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Biomedical subjects

N Matsubara

Publications and source records attributed to N Matsubara.

At least 55 records · Page 3Linked to original sources
PubMed1997

Separation and detection of closely related peptides by micellar electrokinetic chromatography coupled with electrospray ionization mass spectrometry using the partial filling technique.

Closely related peptides such as neurotensin and angiotensin analogues were separated by capillary zone electrophoresis using a nonionic surfactant, sucrose monododecanoate, as a micelle forming reagent. These peptides were detected by an on-line coupled mass spectrometer using an electrospray ionization interface. However, the presence of the micelles in the separation solution drastically reduced the sensitivity of the mass spectrometer. Therefore, a partial filling technique was employed to prevent the micelles from entering the mass spectrometric interface. A part of the capillary from the injection end was filled with the micellar solution in this technique. Analytes passed through the micellar zone during the electrophoresis and when the separated analytes reached the detection end of the capillary, the micellar zone was still behind the analyte zones, because the nonionic surfactant moved very slowly in acidic conditions. Thus the technique was very useful for mass spectrometric detection for CE when the micellar solution was employed for separation. The optimization of separation and detection conditions was investigated.

Amino Acid Sequence↗
PubMed1997

Possible involvement of free radical scavenging properties in the action of tumor necrosis factor-alpha.

Constitutive production of hydroxyl radicals from four established cancer cell lines was detected as spin adducts of 5,5-dimethyl-l-pyroline-N-oxide (DMPO), using an electron spin resonance spectrometer. The generated hydroxyl radicals was decreased in three out of four cancer cell lines when incubated in vitro for 3 h with TNF-alpha No direct scavenging effect of TNF-alpha on hydroxyl radicals or superoxide anions was observed in the in vitro radical generation system. The modulation of intracellular reactive oxygen species of these cancer cells by adding menadione or CuDIPS to the culture medium changed the antiproliferative effect of TNF-alpha on the cells. The ultrastructural localization of the radical-generating sites in cancer cells was visualized using the diaminobenzidine/horseradish peroxide histochemical system at the electron microscopic level. The hydrogen peroxide-dependent formation of electron-dense materials localized at the mitochondrial membranes was decreased after the treatment of the cancer cells with TNF-alpha. These data indicate that the reduction of radical generation in cancer cells by TNF-alpha may be an early mechanism that contributes to the antiproliferative effect of this cytokine on some cancer cells.

Cell Division↗
PubMed1997

The RY sequence is necessary but not sufficient for the transcription activation of a winged bean chymotrypsin inhibitor gene in developing seeds.

A winged bean Kunitz-type chymotrypsin inhibitor (WCI) is expressed in seeds and tuberous roots. In seeds, the expression of WCI is restricted to the period between the mid- and late-maturation stage. To understand the mechanisms that regulate the expression of WCI genes, we analyzed the promoter activity of the upstream region of the WCI-3b gene, which encodes a major WCI protein, in transgenic tobacco plants. By using a series of constructs with 5' deletions in the upstream sequences, the region between -882 and -623, relative to the transcription start site, was shown to contain multiple sequences which are responsible for high level expression in mid-maturation stage seeds. However, when this region was fused to the cauliflower mosaic virus 35S core promoter in both orientations, the chimeric promoters showed only a weak transcription activity in transgenic tobacco plants. Further analyses using internal deletion constructs revealed that the region between -882 and -174 is required for the transcription activation. Disruption of the RY sequence at -517, which is conserved in many seed protein genes, resulted in a drastic reduction of the transcription activity in seeds. These results suggest that sequences necessary for high level induction of the WCI-3b gene transcription in developing seeds are dispersed in the region between -882 and -174, and that the RY sequence is one of these sequences.

Chymotrypsin↗
PubMed1997

Antibacterial activity of quinolones against coagulase-negative staphylococci and the quinolone resistance-determining region of the gyrA genes from six species.

Antibacterial activity of quinolones against three species of coagulase-negative staphylococci was investigated. Tosufloxacin and sparfloxacin exhibited potent activities against Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus saprophyticus compared with other quinolones tested. From the analysis of the DNA sequence in the quinolone resistance-determining region (QRDR), greater than 80% homology was recognized in coagulase-negative staphylococci. A series residue was conserved in all six species at the position corresponding to position 84 in Staphylococcus aureus.

Anti-Infective Agents↗
PubMed1997

Cerebellar ataxia and peripheral neuropathy due to chronic bromvalerylurea poisoning.

A patient with chronic bromvalerylurea poisoning showed cerebellar ataxia and peripheral neuropathy. The patient was a 42-year-old Japanese man who developed consciousness disturbance, diplopia, slurred speech, ataxia and gait disturbance after having taken bromvalerylurea for ten years. Magnetic resonance imaging revealed atrophy of the cerebellum and pontine tegmentum. An electrophysiological study revealed decreased motor nerve conduction velocity and amplitude of compound muscle action potentials of the right tibial nerve. Histological findings of the left sural nerve indicated a slightly decreased large myelinated fiber diameter, which suggested chronic axonal damage.

Adult↗
PubMed1996

A receptor tyrosine kinase, Sky, and its ligand Gas 6 are expressed in gonads and support primordial germ cell growth or survival in culture.

Although a number of growth factors and their receptors are involved in the proliferation and differentiation of primordial germ cells (PGCs), the only factor that has been shown to be active in vivo is Steel factor, a ligand for c-Kit. To identify new growth factor receptors that may be required for PGCs function in vivo, we used an reverse transcription-polymerase chain reaction-based strategy to screen for protein kinase genes expressed in PGC-derived embryonic germ cells. We report here that one such gene encoding the receptor tyrosine kinase, Sky, is expressed in both PGCs and their supporting cells in male genital ridges after 11.5 dpc. Interestingly, Sky expression was not detected in female genital ridges, although transcripts were detected in supporting cells in the developing ovary at later stages. Gas 6, a ligand for Sky, was also expressed in interstitial cells which surround Sky positive cells in genital ridges, and, in addition, it supported PGC growth or survival in culture. After birth, Sky expression in testis was restricted to Sertoli cells, and Gas 6 was detected around peritubular cells and Leydig cells. These results suggest that Gas 6-Sky signaling plays a role in PGC growth, sexual differentiation, and Sertoli cell functions in vivo. Sky expression in Sertoli cells diminished by 3 weeks of age, when haploid germ cells first appear. On the other hand, the expression in Sertoli cells was markedly upregulated in the testis of germ cell-deficient W/Wv and jsd/jsd mice. The results suggest that signals from differentiated germ cells suppress Sky gene expression in Sertoli cells. High-resolution chromosomal mapping of Sky is also reported.

Animals↗
PubMed1996

Mutations of E2F-4 trinucleotide repeats in colorectal cancer with microsatellite instability.

Genetic instability at microsatellites in some colorectal cancer (CRC) have been linked to the defects of human mismatch repair genes, but the targets of these defective genes have been largely unknown. We screened CRC specimens for alteration of E2F-4 gene by analyzing both cDNA and genomic sequences along with replication error (RER+) phenotype. Two out of 20 sporadic CRC patients showed RER+ phenotype. We found tumor-specific copy number alteration in 13 consecutive trinucleotide (CAG) repeats within the coding exon of E2F-4 exclusively in these 2 specimens. Thus, E2F-4 may be a clue of the target gene of defective repair genes in CRC with genetic instability in addition to the TGF-beta type II receptor gene.

Base Sequence↗
PubMed1996

Mutations in the gyrA and grlA genes of quinolone-resistant clinical isolates of methicillin-resistant Staphylococcus aureus.

The mutations in the quinolone-resistance determining regions (QRDR) of the gyrA, gyrB and grlA genes and in the norA gene from five clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) were examined by DNA sequencing. The mutation from Ser84 to Leu in GyrA was associated with relatively high-level resistance to quinolones, whereas the mutation from Glu88 to Gly or Lys in GyrA was associated with low-level resistance to quinolones. Mutations of the grlA gene were observed at codon 80 (Ser80) or 84 (Glu84), independent of the mutations of gyrA. No mutations were observed in either the gyrB or norA genes.

Anti-Infective Agents↗
PubMed1996

Spontaneous complete necrosis of hepatocellular carcinoma.

We report a 69-yr-old female with a liver mass which, when surgically resected, was believed to be the spontaneous complete necrosis of hepatocellular carcinoma. The preoperative level of alpha-fetoprotein was high at 1050 ng/ml, and the mass was grossly an encapsulated nodular lesion with septa. Histologically, no viable tumor cells were demonstrated. The alpha-fetoprotein level decreased to a normal range. The postoperative course was uneventful, and the patient is alive and well 1 yr after surgery.

Aged↗
PubMed1996

[A case of catamenial pneumothorax with elevated level of serum CA 125].

A 40-year old woman had experienced repeated spontaneous pneumothorax on the right side at the beginning of each menstrual period for one year. The level of serum CA125 was high at 60 ng/ml. Right thoracotomy revealed the presence of multiple darkly-pigmented spots and several pinhole like perforations near the central tendon of the diaphragm. The partial resection of the diaphragm was done, endometrial tissues were detected histologically. Tha patient has been asymptomatic after operation and the level of serum CA125 is normal.

Adult↗
PubMed1995

Separation of eleven angiotensin II analogs by capillary electrophoresis with a nonionic surfactant in acidic media.

Eleven angiotension II analogs of same chain length were separated by capillary electrophoresis at pH 2.0 with 200 mM Tween 20. All compounds except one pair of angiotensin II ([Sar1, Gly8]- and [Sar1, Val5, Ala8]-angiotensin II) were baseline-separated, even in the case of peptides with about the same total charge. The migration order of the angiotensin II analogs were determined by the hydrophobicity of the amino acid as long as the difference between amino acids of two peptides is the conservative change. From the study of pH dependency of the separation of these peptides, it was found that the conditions of a low electroosmotic flow under a low pH is effective for the separation of similar peptides.

Amino Acid Sequence↗
PubMed1995

Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes.

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding regions shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythro-leukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the Plk1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage.

Animals↗
PubMed1995

Secretion by Saccharomyces cerevisiae of rat apolipoprotein E as a fusion to Mucor rennin.

As the first step for production of rat apolipoprotein E (rApoE) in Saccharomyces cerevisiae, the rApoE cDNA was cloned and its nucleotide sequence was determined. When the intact rApoE gene including the presequence-encoding region was expressed under the control of the yeast GAL7 promoter, no protein immunoreactive with anti-rApoE antibody was detected either in the culture medium or inside the cells. For the purpose of the extracellular production of rApoE, three fusion genes were constructed in which the mature rApoE-encoding sequence was connected after the pre, prepro, and whole regions of the gene encoding a fungal aspartic proteinase, Mucor pusillus rennin (MPP), since MPP is efficiently secreted from recombinant S. cerevisiae containing the MPP gene. When these three fusion genes were expressed under the control of the GAL7 promoter, only one, encoding the mature rApoE connected to the whole MPP sequence, directed efficient secretion of the fused protein. The maximum yield of the fused protein secreted into the medium reached 11.8 mg/l and the calculated rApoE part was 5.3 mg in the fused protein. The excreted fusion protein was glycosylated at the original two sites in the MPP part. The fused protein was gradually degraded in the medium probably by proteases of the host cell, because no such degradation occurred in a yeast pep4mutant strain.

Amino Acid Sequence↗
PubMed1995

Hereditary motor and sensory neuropathy associated with cerebellar atrophy (HMSNCA): a new disease.

Seven patients with hereditary motor and sensory neuropathy associated with cerebellar atrophy (HMSNCA) are presented. This is the first comprehensive evaluation of what is a unique disorder, half way between the cerebellar atrophies and the hereditary motor and sensory neuropathies. In addition to cerebellar ataxia and peripheral neuropathy, the most frequent features in HMSNCA were nystagmus, dysarthria, mental impairment and tremor. Pyramidal signs or autonomic nerve dysfunction was never revealed. Scoliosis or kyphoscoliosis was not noted. Progression of the disorder was very slow, most of the patients being ambulatory more than 10 years after the onset. Most of the patients had hypoalbuminemia. Half-life periods of serum albumin were normal and decreased synthesis of albumin in the liver was suspected. An autosomal recessive inheritance was strongly suggested, because of healthy consanguineous parents and affected siblings in these families. The segregation ratio was 0.32 +/- 0.10 and was close to the expected ratio of 0.25 in an autosomal recessive inheritance.

Adult↗
PubMed1995

Analysis of the NH2-terminal 83rd amino acid of Escherichia coli GyrA in quinolone-resistance.

Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH2-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues.

4-Quinolones↗