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H Nishio

Publications and source records attributed to H Nishio.

At least 37 records · Page 2Linked to original sources
PubMed2001

Immunolocalization of the mitogen-activated protein kinase signaling pathway in Hassall's corpuscles of the human thymus.

In the present study, we investigated the immunohistochemical localization of mitogen-activated protein kinase (MAPK) signaling pathway in the human thymus. Three members of MAPK, the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK) and the p38 kinase, showed differential expression patterns in the thymus medulla. The phosphorylated form of ERK (p-ERK) was abundantly present in the outer layer of Hassall's corpuscles, and the phosphorylated form of p38 kinase (p-p38 kinase) was present in the entire Hassall's corpuscles. The phosphorylated form of JNK (p-JNK) was expressed in medullary thymocytes. We also examined localization of MAPK kinases (MAPKK or MEK) which specifically activate MAPK. MEK1, an activator of ERK, was found in the outer layer of Hassall's corpuscles where p-ERK was expressed. MEK3, an activator of p38 kinase, was also expressed in the outer layer. MEK4 and MEK7, which are activators of JNK, were present in the entire Hassall's corpuscles. Thus, differential expression of MAPK in the thymus supports the concept that the MAPK signaling pathway controls the specificity of functional thymic responses to extracellular stimuli. Furthermore, the abundant expression of various elements of the pathway in Hassall's corpuscles suggests that the pathway is involved in thymic medullary epithelial maturation.

Child, Preschool↗
PubMed2001

[Primary thalamic hemorrhage penetrating to the lateral ventricle in an infant with abnormal protein S(protein S-Tokushima)].

We experienced a 3 months infant with thalamic hemorrhage penetrating to lateral ventricle with abnormal Protein S. Although the coagulation factor and fibrinogenolysis factors were evaluated, there were no remarkable abnormal laboratory data except for slightly decline of Protein S. The DNA analysis was performed for Protein S, and a missense mutation(A to G transmission) was found, which was resulting in Lys-155 to Glu. The total Protein S antigen was normal level, but co-factor activity for activated Protein C was declined. That mutation is named Protein S-Tokushima, and the patient who has abnormal Protein S tends to suffer recurrent coagulopathy. In our patient, it was interesting that any thrombotic disease had not occurred, but cerebral hemorrhage had occurred.

Cerebral Hemorrhage↗
PubMed2001

[Quantitative determination of lipoprotein by agarose gel electrophoresis and enzymatic staining of cholesterol and triglyceride: effects of age and gender].

290 normal duplicate samples (152 males and 138 females), were subjected to lipoprotein analysis by agarose gel electrophoresis to yield HDL, VLDL and LDL fractions which were respectively stained with Cholesterol and Triglyceride reagents. HDL-Cholesterol were significantly higher in female than male, and VLDL-TG were significantly higher in male than female. Assay C.V.'s varied from 0.96 to 5.75 for cholesterol fractions and 2.00 to 4.34 for triglyceride fractions. Comparison of electrophoretic HDL-Cholesterol and LDL-Cholesterol concentrations with the results of a direct method (HDL-EX and LDL-EX, Denka Seiken) gave correlation coefficients of 0.967 and 0.952 respectively. This method is simple, rapid and can provide the simultaneous assessment of the cholesterol and triglyceride component in each Lipoprotein fraction. Additionally, the method is useful for evaluating lipoprotein assays.

Adult↗
PubMed2000

Alu-mediated phylogenetic novelties in gene regulation and development.

Differential gene expression lies at the heart of biology and is responsible for all developmental processes, including the growth and differentiation of cells. Perhaps even speciation could be defined as a change in differential gene expression over evolutionary time. The present work is a phylogenetic study of four Alu elements known to have gene regulatory functions in the human. The four elements have been shown to regulate the parathyroid hormone (PTH) gene via a negative calcium-response element, the hematopoietic cell-specific FcepsilonRI-gamma receptor gene via a cis-acting positive/negative regulatory element, the CNS-specific nicotinic acetylcholine receptor alpha3 gene via a cis-acting positive/negative control element, and the T-cell-specific CD8alpha gene via a complex transcriptional regulator. The four Alu elements that impact differential gene expression were found to be differentially distributed among seven primate species (human, chimpanzee, gorilla, orangutan, baboon, rhesus, and macaque) in a way that is congruent with an accepted phylogeny of these species. The results establish a link between gene regulation and the divergence of primates. This evolutionary variation in gene regulation also suggests a novel experimental system to study the very complex transcriptional regulation of gene expression, by studying side-by-side the regulation of the same gene from two primate species that differ in the cis-acting regulatory elements of the gene.

Alu Elements↗
PubMed2000

Immunohistochemical study of tyrosine phosphorylation signaling in the involuted thymus.

Thymic involution has been reported to be an important parameter of the degree and duration of child abuse. In the present study, we assessed the status of tyrosine phosphorylation signaling, which is known to play a key role in the physiological function of the thymus, in involuted thymuses of abused children through immunohistological studies performed with anti-phosphotyrosine antibodies. We found that tyrosine-phosphorylated proteins were present in high amounts in Hassall's corpuscles (HC) in the medulla of control thymuses. In involuted thymuses of abused children, expression of tyrosine-phosphorylated proteins was reduced with accompanying morphological changes of HC, such as reduction in size or calcification. These findings lead us to the suggestion that tyrosine phosphorylation signaling is reduced in involuted thymuses of abused children and that reduction of the signaling may be associated with morphological changes of HC as observed in involuted thymuses of abused children. In order to certify the suggestion, we investigated expression of tyrosine-phosphorylated proteins in involuted thymuses of stressed rats as well as in control thymuses. Immunohistochemistry revealed that tyrosine-phosphorylated proteins were expressed in control thymuses, more abundantly in the medulla, and reduced remarkably in involuted thymuses of stressed rats. Further, immunoblot analysis also showed that expression of phosphotyrosine-containing proteins was reduced in thymus extracts of involuted thymuses of stressed rats, thus supporting the suggestion. Our results also raise the possibility that components of tyrosine phosphorylation signaling could be a molecular marker for thymic involution.

Animals↗
PubMed2000

Differences in mediator of nonadrenergic, noncholinergic relaxation of the distal colon between Wistar-ST and Sprague-Dawley strains of rats.

Participation of nitric oxide and vasoactive intestinal peptide (VIP) in electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle and in balloon distension-induced descending NANC relaxation of circular muscle were studied in the distal colon of Wistar-ST and Sprague-Dawley rats. The extent of the nitric oxide-mediated component was approximately 50% in longitudinal and circular muscle of Sprague-Dawley rats, whereas this component was absent in both muscles of Wistar-ST rats. The extent of the VIP-mediated component was approximately 40% in longitudinal muscle of Wistar-ST rats and circular muscle of Sprague-Dawley rats, whereas this component was absent in circular muscle of Wistar-ST rats and longitudinal muscle of Sprague-Dawley rats. In circular muscle of Sprague-Dawley rats, in which participation of both nitric oxide and VIP in the relaxation was suggested, inhibition of descending relaxation by N(G)-nitro-L-arginine (L-NOARG) together with VIP-(10-28) was similar to that by either of the antagonists, and exogenous VIP-induced relaxation was not affected by L-NOARG, but exogenous nitric oxide-induced relaxation was partly inhibited by VIP-(10-28). These results suggest a linkage of the pathways mediated by nitric oxide and VIP. In the immunohistochemical studies, nitric oxide synthase or VIP immunoreactive neurons were seen in the ganglia, primary internodal strands of the myenteric plexus and in the circular muscle layer. However, the overall appearance of immunoreactive cell bodies in the myenteric plexus and the numbers of immunoreactive fibers in the circular muscle layer appeared to be similar in Wistar-ST and Sprague-Dawley rats. These results suggest that mediators of NANC relaxation in the distal colon are different in different strains of rats, i.e., Wistar-ST and Sprague-Dawley, although no such difference was seen in immunohistochemical studies.

Animals↗
PubMed2000

Structural basis for the biological activity of dendrotoxin-I, a potent potassium channel blocker.

A topochemical model to explain the biological activity of dendrotoxin-I (DTX-I), a potent blocker for potassium channels, was developed by searching common spatial arrangements of functionally important residues between DTX-I, alpha-dendrotoxin, dendrotoxin-K, BgK, ShK, and charybdotoxin. The first three are structurally and functionally related to one another, and specifically target to Kv1 type potassium channels. The last three are structurally unrelated to the first three but have the ability to displace (125)I-labeled dendrotoxins on the same types of potassium channels. In order to obtain the correct electronic surface potential, thought to be crucial for the DTX-I function, we determined the three-dimensional solution structure of DTX-I by nmr spectroscopy using its correct amino acid sequence recently determined by our group. The most interesting characteristic of our model is that DTX-I has two binding sites to potassium channels: one is the cationic domain made up of Lys residues at positions 5 in the 3(10)-helix, 28 and 29 in the beta-turn, and the other is the Lys19/Tyr17/Trp37 triad located in the antiprotease domain. The cationic domain and the triad are located at the opposite sides of the molecular structure and are separated by about 25 A between Lys29 Calpha and Tyr17 Calpha. The functional triad is characterized by three distances, d(1) approximately 7.5 A (Lys19 Calpha-the center of the Tyr17 aromatic ring), d(2) approximately 8.1 A (Lys19 Calpha-the center of the 6-membered ring of the Trp37 indole group), and d(3) approximately 7. 3 A (the center of the Tyr17 aromatic ring-the center of the 6-membered ring of the Trp37 indole group). This model should aid in the pharmaceutical design of peptide and nonpeptide drugs with potassium channel blocking potencies, as well as in understanding of the physiology, pharmacology, biochemistry, and structure-function analysis of potassium channels.

Amino Acid Sequence↗
PubMed2000

Combined solid-phase and solution approach for the synthesis of large peptides or proteins.

In the synthesis of large peptides or proteins, highly homogeneous segments are indispensable for a convergent strategy either on a solid-phase resin or in solution. Employing Boc/Bzl chemistry to prepare fully protected segments with a free alpha-carboxyl group from the solid support, base-labile linkers are profitable for practical peptide synthesis since they require no special equipment. For this purpose, an N-[9-(hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS) linker was adopted. Consequently, there must be high compatibility between the protecting groups of the segment and the anchoring group which is cleavable by treatment with morpholine or piperidine in DMF. Instead of using the 2-bromobenzyloxycarbonyl (BrZ) group for the Tyr residue and the formyl (For) group for the Trp residue, both of which are the most susceptible protecting groups under these base-catalysed conditions, the base-resistant 3-pentyl (Pen) and cyclohexyloxycarbonyl (Hoc) groups were introduced to the respective side-chain functional groups. By applying the present strategy, the authors were able to rapidly synthesize homogeneous protected segments for use in the subsequent segment coupling in solution. In the present paper, the utility of the combined solid-phase and solution approach is demonstrated by synthesizing muscarinic toxin 1 (MTX1) which binds to the muscarinic acetylcholine receptors.

Chromatography, High Pressure Liquid↗
PubMed2000

Reduction of tyrosine-phosphorylated proteins in involuted thymuses of stressed rats: a study using immunological methods.

In the present study, we investigated whether tyrosine phosphorylation was involved in thymic involution, which has been reported to correlate well with the effects of various kinds of stresses. Immunohistochemistry using the anti-phosphotyrosine antibody showed that the immunoreactivity decreased remarkably in the involuted thymus of stressed rats as compared with the control thymus. Immunoblot analysis using the anti-phosphotyrosine antibody revealed the tyrosine-phosphorylated proteins with apparent molecular masses of 120, 90, and 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were detected in the control thymus. The immunoreactive band corresponding to the three proteins decreased remarkably in the involuted thymus. Further, we found by immunoprecipitation experiments that the 120 kDa protein was p130cas, a crk-associated src substrate. These findings suggest that tyrosine phosphorylation signaling may be involved in thymic involution.

Animals↗
PubMed2000

Expression of the janus kinases-signal transducers and activators of transcription pathway in Hassall's corpuscles of the human thymus.

The janus kinases (JAK) and signal transducers and activators of transcription (STAT) pathway has been shown to play a key role in cytokine-mediated signal transduction, and to regulate growth, differentiation, and death of both normal and transformed cells. In the present study, we investigated immunohistochemically the distribution of the JAK-STAT pathway in the human thymus. Various elements of the pathway were abundantly expressed in Hassall's corpuscles, located in the thymus medulla and representing terminal stages of the thymic medullary epithelium. Furthermore, the elements of the pathway showed distinct localization in Hassall's corpuscles. JAK1, JAK2, and TYK2 were expressed in high amounts in the entirety of Hassall's corpuscles, whereas JAK3 was in the outer layer. STAT1, STAT2, and STAT6 were abundantly expressed in the entire Hassall's corpuscles, whereas STAT5 was in the outer layer. These findings strongly suggest that the JAK-STAT pathway may play a role in thymic medullary epithelial maturation.

Child, Preschool↗
PubMed2000

Immunolocalization of calcineurin and FKBP12, the FK506-binding protein, in Hassall's corpuscles of human thymus and epidermis.

Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, plays an important role in various physiological functions including T cell activation. This enzyme is a target molecule for the immunosuppressants, cyclosporin A and FK506. In the present study, we investigated immunohistochemical localization of calcineurin and FKBP12, an FK506-binding protein, in human thymus and epidermis. The catalytic subunit (calcineurin A) of calcineurin was abundantly expressed in Hassall's corpuscles which were localized in the thymic medulla and represented the terminal stages of thymic medullary epithelium. The regulatory subunit (calcineurin B) of calcineurin was also expressed in high amounts in Hassall's corpuscles. In the epidermis, which shows similarities to Hassall's corpuscles, both subunits were also abundantly expressed, and their expression increased with the differentiation of keratinocytes. FKBP12 was observed to be expressed abundantly, both in Hassall's corpuscles and the entire epidermis. These findings suggest that the differentiated forms of the two cell types, which are the thymic medullary epithelial cell and the keratinocyte, are the target for pharmacological actions of FK506.

Calcineurin↗
PubMed2000

The C677T mutation in the methylene tetrahydrofolate reductase gene increases serum uric acid in elderly men.

A common mutation, C677T, in the methylene tetrahydrofolate reductase gene (MTHFR) reduces the activity of MTHFR and increases total homocysteine levels in plasma. Increased homocysteine levels are reportedly associated with high serum uric acid levels. The relationship between the MTHFR mutation and uric acid metabolism remains unclear, however. To investigate whether the C677T MTHFR mutation is a risk factor for hyperuricemia, we performed MTHFR genotyping and clinical laboratory determinations, including serum uric acid, in 271 elderly Japanese men (age range, 40-79 years; mean, 52.6 years). The mean uric acid levels for the C/C, C/T, and T/T genotypes were 5.67, 6.00, and 6.39 mg/dl, respectively (P = 0.012). The T/T genotype was more frequent in subjects with high uric acid levels than in those with low uric acid levels (P = 0.038). These findings suggest that the C677T MTHFR mutation contributed to higher uric acid levels in subjects enrolled in this study. In conclusion, the mutation of the MTHFR gene may be a risk factor for hyperuricemia in elderly men.

Adult↗
PubMed2000

Rapid serum vancomycin assay by high-performance liquid chromatography using a semipermeable surface packing material column.

A new isocratic high-performance liquid chromatographic (HPLC) assay has been developed for vancomycin that uses direct injection of microquantities of serum into a separation column filled with octyl-C(8) silica support that has a semipermeable surface. A mixture of disodium hydrogen phosphate buffer (pH 7.0) and acetonitrile is used as the mobile phase, and vancomycin is directly detected at 240 nm. The minimum limit of detection was 0.5 microg/ml at a signal-to-noise ratio of 3:1. Linearity was established from 0 to 100 microg/ml. The coefficient of variation for within-run reproducibility was 1.1-2.7% for a concentration range of 2.9-52.5 microg/ml; for day-to-day reproducibility it was 4.0% and 3.1% for a concentration range of 5.8-26.4 microg/ml, and the recovery rate was 94-105%. There was no interference from 41 antibiotics or other drugs currently in use. The correlation coefficient between the fluorescence polarization immunoassay (x) and this method (y) was 0.995 with a linear equation, y = 1.06x - 0.924. This method is simple, rapid, and provides an economical quantification of serum vancomycin.

Anti-Bacterial Agents↗
PubMed2000

Sibling incest and formulation of paternity probability: case report.

Paternity determination of a fetus whose mother was admitted to an institution for the welfare and health of handicapped persons was requested of us by a doctor and lawyer of the institution. The fetus was recovered by a legal artificial abortion based on the Act on Maternity Health and Welfare (Japan) with the permission of the custodian. Commercially available MCT118, HLADQA, PM, and 9 STRs were tested for DNA samples from the fetus, the mother, her younger brother, her father, her grandfather, and 4 staff members of the institution. Only the brother was not excluded and the paternity probability was estimated at 99.857% on the basis of newly formulated expressions for multiallelic loci on the assumption of sibling incest. We concluded then that the fetus was fathered by the brother. DNA fingerprinting with multilocus and single locus minisatellite probes which were performed to confirm the paternity also support the conclusion. Bandsharing frequencies between the family members, however, did not necessarily reflect their actual kinship, which findings suggest that multilocus DNA fingerprinting requires further accumulation of data for consanguineous cases such as incest. Universal formulation for calculating paternity probability for a sibling incest case on the basis of multiallelic monolocus polymorphisms is also presented.

Journal Article↗
PubMed2000

Solution synthesis and biological activity of human pleiotrophin, a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five disulfide bonds.

Human pleiotrophin (hPTN), a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five intramolecular disulfide bonds, was synthesized by solution procedure in order to demonstrate the utility of our strategy using our newly developed solvent system, a mixture of trifluoroethanol (TFE) and dichloromethane (DCM) or chloroform (CHL). The final protected peptide was synthesized by coupling two larger protected intermediates, Boc-(1-64)-OH and H-(65-136)-OBzl, in CHL/TFE (3:1; v/v) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). After removal of all protecting groups using the HF procedure followed by treatment with Hg(OAc)2, the fully deprotected peptide was subjected to an oxidative folding reaction. The product was confirmed as having the correct disulfide structure by examining the cystine peptides obtained by enzymatic digestions, and as possessing the same biological activities as those of the natural product. The N- and C-terminal half domains (1-64 and 65-136) were also synthesized, and measurement of their biological activities indicated that the C-terminal half domain displays almost all the activities of the full-length molecule, whereas the N-terminal half domain shows almost no activity. From these results, we were able to confirm that the C-terminal half domain is responsible for the expression of biological activities in the same manner as human midkine (hMK), another heparin-binding neurotrophic growth factor.

Amino Acid Sequence↗
PubMed2000

Involvement of cyclic AMP - PKA pathway in VIP-induced, charybdotoxin-sensitive relaxation of longitudinal muscle of the distal colon of Wistar-ST rats.

The intracellular mechanism of vasoactive intestinal peptide (VIP)-induced, charybdotoxin (ChTx)-sensitive relaxation of longitudinal muscle of the distal colon of Wistar-ST rats was studied. A single pulse or 100 pulses at 10 Hz of electrical field stimulation (EFS) induced rapid transient relaxation or that with a subsequent contraction of the longitudinal muscle in the presence of atropine and guanethidine, respectively. Rp-8 bromo cAMPS, an inhibitor of cyclic AMP dependent protein kinase (PKA), at 30 microM inhibited the relaxations induced by EFS with a single or 100 pulses maximally by about 80 or 60%, respectively. It also inhibited VIP (300 nM)-induced relaxation by 82%. VIP (100 nM - 1 microM) increased the cyclic AMP content of longitudinal muscle myenteric plexus preparations obtained from the distal colon. ChTx at 100 nM almost completely inhibited 8 bromo cyclic AMP-induced relaxation of the distal segments. EFS with two or three pulses at 10 Hz induced inhibitory junction potentials consisting of two phases, rapid and subsequent slow hyperpolarization in the membrane potential of longitudinal smooth muscle cells. Rp-cAMPS, another inhibitor of PKA, inhibited the delayed slow hyperpolarization. It also inhibited the exogenously added VIP-induced hyperpolarization of the cell membrane. Thus, the present study suggests that activation of PKA via activation of VIP receptors is associated with activation of ChTx-sensitive K(+) channels in relaxation of longitudinal muscle of the distal colon of Wistar-ST rats. British Journal of Pharmacology (2000) 129, 140 - 146

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Increase in participation of vasoactive intestinal peptide in relaxation of the distal colon of Wistar rats with age.

Changes in participation of vasoactive intestinal peptide (VIP) in nonadrenergic noncholinergic (NANC) relaxation of longitudinal muscle of the distal colon with age were studied in 2- to 50-week-old Wistar rats in vitro. The extent of the VIP-mediated component of the relaxation induced by electrical field stimulation (EFS) was determined by the effect of VIP(10 - 28), a VIP receptor antagonist. In 2-week-old rats, the extent of the VIP-mediated component of the relaxation was scarce, about 10%, whereas the component gradually increase with age and reached the maximum extent 66% at 50-week-old. Since our previous results suggest that VIP induces NANC relaxation via activation of charybdotoxin (ChTx, a blocker of large conductance Ca(2+)-activated K(+) channel)-sensitive K(+) channels with concomitant slow hyperpolarization in the muscle cells, we next studied whether ChTx-sensitive component and slow hyperpolarization changes with age. Extent of ChTx-sensitive component of the relaxation increased with age, showing a very similar pattern to VIP-mediated one. EFS induced monophasic inhibitory junction potentials (i.j.ps) in longitudinal muscle cells of the distal colon of 2- and 4-week-old. EFS also induced biphasic i.j.ps in many longitudinal muscle cells of 8- and 50-week-old: rapid and subsequent slow hyperpolarization. A VIP receptor antagonist selectively inhibited the slow hyperpolarization. Exogenously added VIP induced no appreciable change in the membrane potential of longitudinal muscle cells of 2-week-old, whereas it induced slight slow hyperpolarization of the cell membrane in 4-week-old and magnitude of the hyperpolarization increased with age. On the other hand, relaxant response of the longitudinal muscle to exogenously added VIP was high in younger rats. The present results suggest that the role of VIP in mediating NANC relaxation of longitudinal muscle of the Wistar rat distal colon is very little at neonatal stage, but it increases with age.

Age Factors↗