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H Shibata

Publications and source records attributed to H Shibata.

At least 397 records · Page 22Linked to original sources
PubMed1993

Molecular cloning of polymorphic markers on RLGS gel using the spot target cloning method.

A new method for target cloning of DNA fragments corresponding to spots on the two-dimensional restriction landmark genomic scanning (RLGS) profile has been developed (targeted spot cloning). We used a Not I restriction trapper to select target DNA fragments from Not I, Eco RV double digests of genomic DNA. The use of the restriction trapper substantially reduces the background clones that are established from the direct recovery of RLGS spot DNA from the two-dimensional gels. Genomic DNA clones were isolated in this study as mouse genome markers for 58 spot loci that were previously characterized using RLGS spot mapping. This method provides a powerful tool for isolating DNA clones after their identification by RLGS system.

Animals↗
PubMed1993

[Adrenal incidentaloma].

Recent increased use of ultrasonography and computed tomography (CT) has detected a substantial number of incidentally discovered adrenal tumor (incidentaloma). In our institute the discovery rate of adrenal incidentaloma per number of abdominal CT examinations was 0.43%. Those incidentalomas were incidentally found during examination for abdominal discomfort and lumbar pains or evaluation of the cause of hypertension. In the group study by the research committee on "Disorders of Adrenal Steroid Hormones" under the sponsorship of the Ministry of Health and Welfare of Japan, in 53 of 149 patients with incidentaloma, surgical resection was done during 5 years between 1983 and 1989. The pathological examination of those tumors revealed 67.9% of non-hyperfunctioning adrenocortical adenoma, 9.4% of adrenal cancer, 9.4% of ganglioneuroma, 5.7% of pheochromocytoma, 5.7% of adrenal cyst and 1.9% of myelolipoma. The criteria for resection of adrenal incidentaloma have not been established. However, tumors having a diameter of over 5 cm are generally excised in most institutes. Tumors having a diameter of between 3 cm and 5 cm are usually resected if the shape of the tumor is ambiguous and the margin of the tumor is irregular. Tumors having a diameter of less than 3 cm should be carefully followed up by abdominal CT examination every 6 months.

Adrenal Gland Neoplasms↗
PubMed1993

Role of calcium entry and protein kinase C in the progression activity of insulin-like growth factor-I in Balb/c 3T3 cells.

We previously reported that insulin-like growth factor-I (IGF-I) stimulated calcium entry (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1988) J. Biol. Chem. 263, 16561-16567) and production of 1,2-diacylglycerol in IGF-responsive "primed competent" Balb/c 3T3 cells (Kojima, I., Kitaoka, M., and Ogata, E. (1990) J. Biol. Chem. 265, 16846-16850). The present study was conducted to determine a role of protein kinase C (PKC) in the progression activity of IGF-I. To monitor the activity of PKC in intact cells, we measured phosphorylation of a synthetic KRTLRR peptide, a substrate of PKC, immediately after the permeabilization of the cells with digitonin. When 1 nM IGF-I was added to primed competent cells, KRTLRR peptide phosphorylation was augmented. IGF-I induced more than 2-fold increase in KRTLRR peptide phosphorylation that was blocked by PKC19-36, a pseudosubstrate of PKC, which blocks the activity of the kinase, and Ro31-8220, an inhibitor of PKC. The phosphorylation remained elevated for up to 6 h. To assess the role of PKC in cell cycle progression, IGF-I-induced nuclear labeling was measured in the presence of Ro31-8220. Ro31-8220 reduced the rate of entrance into S phase when added in the first quarter of the G1 phase, but did not affect cell cycle progression when added at the second quarter or later. In contrast, reduction of extracellular calcium completely blocked cell cycle progression when done in the first, second, and third quarter but had no effect in the last quarter. These results indicate that IGF-I persistently activates PKC in primed competent cells, but the activation is required only for the initiation of progression. We conclude that IGF-I promotes cell cycle progression by calcium-dependent mechanisms that are largely independent of PKC.

3T3 Cells↗
PubMed1993

Efferent projections from the anterior thalamic nuclei to the cingulate cortex in the rat.

The organization of projections from the anterior thalamic nuclei to the cingulate cortex was analyzed in the rat by the anterograde transport of Phaseolus vulgaris-leucoagglutinin. The rostral part of the anteromedial nucleus projects to layers I, V and VI of the anterior cingulate areas 1 and 2, layers I and III of the ventral orbital area, layers I, V and VI of area 29D of the retrosplenial area, and layers I and V of the caudal part of the retrosplenial granular and agranular areas. In contrast, the caudal part of the anteromedial nucleus projects to layer V of the frontal area 2, and layers I and V of the rostral part of the retrosplenial granular and agranular areas. The interanteromedial nucleus projects to layers I, III and V of the frontal area 2, layer V of the agranular insular area, and layers I, V and VI of area 29D. The anteroventral nucleus projects to layers I and IV of the retrosplenial granular area, whereas the anterodorsal nucleus projects to layers I, III and IV of the same area. Projections from the anteroventral and anterodorsal nuclei were, furthermore, organized such that their ventral parts project to the rostral part of the retrosplenial granular area, whereas their dorsal parts project to the more caudal part. The results suggest that the anterior thalamic nuclei project to more widespread areas and laminae of the cingulate cortex than was previously assumed. The projections are organized such that the anteromedial and interanteromedial nuclei project to layer I and the deep layers of the anterior cingulate and retrosplenial cortex, whereas the anteroventral and anterodorsal nuclei project to the superficial layers of the retrosplenial cortex. These thalamocortical projections may play important roles in behavioral learning such as discriminative avoidance behavior.

Animals↗
PubMed1993

Restriction landmark genomic scanning method and its various applications.

We have developed a new genome scanning method (restriction landmark genomic scanning (RLGS), based on the new concept of using restriction enzyme sites as landmarks. RLGS employs direct end labeling of the genomic DNA digested with a restriction enzyme and two-dimensional electrophoresis with high-resolution. Its advantages are: (i) high-speed scanning ability, allowing simultaneous scanning of thousands of restriction landmarks; (ii) extension of the scanning field using different kinds of landmarks in an additional series of electrophoresis; (iii) application to any type of organism because of direct-labeling of restriction enzyme sites and no hybridization procedure; and (iv) reflection of the copy number of the restriction landmark by the spot intensity which enables distinction of haploid and diploid genomic DNAs. The RLGS method has various applications because it can be used to scan for physical genomic DNA states, such as amplification, deletion and methylation. The copy number of the locus of a restriction landmark can be estimated by the spot intensity to find either an amplified or deleted region. The methylation state of genomic DNA can also be discovered by use of a methylation-sensitive restriction enzyme sites as a restriction landmark (restriction landmark genomic scanning for screening methylated sites, RLGS-M). This article introduces the basic principle of RLGS and its applications to the analysis of cancer, mouse mutant DNAs and tissue-specific methylation, showing the usefulness of RLGS for a variety of biological fields.

Animals↗
PubMed1993

Modulation of growth of vascular smooth muscle cells by activin A.

Activin A is a homodimeric protein structurally related to transforming growth factor-beta. The present study was conducted to examine the effect of activin A on the growth of rat vascular smooth muscle cells (VSMC) in culture. When serum-deprived cells were incubated with activin A, nuclear labeling of bromodeoxyuridine occurred after a 12-h lag period. The effect of activin A on nuclear labeling was dose-dependent, being maximal at 10(-9) M. Activin A also increased the number of VSMC after 30 h of incubation. Insulin-like growth factor-1 (IGF-1) had only a small effect on nuclear labeling by itself but the effects of IGF-1 and activin A were additive. When quiescent VSMC were treated with activin A for 4 h, the effect of subsequent IGF-1 was markedly enhanced. Furthermore, activin A induced an autocrine production of IGF-1 in VSMC. In contrast to these positive effects on cell growth, activin A was rather inhibitory to the action of IGF-1 in activin-primed cells. In addition, activin A inhibited platelet-derived growth factor-induced nuclear labeling. These results indicate that activin A modifies growth of VSMC by complex mechanisms involving autocrine production of IGF-1 and modification of the action of IGF-1.

Activins↗
PubMed1993

Topographic relationship between anteromedial thalamic nucleus neurons and their cortical terminal fields in the rat.

The present study has examined the topographic relationship between cells in the anteromedial thalamic nucleus (AM) and their cortical terminal fields, with retrograde transport of Fluoro Gold in the rat. Projections to the frontal area 2 originate from the ventrolateral part of the AM and the entire interanteromedial nucleus (IAM). Projections to the anterior cingulate area originate from the peripheral part of the rostral AM and the entire IAM. Fibers to the rostral retrosplenial area arise from the caudodorsal part of the AM, whereas those to the caudal retrosplenial area arise from the rostralmost and the rostrodorsomedial parts. Fibers to the rostral area 29D originate from the rostrocentral part of the AM, whereas those to the caudal area 29D originate from the rostroventrolateral and the ventromedial parts. Projections to the medial half of the entorhinal area originate from the rostrodorsomedial part of the AM. In contrast, projections to the lateral half of the entorhinal area originate from the IAM and the central part of the AM. The results show a complex topographic relationship between cells of origin of the AM and their cortical terminal fields, suggesting complex functional roles played by the AM in learning behavior such as discriminative avoidance behavior.

Animals↗
PubMed1993

Tension inhibitory effect of lead ions and lead uptake in ileal longitudinal muscle of the guinea-pig.

1. In ileal longitudinal smooth muscle of the guinea-pig, lead ions (Pb2+) inhibited the phasic response to high-K+ (60 mM) solution more strongly than the tonic response. 2. Pb2+ displaced Ca2+ concentration-response curves to the right. Moreover, Pb2+ inhibited the 45Ca binding at high affinity sites determined by the La3+ method than low affinity sites during K(+)-induced contraction. 3. After treatment with Pb2+, the tonic tension caused by high-K+ was fairly restored by a wash with normal medium. The lead bound to the muscle cells was almost eliminated after 20-30 min with a normal medium. 4. We suggest that Pb2+ binds loosely to the surface membrane of ileal longitudinal muscle. Pb2+ probably reduced the tension in response to the high-K+ mainly by inhibiting Ca2+ release rather than by affecting Ca2+ influx.

Animals↗
PubMed1993

Inhibitory effects of hexavalent chromium ions on the contraction in ileal longitudinal smooth muscle of guinea-pig.

1. The high-K (60 mM)-induced ileal tonic tensions were not affected after 5 min with (0.01-1 mM) Cr6+. However, they were inhibited dose-dependently by Cr6+ after 30 min application. 2. Cr6+ inhibited the tonic response to K more strongly than the phasic response. 3. Cr6+ did not displace Ca(2+)-concentration-response curves to the right, although it reduced the Ca2+ response size. 4. The low affinity binding sites of Ca2+ and the tissue ATP concentration during K contraction was not entirely inhibited after 5 min with 0.5 mM Cr6+. However, they were fairly inhibited after 30 min with the Cr6+. 5. We suggest that Cr6+ probably reduced tension by the interference of Ca2+ influx at the cell membrane resulting from the inhibitory action on mitochondria.

Animals↗
PubMed1993

Blockade by nickel ions of phasic contraction to K+ and high affinity calcium of ileal longitudinal muscle of guinea-pig.

1. Preincubation with 1 or 2 mM Ni2+ inhibited dose-dependently the ileal phasic response to K+ (60 mM) without appreciable effects on the tonic response. Ni2+ above 3 mM inhibited the tonic response. 2. Ni2+ inhibited the high affinity Ca2+ sites than the low affinity sites during K+ contraction. 3. After treatment with Ni2+, the K+ response was fairly restored by a wash with normal medium. The nickel bound to the ileal cells was almost eliminated with the washing. 4. This probably indicates that Ni2+ mainly inhibited the K(+)-induced phasic tension by reducing Ca2+ release rather than Ca2+ influx.

Animals↗
PubMed1993

Purification and characterization of endogenous protein activator of human platelet proteasome.

An endogenous activator of 20S proteasome was purified from human platelets and its effect on three peptidase activities of proteasome was studied. This activator had a molecular weight of 170 kDa, and was composed of 32 kDa polypeptides as determined by SDS-PAGE. It was highly labile upon heat treatment (56 degrees C, 20 s) and proteinase (pronase CB) digestion. Suc-LLVY-MCA degrading activity of the platelet proteasome showed positive cooperativity between two or more catalytic sites because the coefficient was 1.54 when analyzed by use of the Hill plot. The endogenous activator increased Vmax and caused a loss of cooperativity. The plot of reaction velocity as a function of activator concentration yielded a saturation curve, implying the binding of the activator to proteasome. Boc-LTR-MCA degrading activity followed Michaelis-Menten kinetics. The activator enhanced the activity by increasing Vmax and decreasing Km. In contrast, CBz-LLE-2NA degrading activity could not be analyzed according to any kinetic scheme reported so far. The activator stimulated this activity at lower substrate concentrations (below 200 microM), while it inhibited the activity at higher substrate concentrations (400-800 microM). It is concluded from these findings that the endogenous protein activator may regulate the intracellular proteasome activity by functioning as a positive allosteric effector.

Blood Platelets↗
PubMed1993

Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) on germination of Bacillus cereus T spores.

Effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), a calcium antagonist, on germination of Bacillus cereus T spores induced by L-alanine and inosine was investigated. TMB-8 had no effect on the germination of heat-activated spores, whereas it inhibited that of nonactivated spores. The TMB-8 inhibitory effect was antagonized competitively by inosine, but not by L-alanine. Addition of Ca2+ reversed the inhibitory effect of TMB-8 in a dose-related fashion. Based on the results, a role of inosine and a site(s) for inhibitory action of TMB-8 in the process leading to the germination of nonactivated spores were discussed.

Alanine↗
PubMed1993

Role of calcium in biphasic germination of Bacillus cereus T spores sensitized to lysozyme.

Biphasic germination induced by inosine in the presence of Ca2+ was examined using Bacillus cereus T spores treated with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) at pH 10. The first phase of the germination was stimulated by Ca2+ in the concentration-dependent manner, showing the optimal concentration at 0.5-1.0 mM. The second phase appeared to be insensitive to the cation. The optimal temperatures for the first and the second phase were 25 C and 40 C, respectively; the optimal pHs for the two phases were 7-9 and around 7.5, respectively. Heat resistance and dipicolinic acid of the SDS-DTT-treated spores were lost mostly during the first phase. A Ca(2+)-specific chelator, glycoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), inhibited the first phase evoked by Ca2+, while it had no inhibitory effect on the second phase. In contrast, the divalent cations examined, except Mg2+ and Sr2+, affected not only the first phase but also the second phase. The order of inhibitory effect on the first phase was Hg2+ > Zn2+ > Ba2+, Co2+, Cu2+ > Mn2+; on the second phase, it was Hg2+ > Cu2+ > Zn2+ > Co2+ > Mn2+ > Ba2+.

Bacillus cereus↗
PubMed1993

[Detection and identification of HBV DNA and DNA subtypes of HBs-antigen by the use of the polymerase chain reaction and non-radioactive probes].

A technique of nested polymerase chain reaction (PCR) and hybridization with non-radioactive probes was developed for the detection and identification of HBV DNA and HBs-subtypes in very small volumes of human sera. Four oligonucleotide primers (20 mer) complementary to DNA sequences in the S region of HBV and probes (18 or 20 mer) conjugated to alkaline phosphatase were used for the present PCR assay. The results of the PCR assay coincide with those of enzyme immunoassay (EIA) in 14 HBe-positive and 59 HBe-negative samples with 98.6% of specificity. The HBV subtypes adr and adw were identified using an 18-mer DNA probe in 30 samples with an accuracy of 100%. Further, the DNA subtypes were clearly demonstrated in 3 samples where HBs-antigen was undetectable. These results indicate that amplification of HBV DNAs by PCR and their detection with non-radioactive probes is a reliable tool for diagnosis of HBV infection in clinical laboratories.

Base Sequence↗
PubMed1993

Coexisting undifferentiated thymic carcinoma and thymic carcinoid tumor.

Thymic carcinoma is an unusual neoplasm, and the undifferentiated type is rare. Thymic carcinoid is also rare. This report describes a patient with coexisting undifferentiated thymic carcinoma and a carcinoid tumor. Both lesions were completely excised. The carcinoid cells showed argyrophilic granules by Grimelius' method and immunoreactivity against neuron-specific enolase, whereas the undifferentiated carcinoma cells were negative for argyrophilic stain and immunostaining against neuron-specific enolase.

Carcinoid Tumor↗
PubMed1993

Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes.

The present study was conducted to examine the effect of activin A on growth of rat hepatocytes. EGF induced a 10-fold increase in DNA synthesis as assessed by [3H]thymidine incorporation in cultured hepatocytes. When activin A was added together with EGF, DNA synthesis induced by EGF was markedly inhibited. Inhibition was detected at a concentration of 10(-10) M, and 5 x 10(-9) M activin A almost completely blocked EGF-mediated DNA synthesis. Similarly, activin A completely blocked DNA synthesis induced by hepatocyte growth factor/scatter factor. Activin A was capable of inhibiting EGF-mediated DNA synthesis, even when added 36 h after the addition of EGF. With the same time interval, TGF-beta also blocked EGF-induced DNA synthesis. Although both activin A and TGF-beta inhibited growth of hepatocytes in a similar manner, either activin A or TGF-beta did not compete with each other in their binding when assessed by competitive binding using an iodinated ligand. When hepatocytes were incubated with EGF, release of bioactivity of activin A into culture medium was detected after 48 h or later. Activity of activin A was released from parenchymal cells but not from nonparenchymal cells. mRNA for beta A subunit of activin was detected only slightly in unstimulated hepatocytes, but markedly increased at 48 h after the addition of EGF. To determine whether endogenously produced activin A affects DNA synthesis, we examined the effect of follistatin, an activin-binding protein that blocks the action of activin A. An addition of follistatin significantly enhanced EGF-induced DNA synthesis. Finally, in partial hepatectomized rat, expression of mRNA for beta A subunit in liver was markedly increased 24 h after the partial hepatectomy. These results indicate that activin A inhibits initiation of DNA synthesis in hepatocytes by acting on its own receptor and that activin A acts as an autocrine inhibitor of DNA synthesis in rat hepatocytes.

Activins↗
PubMed1993

Existence of activin-A in A- and D-cells of rat pancreatic islet.

Activin-A, a member of the transforming growth factor-beta supergene family, stimulates insulin secretion in rat pancreatic islets and causes glycogenolysis in isolated rat hepatocytes. These observations prompted us to determine whether activin-A existed in rat pancreas by using an immunocytochemical method. Cells in pancreatic islets were stained by antibody against activin-A, whereas no immunoreactivity was observed in exocrine pancreas. Cells localized in the mantle of the islets were densely stained by the antibody. Immunoelectron microscopic study showed that activin-A existed in secretory granules in both A- and D-cells. Furthermore, studies using a double labeling method revealed that activin-A coexisted with glucagon in secretory granules in A-cells and with somatostatin in D-cells. Antibody against inhibin-A weakly stained cells in both the core and mantle of the islets only when the rat was pretreated with colchicine. Subtypes of activin subunit in islets were identified to be beta A by a reverse transcription-polymerase chain reaction method. In addition, mRNA for inhibin alpha-subunit was expressed in islets. However, mRNA for these inhibin subunits was not detected in exocrine pancreas. To further examine the action of activin-A on insulin secretion, we examined the effect of activin-A in a flow-through perifusion system. Activin-A induced a biphasic insulin secretory response in the presence of 2.8 mM glucose, and a low concentration of activin-A, which does not stimulate insulin secretion by itself, markedly enhanced glucose-mediated insulin secretion at concentrations above 2.8 mM glucose. Inhibin-A did not affect insulin secretion. These results suggest the existence of activin-A in A- and D-cells of rat pancreatic islets and raise the possibility that activin-A acts as a physiological regulator of carbohydrate metabolism.

Activins↗