pairing within RepZ mRNA between two short complementary sequences located in the repY and inc gene regions. In addition, repZ translation can be negatively regulated by Inc RNA, the product of the inc gene and a countertranscript to RepZ mRNA. Here we present evidence indicating that a stable secondary structure of RepZ mRNA, designated as structure III, sequesters one of the complementary sequences and the ribosome-binding site, thereby preventing repZ translation. When site-directed mutagenesis was used to destabilize structure III without changing the ribosome-binding site, a significant level of repZ expression was observed even in the absence of repY translation. Under these conditions, however, repZ expression could be substantially reduced by additional mutations that directly diminished the intergenic base pairing at the mRNA level between the two complementary sequences. These results indicate that repY translation is essential for the disruption of structure III by inducing the formation of a new secondary structure through the intergenic base pairing, and more importantly, that this new structure enhances repZ translation. We also found that the site of repY translation termination played a critical role in the intramolecular conformational alteration of structure III.

Bacteriocin Plasmids↗
PubMed1991

Positive and negative regulations of plasmid CoLIb-P9 repZ gene expression at the translational level.

Expression of the repZ gene involved in DNA replication of the ColIb-P9 plasmid depends on translation of a transcribed repZ leader sequence (repY) and is negatively regulated by Inc RNA, the product of the inc gene and a countertranscript to RepZ mRNA. To further understand the regulatory loop of repZ expression, we isolated and characterized replication-defective ColIb-P9 mutants that affected the level of repZ expression. Here we report that mutations occurring in two complementary sequences, one (5'GGCG3') in the inc region and one in the repY region, reduce the level of repZ expression without affecting transcription. The mutations in one complementary sequence were suppressed by compensatory base changes in the other sequence, restoring the ability of repZ expression. These results indicated that interaction by base pairing between the two complementary sequences of RepZ mRNA was essential for repZ translation. The two sequences, separated by 107 bases from each other, have a potential to form a novel pseudoknot in the RepZ mRNA leader. We also found that some mutations in the 5'GGCG3' sequence altered the specificity of Inc RNA, thereby reducing significantly its regulatory activity. Thus, this single specific sequence is involved in both positive and negative regulations for repZ expression. Possible regulatory mechanisms of repZ expression are discussed.

Amino Acid Sequence↗
PubMed1990

Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid.

The frequency of replication initiation of the ColIb-P9 plasmid depends on the level of repZ expression, which has been shown to be negatively regulated by inc RNA, the approximately 70-base-long product of the inc gene. To further understand the regulatory mechanism of repZ gene expression, we isolated mutants defective in ColIb-P9 replication using a lambda:ColIb-P9 hybrid phage. Among six mutants isolated, one amber mutant, rep57, failed to synthesize the RepZ protein. The mutation occurred in the repZ leader sequence that encodes a 29-amino-acid reading frame, designated as repY. We also isolated mutants that suppressed the rep57 phenotype. These mutations were single base insertions between the repY initiation codon and the rep57 mutation site and resulted not only in a frame shift of repY but also in the formation of repY-repZ fusions without changing the amino acid sequence of RepZ. Thus, repY is not directly involved in the replication reaction but rather functions as a positive regulator for repZ expression. We propose that repZ expression is coupled with repY translation, which acts to disrupt a secondary structure sequestering the repZ translation initiation signal. The positive and negative regulations of repZ expression were discussed. The other mutants were mapped in repZ, confirming that repZ is essential for ColIb-P9 replication.

Base Sequence↗
PubMed1990

Organization of the replication control region of plasmid ColIb-P9.

We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair IncI alpha group ColIb-P9 plasmid. Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ and inc. The repZ gene encodes a protein with a molecular weight of 39,000, which probably functions as an initiator for the ColIb-P9 replicon. The inc gene that phenotypically governs the incompatibility encodes an RNA with a size of about 70 bases. This small RNA acts in trans to repress the expression of repZ, thereby functioning to maintain a constant copy number of the ColIb-P9 replicon in host cells.

Amino Acid Sequence↗
PubMed1990

Posttranscriptional control of plasmid ColIb-P9 repZ gene expression by a small RNA.

The replication frequency of plasmid ColIb-P9 depends on the level of repZ gene expression, which is negatively regulated by the action of the inc gene (C. Hama, T. Takizawa, H. Moriwaki, Y. Urasaki, and K. Mizobuchi, J. Bacteriol. 172:1983-1991, 1990). To further understand the mechanism of this regulation, we analyzed transcripts of the ColIb-P9 replication control region. Four RNA species, designated RNAI to RNAIV, were observed in plasmid pCH11, which contained the whole inc gene region and the 5' portion of the repZ gene. RNAII, RNAIII, and RNAIV, with sizes of approximately 200, 500, and 1,500 bases, respectively, were identified as rightward transcripts that shared common transcription initiation sites; RNAIV was determined to be equivalent to a part of repZ mRNA, which was observed in pCH10, a plasmid that contained sufficient information for replication and control of ColIb-P9. Conversely, RNAI, with a size of about 70 bases, was transcribed leftward and was identified as the product of the inc gene and hence equivalent to inc RNA detected by in vitro RNA synthesis. This small RNA was found to be complementary to a part of repZ mRNA. These results and quantitative analyses of the transcripts in Inc- mutants indicate that the inc RNA negatively regulates repZ expression mainly at the posttranscriptional level through the possible formation of an inc RNA-repZ mRNA hybrid in the host cells.

Bacterial Proteins↗
PubMed1989

The organization of the purL gene encoding 5'-phosphoribosylformylglycinamide amidotransferase of Escherichia coli.

Escherichia coli 5'-phosphoribosylformylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) encoded by the purL gene catalyzes the conversion of FGAR to formylglycinamidine in the presence of glutamine and ATP for the de novo purine nucleotide biosynthesis. On the basis of the nucleotide sequence of purL, the enzyme was dissected along the polypeptide chain into at least three discrete regions, designated as domains I, II, and III, by genetic complementation tests. Domain III (255 amino acids), which resides in the C-terminal region, is similar in amido acid sequence to several glutamine amidotransferases and exerts the transfer of the amide nitrogen of glutamine. Domain I (791 amino acids) resides in the N-terminal region and contains a potential ATP binding motif. Domain II (249 amino acids) locates between domains I and III and is composed of an alternating structure of at least eight predicted beta-strand and alpha-helix elements, as has been observed in the family of triosephosphate isomerases. The functions of domains I and II have been discussed in relation to the transfer of the carbonyl oxygen of FGAR into the gamma-phosphorus moiety of ATP. These results support a model that the E. coli purL gene is a fused gene of at least three different gene families. The highly repetitive sequences of the E. coli genome appeared to play an important role in the process of the gene fusion.

Amino Acid Sequence↗