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Biomedical subjects

K Kishi

Publications and source records attributed to K Kishi.

At least 325 records · Page 18Linked to original sources
PubMed1995

Thalamocortical synapses between axons from the mediodorsal thalamic nucleus and pyramidal cells in the prelimbic cortex of the rat.

A combined anterograde axonal degeneration and Golgi electron microscopic (Golgi-EM) study was undertaken to identify thalamocortical synaptic connections between axon terminals from the mediodorsal thalamic nucleus (MD) and pyramidal cells in layers III and V of the agranular prelimbic cortex in the rat. The morphological characteristics of thalamocortical synapses from MD were also examined by labeling axon terminals with anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). WGA-HRP labeled axon terminals from MD to the prelimbic cortex were small in size (0.5-1 microns in diameter), contained round synaptic vesicles, and formed axospinous synapses with asymmetrical membrane thickenings. With Golgi-EM methods, gold-toned apical dendrites in layer III were analyzed by reconstruction of serial ultrathin sections. Following lesions in the thalamus, degenerating thalamocortical axon terminals formed asymmetrical contacts exclusively on dendritic spines of the identified apical dendrites. More thalamocortical synapses were found on apical dendrites of layer V pyramidal cells than on apical dendrites of layer III pyramidal cells. In addition to thalamocortical synapses, a very few unlabeled symmetrical synapses were found on apical dendrites and somata of pyramidal cells, but they were not quantified and their sources are unknown.

Animals↗
PubMed1995

Activity staining of mammalian ribonuclease inhibitors after electrophoresis in sealed vertical slab polyacrylamide gels.

A method for detecting the activity of ribonuclease inhibitors (RIs) after nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing was developed. Both types of electrophoresis were performed using vertical slab polyacrylamide gels in the presence of dithiothreitol and in a sealed system. In each system, purified 50 kDa human RI was visualized as a single band by immunoblotting with a specific antibody. RI activity in the polyacrylamide gel slab was detected by sandwiching the gel slab between a cellulose acetate membrane moistened with a solution of bovine pancreatic ribonuclease A and a dried agarose film sheet containing substrate yeast RNA plus ethidium bromide and incubating at 37 degrees C. The ribonuclease penetrated the polyacrylamide gel and digested the substrate RNA in the agarose film. However, if an RI was present in the gel, the enzyme was inactivated by complex formation. Fluorescent bands corresponding to RIs were observed on a dark background under ultraviolet light. This activity staining had a high sensitivity allowing detection of less than 0.6 units of mammalian RIs (corresponding to 5 ng of purified human RI) and produced a sharp band which compared favorably with that obtained on immunoblotting. These electrophoretic techniques appear useful for the investigation of RIs in heterogeneous biological samples.

Animals↗
PubMed1995

Electron microscopic evidence that axon terminals from the mediodorsal thalamic nucleus make direct synaptic contacts with callosal cells in the prelimbic cortex of the rat.

A combined study of anterograde axonal degeneration and HRP retrograde labeling has shown that there exist monosynaptic connections between afferent fibers from the mediodorsal thalamic nucleus (MD) and callosal cells in the prelimbic cortex of the rat. Degenerating axon terminals from MD made asymmetrical synaptic contacts with dendritic spines from apical dendrites of layer III pyramidal cells that were retrogradely labeled with HRP after its injection into the prelimbic cortex contralateral to MD lesions.

Afferent Pathways↗
PubMed1995

Two classes of cortical neurones labelled with Vicia villosa lectin in the guinea-pig.

Cell surface substance(s) containing terminal N-acetylgalactosamine (GaINAc) was localized in the cerebral cortex of the guinea-pig. Vicia villosa and Glycine max, both specific to GaINAc, labelled the surface of the cell body of multipolar and triangular neurones. The labelling extended to the apical dendrite and axon initial segment in the triangular neurones. They were distributed exclusively in layer 5. Most of the labelled multipolar neurones contained a calcium-binding protein, parvalbumin (Pv), while none of the triangular neurones did. The findings indicate that, in the guinea-pig, pyramidal neurones as well as non-pyramidal interneurones are enwrapped by GaINAc-containing substances and suggest that this enwrapping does not seem to be linked to Pv production.

Aging↗
PubMed1995

The molecular basis for genetic polymorphism of human deoxyribonuclease I: identification of the nucleotide substitution that generates the fourth allele.

In addition to the three alleles commonly responsible for the protein polymorphism of human deoxyribonuclease I, a mutation encoded by a fourth allele, DNASE1*4, was detected by isoelectric focusing. All 8 exons covering the entire open reading frame of the human DNase I gene were amplified by the polymerase chain reaction and subjected to direct sequencing. Only one nucleotide substitution, a C-to-G transition (CAG-->GAG), in the codon for amino acid 9 of the mature enzyme was found. This substitution resulted in the replacement of Gln with Glu (Q9E).

Alleles↗
PubMed1995

Cytopathological features of undifferentiated carcinoma of the ovary.

Cytopathological characteristics of undifferentiated carcinoma of the ovary were examined and reviewed. Seven cases of undifferentiated carcinoma were encountered among 202 malignant ovarian neoplasms in the Saitama Cancer Center. Histologically, two cases contained a minor component of usual Müllerian carcinoma and five others were purely of solid architecture without differentiation. Nevertheless, six cases, four of which were totally undifferentiated histologically, showed cytological features characteristic of adenocarcinoma. The results indicate that even carcinomas histologically diagnosed as undifferentiated may have cytological features of glandular differentiation, which may be Müllerian in origin.

Aged↗
PubMed1995

Genotyping of human deoxyribonuclease I polymorphism by the polymerase chain reaction.

We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.

Alleles↗
PubMed1995

Expression of the c-kit gene product in normal and neoplastic mast cells but not in neoplastic basophil/mast cell precursors from chronic myelogenous leukaemia.

The expression of the c-kit gene product has been examined in normal mast cells, mast cell neoplasms, and basophil/mast cell precursors obtained from patients with chronic myelogenous leukaemia (CML). Formalin-fixed, paraffin-embedded sections or smears fixed with formalin vapour were studied by immunohistochemical methods, using a polyclonal antibody against the c-kit gene product. Normal and neoplastic mast cells showed a positive immunoreaction for c-kit gene product, but neoplastic basophil/mast cell precursors from CML patients lacked c-kit gene product by immunohistochemical and flow cytometric methods, even in cells having mast cell granules, together with or without basophil granules. Mast cell tryptase was, however, expressed in normal and neoplastic mast cells and basophil/mast cell precursors containing mast cell granules. In addition, cells of monocyte/macrophage lineage lacked c-kit gene product. These findings indicate that the c-kit gene product may play an important role in the development and function of mast cell but not of cell of basophil and monocyte/macrophage lineage.

Animals↗
PubMed1995

Ribonuclease inhibitors in human blood: comparative studies on the inhibitors detected in erythrocytes, platelets, mononuclear leukocytes and granulocytes.

The erythrocyte ribonuclease inhibitor (RI) and platelet RI were separately purified to homogeneity and showed similar properties to those of human brain and placental RIs. The same type of RI seemed to be present in mononuclear leukocytes and granulocytes. The RI contents of these cells were detectable by immunoblot analysis using anti-human placental RI antibody. Neither RI activity nor the molecule cross-reactive with the anti-human placental RI antibody was detectable in any plasma sample. It is intriguing to detect active RI in mature erythrocytes, where no nucleus exists and RNA metabolism is unlikely to occur.

Amino Acid Sequence↗
PubMed1995

Deoxyribonuclease I from rat urine: affinity purification, characterization, and immunochemical studies.

Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-fold to apparent homogeneity with a 14% yield by affinity chromatography utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases. Isoelectric focusing electrophoresis revealed that all six isoelectric forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-terminal amino acid sequencing were performed on the purified DNase I. The N-terminal sequence up to the 15th residue of the enzyme was identical to that of rat parotid DNase I. The enzyme was found to be a glycoprotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule. The isoelectric multiplicity of the enzyme was partly due to differences in the sialic acid content of the isoforms. Gel filtration on Superose 12 and electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicated an approximate molecular mass for DNase I of 32 kDa. The enzyme had an optimum pH of 6.5 and required divalent cations such as Ca2+ for its activity. Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin. An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the activity of the purified enzyme.

Amino Acid Sequence↗
PubMed1995

Proliferative and apoptotic status in endometrial adenocarcinoma.

The contribution of cell proliferation and apoptosis to growth patterns in endometrial adenocarcinoma were investigated. Immunohistochemical staining was carried out by an antibody for Ki-67 proliferative antigen, Ley apoptotic antigen, and oncogene products bcl-2 and p53. Forty cases of endometrial adenocarcinoma were classified as exophytic, endophytic, and mixed exo- and endophytic in light of their vertical growth pattern, and, in each case, the carcinomatous area was divided into three layers by its vertical axis. In all but one case, no zonal distribution of the antigen expression was observed. In one case, an exophytic tumor, Ki-67 expression was intense in the surface layer and Ley expression in the deep layer was also intense, suggesting a correlation between macroscopic growth pattern and cellular growth and apoptotic potential. However, in general, zonal distribution of cell proliferation and apoptosis could not explain the growth morphology of endometrial adenocarcinoma of the uterus and it was suggested that factor(s) other than cell proliferation and apoptosis determine macroscopic growth patterns.

Adenocarcinoma↗
PubMed1995

Molecular analysis of the third allele of human deoxyribonuclease I polymorphism.

In addition to common phenotypes 1, 1-2 and 2 of human deoxyribonuclease I (DNase I), phenotypes 1-3 and 2-3, encoded by a third allele DNASE1*3, have been found by means of isoelectric focusing. The main objective of this study was to identify the mutation site(s) underlying phenotype 3. All eight exons covering the entire open reading frame of the human DNase I structural gene were amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequencing. When the entire 780-bp coding region and exon/intron junctions of the DNase I gene of two individuals with phenotypes 1-3 and 2-3 were sequenced, only one nucleotide substitution, a C-G transition (CCC-->GCC), in the codon for amino acid 132 of the mature enzyme located in exon VI was found that resulted in the replacement of proline with alanine (P132A). The mutation was confirmed by allele-specific amplification of genomic DNA. The replacement of the amino acid residue may reduce the hydrophobicity of the enzyme and thus increase the pI value of the type-3 isozyme compared with that of type 1, as increasing the hydrophobicity of a protein is known to decrease its pI value. The specific PCR-amplifications of exons and alleles developed in this study may provide a new tool suitable for rapid screening of DNase I variants.

Alleles↗
PubMed1995

Structure of the human deoxyribonuclease I (DNase I) gene: identification of the nucleotide substitution that generates its classical genetic polymorphism.

The objectives of this study were to elucidate the structural organization of the gene for human deoxyribonuclease I (DNase I) and to identify the mutation site underlying its classical genetic polymorphism. In order to determine the organization of this gene, we utilized a combination of direct polymerase chain reaction (PCR)-amplification of human genomic DNA and isolation of the overlapping clones from a cosmid human genomic library. Restriction endonuclease mapping, Southern blotting and DNA sequencing showed that the DNase I gene was approximately 3.2 kilobases long, it comprised 9 (I-IX) exons separated by eight introns and its complete sequence was determined. The first exon contained only the non-translated sequences of mRNA. In addition to several putative regulatory elements, TATA-like and CAAT-like sequences were observed in the region upstream of the translation initiation codon. These results provide information that will help to understand the expression and regulation of DNase I. The isoelectric focusing patterns of human DNase I showed that it exhibits classical genetic polymorphism (Kishi et al. 1989, 1990). A comparison of the entire translated sequences of the DNase I gene from two pairs of individuals with common DNase I phenotypes 1 and 2 revealed only one nucleotide residue difference in exon VIII, A for phenotype 1 and G for phenotype 2, thus producing Gln and Arg amino acid substitutions respectively at position 222 from the NH2-terminus of the mature enzyme. The predicted charge changes attributable to these amino acid substitutions are entirely consistent with the isoelectric focusing profiles of these two DNase I isozymes. We conclude that this substitution is solely responsible for the classical polymorphism of DNase I protein.

Adult↗
PubMed1995

Chromosomal assignment of the human deoxyribonuclease I gene, DNASE 1 (DNL1), to band 16p13.3 using the polymerase chain reaction.

To localize the human deoxyribonuclease I (DNase I) gene, DNASE1 (DNL1), we performed a polymerase chain reaction (PCR) using DNA extracted from a panel of cloned human x rodent hybrid cell lines carrying different human chromosomes and screened for the presence of the expected PCR products. Two different sets of oligonucleotide primers specific for human DNase I cDNA sequences were used to amplify unique fragments in the human DNase I gene. Based on this work, DNL1 could be assigned to human chromosome 16. Furthermore, regional localization of the gene to 16p13.3 was performed by PCR analysis of a high-resolution mouse x human somatic cell hybrid panel that contained defined portions of human chromosome 16.

Animals↗
PubMed1995

Regulation of rat placental lactogen (rPL)-II secretion: cAMP inhibits rPL-II secretion in vitro.

We examined whether epidermal growth factor (EGF), interleukin (IL)-1, IL-6 and cAMP analogs, which regulate mouse placental lactogen II secretion, affect rat placental lactogen (rPL)-II secretion using a rat choriocarcinoma cell line, Rcho-1. EGF, IL-1 and IL-6 did not affect rPL-II secretion, but 8-bromo-cAMP and forskolin inhibited rPL-II secretion by the 8th day of culture. The effects were dose dependent and the lowest concentrations of 8-bromo-cAMP and forskolin that significantly inhibited rPL-II secretion were 125 and 5 mumol/l, respectively. Cholera toxin also inhibited rPL-II secretion. The reverse hemolytic plaque assay for rPL-II indicated that the cells releasing rPL-II in the culture were giant cells and that 8-bromo-cAMP decreased the number of rPL-II-releasing cells. Western blot analysis of rPL-II yielded a single band at approximately 24.5 K, and 8-bromo-cAMP treatment significantly reduced the band intensity. Northern blot analysis of rPL-II indicated that 8-bromo-cAMP also reduced rPL-II gene expression. These findings suggest that the increase of intracellular cAMP accumulation results in inhibition of rPL-II secretion by decreasing rPL-II gene expression and inhibiting giant cell differentiation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Potential parameters of male reproductive toxicity: reproductive performance, histopathology and sperm evaluation in SD rats given nitrazepam.

The present study was designed to elucidate the correlation between findings from reproductive performance testing and those from histopathological examination of the testis and sperm analysis in rats given a benzodiazepine derivative, nitrazepam, for 2 and 4 weeks. The mechanisms of toxicological action of nitrazepam on the male reproductive organs were also investigated. Nitrazepam was given orally to Sprague-Dawley male rats (6-week-old) at a daily dose of 80 mg/kg for 2 weeks or at daily doses of 20, 40 or 80 mg/kg for 4 weeks. Treated males were mated to examine reproductive performance with untreated females after each dosing period, and after 4 and 9 week of recovery periods. Necropsy was performed for histopathological examination of the testis and epididymis and for sperm analysis after each dosing period and the final mating trial (total of 11 weeks recovery). In the findings from reproductive performance testing, significant decrease in the fertility index was observed in the 80 mg/kg group even after 2 weeks dosing and thereafter until 4 weeks recovery, though the mating index did not significantly differ from that of controls through the experiment. In the histopathological examination and sperm analysis, testicular signs of toxicity, decrease in number of sperm heads in the testis and increase in number of sperm with abnormal heads in the seminiferous tubules were noted in the 80 mg/kg group after 2 weeks dosing and in the 40 and 80 mg/kg groups after 4 weeks dosing. Concentrations of plasma testosterone and content of testis testosterone in nitrazepam-treated groups were not significantly different from those of controls. Plasma FSH concentration was significantly elevated in the 80 mg/kg group through the experiment, although significant elevation of plasma LH was observed only after 2 weeks dosing. These results indicate that histopathological examination is the most reliable approach to detect male reproductive adverse effects induced by nitrazepam rather than using parameters from mating trials. The four-week-dosing period is appropriate for their detection. Hypospermatogenesis induced by nitrazepam is suggested to be caused by direct action of nitrazepam on germ cells and/or Sertoli cells rather than by indirect action through inhibition of testosterone secretion.

Animals↗
PubMed1995

Alteration of the respiratory burst and phagocytosis of macrophages under protein malnutrition.

To understand the role of macrophages in impaired host defense under protein malnutrition (PM), we examined the activities of the respiratory burst and phagocytosis of resident peritoneal macrophages from weaning female mice fed 5% casein or 5% soy protein isolate (SPI) diet for 14 days. Resident macrophages from the low-protein diet groups released larger amounts of superoxide anion (O2-) after stimulation by phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan than those from the 20% casein and 20% SPI diet groups. Activation of macrophages from protein-deficient mice in vitro with lipopolysaccharide (LPS) or macrophage colony-stimulating factor (M-CSF) under LPS-free conditions did not further enhance O2- production. In spite of the increased O2- production with opsonized zymosan, macrophages from protein-deficient mice did not show any acceleration of phagocytosis of Candida albicans in the presence of normal serum. Our results confirm that the phagocytic function of macrophages is susceptible to PM, and suggest that functional alterations of macrophages may be involved in the failure of development of a specific immune response under PM. Furthermore, the enhanced production of oxygen intermediates by macrophages may augment tissue damage under PM.

Animals↗