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Biomedical subjects

K Chihara

Publications and source records attributed to K Chihara.

At least 181 records · Page 10Linked to original sources
PubMed1995

Fasting increases the expression of basic fibroblast growth factor (FGF-2) messenger ribonucleic acid in rat hypothalamus.

Basic fibroblast growth factor (FGF-2) has been isolated from the brain, but the regulation of FGF-2 synthesis in the brain is not yet fully understood. Since exogenously administered FGF-2 has been reported to suppress food intake as well as the secretion of gastric acid and pepsin in rats, we examined the effect of fasting on FGF-2 mRNA levels in the hypothalamus and the cerebral cortex of male rats, using RNase protection assay. Fasting for 72 h resulted in an approximately 2-fold increase in FGF-2 mRNA level in the hypothalamus but did not affect FGF-2 mRNA level in the cerebral cortex significantly. These findings support the hypothesis that FGF-2 plays a significant role in regulation of hypothalamic function.

Adrenalectomy↗
PubMed1995

Establishment of a myeloid leukaemic cell line (SKNO-1) from a patient with t(8;21) who acquired monosomy 17 during disease progression.

A novel cell line SKNO-1 was established from the bone marrow cells of a 22-year-old male suffering from acute myeloblastic leukaemia (AML) M2 with t(8;21) whose disease became resistant to chemotherapy after acquisition of 17 monosomy. SKNO-1 has been maintained for more than 36 months as a granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent line. Morphologically, SKNO-1 cells were myeloblasts somewhat matured. The cells grow in suspension with a doubling time of 48-72 h. The survival and growth of SKNO-1 cells was absolutely dependent on granulocyte-macrophage colony stimulating factor (GM-CSF). SKNO-1 cells possessed t(8;21) and monosomy 17 which were observed in original leukaemic cells. We confirmed that the AML1 gene, located on chromosome 21, was rearranged and the AML1-MTG8 fusion transcript was expressed in SKNO-1 cells. Over-expression and mutation of the p53 gene were also detected in SKNO-1. It is likely that alterations of AML1 or MTG8 gene and p53 gene contribute to a disease progression in this case. Since t(8;21) translocation is a common chromosome abnormality in AML, and inactivation of the p53 gene may play a crucial role in disease progression in AML, SKNO-1 would be a useful tool for analysing the molecular mechanisms in myeloid leukaemogenesis.

Adult↗
PubMed1995

Human TRH receptor messenger ribonucleic acid levels in normal and adenomatous pituitary: analysis by the competitive reverse transcription polymerase chain reaction method.

OBJECTIVE: Little is known about the mechanism of diversity in in-vivo hormonal responsiveness to TRH in patients with functional pituitary adenomas. In order to clarify the relation between the responsiveness to TRH and TRH receptor messenger ribonucleic acid (mRNA) expression, we attempted to measure TRH receptor mRNA levels in human pituitary adenoma tissues by competitive reverse transcription polymerase chain reaction (RT-PCR) method. PATIENTS: Pituitary tissue samples were obtained at autopsy from 5 patients without pituitary disease. Pituitary adenoma tissue samples were obtained at surgery from 18 patients with pituitary adenoma (4 non-functioning, 8 prolactinoma, 4 acromegaly, 1 Cushing's disease and 1 FSH producing adenoma). METHODS: Partial TRH receptor cDNA from a human GH producing adenoma cDNA library was amplified by PCR under low stringency conditions using primers encoding the transmembrane domains III and VI of pituitary TRH receptor cDNA. The partial sequence of the amplified cDNA determined by a dideoxy-chain termination method was identical to the corresponding sequence of human TRH receptor cDNA. A competitor was generated by deleting the inner 111 bp from the amplified TRH receptor cDNA and subcloning. RNA extracted from human pituitary was reverse transcribed and co-amplified with competitor by PCR under higher stringency conditions. The TRH receptor mRNA levels, expressed as the relative intensity against the amplified levels of competitor, were compared among various pituitary tissues. RESULTS: The relative TRH receptor mRNA levels of pituitary tissues in patients without pituitary disease were detectable and variable (M +/- SD) (0.370 +/- 0.231, n = 5), and slightly but not significantly lower than those in patients with pituitary tumours (0.598 +/- 0.265, n = 18). In patients with prolactinoma, the relative levels of TRH receptor mRNA were quite variable (0.02-1.170, 0.604 +/- 0.358, n = 8) and not correlated with PRL responsiveness to TRH (responder 0.457; non-responder 0.340-0.950). In patients with acromegaly, TRH receptor mRNA was detectable not only in the paradoxical GH responder to TRH (0.718) but also in the non-responder (0.758 and 0.765). In one patient with Cushing's disease, a relatively low level of TRH receptor mRNA could be detected (0.415). In the patient with a FSH producing tumour whose plasma FSH did not respond to TRH, a small amount of TRH receptor mRNA was detectable (0.447). CONCLUSIONS: In patients with functioning pituitary adenomas, hormonal responsiveness to TRH in vivo might not be assessable by TRH receptor mRNA levels in the adenoma cells.

Adenoma↗
PubMed1995

Central glucopenia induced by 2-deoxy-D-glucose stimulates somatostatin secretion in the rat.

The mechanisms involved in 2-deoxy-D-glucose (2-DG)-induced growth hormone (GH) suppression in the rat were examined. Conscious male rats were given 2-DG by intracerebroventricular (icv) injection and the pulsatile GH secretion was monitored for 6 h. The single icv injection of 2-DG (8 mg/rat) eliminated pulsatile GH secretion in conscious rats. Pretreatment with somatostatin (SS) antiserum completely restored the suppressed GH secretion in the 2-DG treated rats. Hypothalamic GH-releasing hormone (GRH) and SS mRNA levels were not altered by single and multiple icv injections of 2-DG. These findings suggest that 2-DG-induced GH suppression is primarily due to hypersecretion of SS without a significant change at the transcription level in the rat.

Animals↗
PubMed1995

Regional distribution of growth hormone-releasing hormone (GHRH) receptor mRNA in the rat brain.

We examined the tissue distribution of growth hormone-releasing hormone (GHRH) receptor mRNA in the rat brain because several lines of evidence have suggested that GHRH plays a functional role in the brain. GHRH receptor mRNA was detected in the hypothalamus as well as pituitary, but not in olfactory bulb, caudate putamen, cerebral cortex, hippocampus, cerebellum, or brainstem by RNase protection assay. To clarify the precise localization of GHRH receptor mRNA in the hypothalamus, reverse transcription-polymerase chain reaction (RT-PCR) was used. A PCR product of the predicted size (564 bp) was detected in the periventricular, arcuate, and ventromedial nuclei, and the anterior hypothalamic area, but not in the paraventricular nucleus in the hypothalamus. These areas where GHRH receptor mRNA was detected are possible sites of GHRH action. Another band, smaller in size than that of the predicted PCR amplification product, was detected in the anterior hypothalamic area and arcuate nucleus, respectively. Hybridization analysis with a cDNA probe for GHRH receptor demonstrated that the smaller bands as well as that of the predicted size corresponded to GHRH receptor cDNA. The role of the short form of the GHRH receptor remains unknown.

Animals↗
PubMed1995

Carboxyl-terminal peptides from parathyroid hormone-related protein stimulate osteoclast-like cell formation.

The role of the carboxyl (C)-terminal portion of PTH-related protein (PTHrP) in bone resorption continues to be controversial. The present study was performed to examine the effect of C-terminal PTHrP peptides on osteoclast-like cell formation as well as bone resorption in mice. C-Terminal PTHrP peptides [human (h) PTHrP-(107-139) and hPTHrP-(107-111); 10(-10)-10(-8) M] stimulated osteoclast-like cell formation in a concentration-dependent manner in osteoblast-containing mouse bone cell cultures. Moreover, osteoclast-like cells newly formed by these peptides possessed the ability to form pits on the dentine slices. The conditioned medium from UMR-106 cells and MC3T3-E1 cells pretreated with the C-terminal peptides did not affect osteoclast-like cell formation from mouse hemopoietic blast cells derived from spleen cells. The C-terminal peptides as well as hPTHrP-(1-34) stimulated osteoclast-like cell formation from mouse hemopoietic blast cells in the absence of osteoblasts, although both amino- and C-terminal peptides were unable to support hemopoietic blast cells. Protein kinase-C inhibitors (H-7 and staurosporine) almost completely inhibited the stimulation of osteoclast-like cell formation by the C-terminal peptides in both the presence and absence of osteoblasts. The C-terminal peptides did not affect bone resorption by mature osteoclasts in osteoblast-containing mouse bone cell cultures. The present study indicates that C-terminal PTHrP peptides possess the ability to stimulate osteoclast-like cell formation in both the presence and absence of osteoblasts, possibly through the pathway involving protein kinase-C activation.

Animals↗
PubMed1995

Effects of 22-oxacalcitriol on bone metabolism in vitro: comparison with calcitriol--effects of 22-oxacalcitriol on osteoclast-like cell formation and bone-resorbing activity.

22-Oxacalcitriol (OCT), a synthetic vitamin D3 analog, can mimic the ability of calcitriol to differentiate leukemia and skin cells, to enhance the immune response and to suppress parathyroid hormone secretion, but has much less calcemic activity than that of calcitriol. The mechanism of this selective action remains not fully understood, and the actions of OCT on bone metabolism are little known. The present study was, therefore, designed to investigate the effects of OCT and calcitriol on: the proliferation and functions of osteoblastic MC3T3-E1 cells; osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells as well as from unfractionated bone cells in the presence of stromal cells; bone resorption; and the proliferation of MC3T3-E1 cells via monocytes. 22-Oxacalcitriol and calcitriol inhibited [3H]thymidine (TdR) incorporation, alkaline phosphatase activity and collagen synthesis of MC3T3-E1 cells to a similar degree. Both OCT (10(-10)-10(-8) mol/l) and calcitriol significantly and similarly stimulated osteoclast-like cell formation from both hemopoietic blast cells and unfractionated bone cells. 22-Oxacalcitriol (10(-10) and 10(-8) mol/l) significantly stimulated bone resorption, although to a slightly lesser degree than did calcitriol. Human monocyte-conditioned medium (CM) significantly stimulated TdR incorporation into MC3T3-E1 cells. On the other hand, CM obtained from monocytes treated with calcitriol (10(-10)-10(-8) mol/l) significantly inhibited TdR incorporation in a dose-related fashion, whereas CM obtained from monocytes treated with OCT (10(-10)-10(-8) mol/l) significantly stimulated TdR incorporation in a dose-related fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1995

Role of alternatively spliced beta E-globin mRNA on clinical severity of beta-thalassemia/hemoglobin E disease.

In spite of seemingly identical genotypes, severity of beta-thalassemia/hemoglobin (Hb) E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. Since mutation in codon 26 of the beta E-globin gene can lead to an alternative splicing, Hb E acts like a mild beta(+)-thalassemia. Variation in the amount of beta E-globin mRNA may also govern the difference in severity of anemia in beta-thalassemia/Hb E patients who otherwise have the same genetic determinants. We have determined the percentage of the alternatively spliced beta E-globin mRNA by the RT-PCR technique in 14 patients and found that the amount of abnormal spliced beta E-globin mRNA in those patients with severe symptoms ranged between 2.9 to 6.1%, whereas those with milder symptoms had the values which ranged between 1.6 to 2.6%. The extent of beta E-globin mRNA cryptic splicing was better associated with clinical severity of the patients than did the patterns of the Xmn I polymorphism at position -158 of the G gamma-globin gene or levels of Hb F.

Adolescent↗
PubMed1994

Persistent expression of proto-oncogene c-fos stimulates osteoclast differentiation.

We analyzed c-fos mRNA expression by northern blotting analysis in chicken osteoclast precursors which spontaneously differentiate to multinucleated osteoclasts in 5-6 days. Osteoclast precursors as well as mature multinucleated osteoclasts showed constitutive expression of c-fos mRNA which is not found in osteoblasts. The c-fos expression was enhanced transiently by serum, dibutyryl cAMP (10(-4) M) and phorbol 12-myristate 13-acetate (TPA) (5 x 10(-7) M). To clarify the role of c-fos in osteoclast differentiation, c-fos DNA was transfected into osteoclast precursors. Greater than 2 fold increases in tartrate resistant acid phosphatase (TRAP) and bone resorptive activity were observed in the transfected cells compared to controls 3 days after transfection, suggesting that prolonged expression of c-fos caused enhanced osteoclast differentiation.

Acid Phosphatase↗
PubMed1994

Somatostatin receptor subtype SSTR2 mediates the inhibition of high-voltage-activated calcium channels by somatostatin and its analogue SMS 201-995.

Somatostatin and its analogue SMS 201-995 inhibit high voltage-activated (HVA) Ca2+ currents in the rat insulinoma cell line RINm5F which stably express cloned human somatostatin receptor subtype 2 (hSSTR2). In contrast, neither somatostatin nor SMS 201-995 suppresses the HVA Ca2+ currents in RINm5F which stably express cloned hSSTR1. These results suggest that somatostatin-induced inhibition of HVA Ca2+ currents is mediated by a specific receptor subtype and that inhibition of calcium influx through HVA Ca2+ channels is one of the mechanisms of SMS 201-995 action on inhibitory processes of hormone secretion and cell proliferation.

Animals↗
PubMed1994

Protein kinase C is crucial for the stimulation of sodium-dependent phosphate transport by parathyroid hormone-related peptide in osteoblast-like cells.

In the present study, we investigated the role of parathyroid hormone-related peptide (PTHrP)-responsive dual signal transduction systems in the regulation of sodium-dependent phosphate (Pi) transport by PTHrP in UMR-106 cells. Exposure of the cells to 10(-7) M human (h) PTHrP-(1-34) induced a significant increase in Pi uptake within 15 min of incubation. The peptide stimulated Pi uptake dose-dependently at the range of 10(-11)-10(-7) M. Activation of protein kinase C (PKC) by 12-O-Tetradecanoyl phorbol-13-acetate (TPA) also increased Pi uptake in time- and dose-dependent manners similar to PTHrP. In contrast, neither activation of adenylate cyclase by 10(-5) M forskolin nor calcium ionophore treatment with 10(-7) M A23187 affect Pi uptake. These agents failed to influence on Pi uptake even in combined treatment with TPA. The PTHrP-induced increase in Pi uptake was strongly inhibited by pretreating cells with PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) (50 microM), and by down-regulating PKC with a prolonged TPA pretreatment. These results indicate that the messenger system mediated by PKC, rather than adenylate cyclase or cytosolic calcium, plays a crucial role in the regulation of sodium-dependent Pi transport by PTHrP in the osteoblast-like cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Distribution of mRNA for CCK-B receptor in the brain of Mastomys natalensis: abundant expression in telencephalic neurons.

The distribution of cholecystokinin B (CCK-B) receptors in the Mastomys brain was studied using Northern blot analysis and in situ hybridization technique. By Northern blot analysis using 32P-labeled cDNA probe, the cortex had the highest hybridization signal of CCK-B receptor mRNA in the brain. The olfactory bulb and hippocampus showed a moderate level of signals. In situ hybridization using 35S-labeled cRNA probes revealed a wide and region-specific distribution of CCK-B receptor mRNA in the telencephalon. Throughout the cerebral cortex, labeled cells were found in all layers, with higher intensities in layers II, V and VI. Pyramidal cells of the layer II of the piriform cortex showed the highest level of signals in the brain. In the hippocampus, most of the pyramidal cells of the Ammon's horn were labeled, although labeled cells were not detected in other layers. Distinct signals were also detected in the various amygdaloid nuclei, caudate-putamen, reticular thalamic nucleus, hypothalamic ventromedial nucleus and inferior colliculus. This distribution pattern may further support the prominent existence of CCK-B receptors in the brain particularly in the telencephalon.

Animals↗
PubMed1994

Calcitonin directly acts on mouse osteoblastic MC3T3-E1 cells to stimulate mRNA expression of c-fos, insulin-like growth factor-1 and osteoblastic phenotypes (type 1 collagen and osteocalcin).

The present study was performed to examine whether calcitonin directly acted on mouse osteoblastic MC3T3-E1 cells to stimulate the mRNA expression of insulin-like growth factor-1 (IGF-1) and c-fos, followed by an increase in their proliferation and differentiation. Eel calcitonin increased [3H]thymidine incorporation and alkaline phosphatase activity as well as the mRNA expression of type 1 collagen and osteocalcin which were characteristic of osteoblasts. Eel calcitonin (10(-8)M) induced c-fos mRNA transiently after its addition, followed by gene expression of IGF-1, an important autocrine/paracrine growth factor in the regulation of osteoblastic proliferation. We first demonstrated that calcitonin directly acted on osteoblasts to stimulate transcription of c-fos and IGF-1 genes as well as functional phenotypes including type 1 collagen and osteocalcin.

Animals↗
PubMed1994

Direct involvement of cAMP-dependent protein kinase in the regulation of alkaline phosphatase activity by parathyroid hormone (PTH) and PTH-related peptide in osteoblastic UMR-106 cells.

The present study was performed to characterize the participation of parathyroid hormone (PTH)- and PTH-related peptide (PTHrP)-responsive dual signal transduction systems [cAMP-dependent protein kinase (PKA) and Calcium/protein kinase C (Ca/PKC)] in the regulation of alkaline phosphatase (ALP) activity in osteoblastic osteosarcoma cells (UMR-106). Both human (h) PTH-(1-34) and hPTHrP-(1-34) at 10(-8) M stimulated ALP activity to the similar degree. Dibutyryl, cAMP (dbcAMP) (10(-5), 10(-4) M) and Sp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Sp-cAMPS), a direct stimulator of PKA (10(-4) M) also stimulated its activity. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, (10(-7), 10(-6) M) did not affect its activity, while calcium ionophores, A23187 and ionomycin (10(-7), 10(-6) M) inhibited it. Although Rp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), a direct inhibitor of PKA, (10(-4) M) did not affect ALP activity by itself, it significantly antagonized not only Sp-cAMPS-induced increase in ALP activity, but also PTH- and PTHrP-induced one. The present study first indicated that the activation of PKA was directly involved and acted as a main pathway in the regulation of ALP activity by PTH and PTHrP in osteoblasts.

Alkaline Phosphatase↗
PubMed1994

Alternative splicing generates two distinct transcripts for the Drosophila melanogaster fibroblast growth factor receptor homolog.

We screened Drosophila melanogaster genomic and cDNA libraries by low-stringency hybridization with a probe representing the protein tyrosine kinase (TyK) domain encoded by a human alpha-platelet-derived growth factor receptor-encoding cDNA. The complete sequences of the open reading frames and 3'-untranslated regions (UTR) of some cross-hybridizing clones were identical to the recently published sequence of DFR1, encoding the novel D. melanogaster fibroblast growth factor receptor homology. However, two species of DFR1 cDNAs were isolated that differed with respect to their 5'-UTR. Analysis of the genomic organization revealed that DFR1 is composed of three exons. The entire coding region is contained within the third exon. S1 mapping and RNase-protection assays demonstrated that two distinct DFR1 transcripts possessing either the first or the second exon in combination with the third exon are generated by alternative splicing. This suggests that the transcriptional, as well as posttranscriptional, regulation of fibroblast growth factor receptor (FGFR)-encoding genes during D. melanogaster development is likely to be complex.

Alternative Splicing↗
PubMed1994

Cross-talk of parathyroid hormone-responsive dual signal transduction systems in osteoblastic osteosarcoma cells: its role in PTH-induced homologous desensitization of intracellular calcium response.

The present study was designed to characterize the cross-talk of parathyroid hormone (PTH)-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [PKC]) and its participation in PTH-induced homologous desensitization of intracellular calcium ([Ca2+]i) in osteoblastic UMR-106 cells. Although our recent study revealed that prolonged (more than 2 h) pretreatment with PKC-activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) significantly decreased the PTH-stimulated cAMP production, pretreatment with PMA (10(-7) and 10(-6) M) but not 10(-6) M 4 alpha-phorbol 12,13-didecanoate (PDD), incapable of activating PKC for 30 min significantly augmented 10(-7) M hPTH-(1-34)-stimulated cAMP production. H-7 (50 microM), a PKC inhibitor, significantly antagonized this PMA-induced effect. Pretreatment with 10(-6) M PMA for 30 min did not affect PTH receptor binding but significantly augmented a cAMP responsiveness to 10(-5) M forskolin and 1 microgram/ml cholera toxin. Pertussis toxin (0.5 microgram/ml) did not affect the PMA-induced augmentation of the PTH-stimulated cAMP production. PTH caused a complete homologous desensitization of [Ca2+]i response within 30 min. Pretreatment with 10(-4) M dibutyryl cAMP for 30 min and 6 h significantly reduced and completely blocked the PTH-induced increase in [Ca2+]i, respectively. Pretreatment with 10(-4) M Sp-cAMPs, a direct PKA activator, for 30 min completely blocked the PTH-induced increase in [Ca2+]i. Rp-cAMPS (10(-4) M), an antagonist of PKA, slightly but significantly antagonized the PTH-induced homologous desensitization of [Ca2+]i response. The present study indicates that the time of exposure to PKC activation is a critical determinant in modulating the cAMP system, while PKA activation counterregulatorily acts on the [Ca2+]i system, and that PKA activation is linked to the PTH-induced homologous desensitization of [Ca2+]i response.

Adenylyl Cyclases↗