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Biomedical subjects

K Chihara

Publications and source records attributed to K Chihara.

At least 91 records · Page 5Linked to original sources
PubMed1999

Response to granulocyte colony-stimulating factor in an autoimmune neutropenic adult.

Clinical value of granulocyte colony-stimulating factor (G-CSF) for autoimmune neutropenia (AIN) has not been well established. We experienced an adult case of AIN which showed an excellent response to G-CSF. A 75-year-old female was admitted with high-grade fever. Her neutrophil count was remarkably low (neutrophil 0.09 x 10(9)/l). Antigranulocyte autoantibody was demonstrated in her serum by an immunofluorescence method and she was diagnosed as AIN. Administration of G-CSF (filgrastim 5 microgram/kg) gave a rapid increase of neutrophils (from 0.11 x 10(9)/l to 2.10 x 10(9)/l on the second day), which has enabled us to preserve the use of G-CSF for emergency, that is, for overt serious infection.

Aged↗
PubMed1999

Serum concentration of 20K human growth hormone (20K hGH) measured by a specific enzyme-linked immunosorbent assay. Study Group of 20K hGH.

Several GH isoforms have been identified in pituitary and serum, the most abundant of which is the 22K human GH (hGH) isoform. The 20K hGH isoform is produced by alternative splicing of GH messenger ribonucleic acid and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined, partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunosorbent assays (ELISAs) specific to 20K and 22K hGH. To determine whether regulation of 20K hGH secretion is the same as that for 22K hGH, we measured serum concentrations of both species of hGH in normal subjects and patients with a variety of endocrine disorders. The serum levels of 20K hGH after overnight fasting was 118 +/- 178 pg/mL (n = 282) in normal women, significantly higher than that in normal men (64 +/- 170 pg/mL; n = 226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 +/- 2.6%, mean +/- SD; n = 176) and women (6.3 +/- 2.1%; n = 263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight, or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P < 0.001) higher in patients with active acromegaly (9.2 +/- 2.2%; n = 33) and patients with anorexia nervosa (9.0 +/- 1.9; n = 8), both of which are characterized by chronic elevation of circulating GH levels. The proportion of 20K hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or that a chronic excess of 22K hGH alters the MCR of 20K hGH. The values in patients with adult GH deficiency, hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests, such as the insulin tolerance test and the GHRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH. This new assay may provide a tool for understanding the physiological or pathophysiological role of the 20K hGH isoform.

Acromegaly↗
PubMed1999

The C422F mutation of the growth hormone receptor gene is not responsible for short stature.

A missense mutation, C422F, was identified in the intracellular domain of GH receptor (GHR) in a Japanese short boy. Although this mutation was previously reported in a patient with GH insensitivity syndrome (GHIS), it has not been clear whether this mutation causes GH insensitivity. To clarify the effect of this mutation on GH signal transduction, mutant GHR was expressed in CHO cells, and its functional properties were investigated. There were no significant differences in GH-induced tyrosine phosphorylation of STAT5b (signal transducer and activator of transcription) between wild-type GHR (GHR-wt)- and mutant GHR (GHR-C422F)-expressing cells. Moreover, STAT5-mediated transcriptional activation of GHR-C422F-expressing cells was comparable to that of GHR-wt-expressing cells. These findings indicated that the C422F mutation of GHR affected neither GH-induced tyrosine phosphorylation nor the transcriptional activation of STAT5. In addition, the analysis of genotypes and phenotypes of his family revealed that body heights of family members with heterozygous or homozygous C422F mutations were all within normal ranges, with the single exception of the proband. These in vitro and in vivo results indicate that the C422F missense mutation of GHR is a polymorphism that does not result in GHIS.

Animals↗
PubMed1999

Functional characterization of truncated growth hormone (GH) receptor-(1-277) causing partial GH insensitivity syndrome with high GH-binding protein.

We have previously reported a novel heterozygous donor splice site mutation in intron 9 of the GH receptor (GHR) gene in Japanese siblings who showed partial GH insensitivity and high serum GH-binding protein (GHBP) levels. This mutation caused the splicing abnormality and produced the truncated GHR consisting of 277 amino acids (GHR-277), which lacked most of the intracellular domain of GHR, including both boxes 1 and 2. In this study, we have characterized the function of GHR-277 expression in COS-7 and CHO cells in vitro. Scatchard analysis revealed that GHR-277 possessed approximately 1.5 times higher affinity to GH and twice the number of binding sites compared to wild-type full-length GHR (GHR-fl). The GHBP level in culture medium of GHR-277-expressing cells was approximately 3 times higher than that in GHR-fl-expressing cells. Interestingly, the ligand-induced internalization of GHR-277 was significantly reduced compared with that of GHR-fl. Moreover, in GH-induced tyrosine phosphorylation of signal transducer and activator of transcription-5 (STAT5), GHR-277 exerted a dominant negative effect when GHR-277 and GHR-fl were cotransfected. These in vitro data would well explain the clinical characteristics in our patients showing high serum GHBP levels and development of short stature despite a heterozygous mutation of the GHR gene.

Adolescent↗
PubMed1999

[Elderly dyserythropoietic anemia first diagnosed after presentation of hemorrhagic gastric ulcer].

A 71-year-old man presented with epigastralgia and tarry stool. Laboratory examination showed anemia (Hb 7.1 g/dl) due to hemorrhagic gastric ulcer and positive Coombs' test without features of hemolysis. Initial bone marrow smears disclosed normal granulocytes and megakaryocytes, but only erythroid hyperplasia with multinuclearity and megaloblastosis was identified. Cytogenetic studies revealed normal karyotype. Congenital dyserythropoietic anemia (CDA) type II was initially suspected. Serologically, however, acid hemolysis and anti-I * i tests were negative as were the results of another Coombs' test. The second bone marrow aspiration disclosed a marked decrease in multinucleated erythroblasts together with an increase in those that had internuclear chromatin bridges. Electron microscopy of bone marrow specimens demonstrated morphological features of CDA types I and II, thus yielding a final diagnosis of elderly dyserythropoietic anemia, which is similar to both CDA types. It was suggested that the reactive secretion of erythropoietin due to bleeding played a role in these pathologic changes.

Aged↗
PubMed1999

Leptin stimulates basal and GHRH-induced GH release from cultured rat anterior pituitary cells in vitro.

Leptin, the product of ob gene, is secreted from adipocytes and appears to regulate food intake and energy expenditure through its receptor in the hypothalamus. In addition, leptin is reported to modulate pituitary hormones including LH, FSH and ACTH, probably via the hypothalamus. In obesity, growth hormone (GH) secretion is impaired, while serum leptin levels are elevated. To investigate the possibility that leptin serves as a metabolic signal that influences GH secretion from the pituitary, we studied the effect of leptin on GH secretion from primary monolayer cultures of rat anterior pituitary cells. Twenty micrograms/ml leptin increased GH secretion, but did not increase GH release at a physiological concentration up to 200 ng/ml. However, 200 ng/ml leptin stimulated GH release in the presence of GH-releasing hormone (GHRH) ranging from 10(-8) to 10(-7) M. Twenty ng/ml leptin tended to increase 10(-7) M GHRH-induced GH release, whereas 20 ng/ml leptin did not increase either 10(-9) or 10(-8) M GHRH-induced GH release. This result suggests that leptin has a direct effect on the pituitary to enhance GHRH-induced GH secretion.

Analysis of Variance↗
PubMed1998

Cloning and characterization of the 5'-flanking region of the human growth hormone secretagogue receptor gene.

Recently, the growth hormone secretagogue receptor (GHS-R) cDNA has been isolated from the pituitary and hypothalamus. To evaluate the regulation of human (h) GHS-R gene expression, we cloned the hGHS-R gene containing the 5'-flanking region of 0.6-2.9 kilobase pairs. Analysis of the hGHS-R transcripts with 5'-rapid amplification of cDNA ends suggested that the putative transcription initiation site was approximately -453 base pairs upstream of the translation initiation site (+1). There is no typical TATA, CAAT, or GC box but an initiator-like sequence and putative binding sites for several transcription factors around the putative transcription start site. The 5'-flanking region inserted into a luciferase reporter vector had promoter activity in GH3 cells but had activity indistinguishable from background in HeLa or EP1 cells. The hGHS-R promoter activity in GH3 cells increased by deletion of nucleotides from -1224 to -734, whereas it was decreased by further deletion from -734 to -608. Knowledge of the promoter region of the hGHS-R gene will facilitate elucidation of its transcriptional control.

Base Sequence↗
PubMed1998

Possible involvement of the inactivation of the Rho-Rho-kinase pathway in oncogenic Ras-induced transformation.

Recent evidence has strongly suggested the involvement of Rho family small guanosine triphosphatases (GTPases) in Ras-induced transformation. To further clarify the role of Rho family GTPases in Ras-induced transformation, we examined the effects of dominant active or dominant negative forms of Rho family GTPases on the morphological changes induced by oncogenic Ras (RasV12) in Rat1 fibroblasts. The cells expressing RasV12 showed the severe disruption of actin stress fibers and cell adhesions. The coexpression of dominant active form of Rho (RhoV14) reverted not only the formation of stress fibers and focal adhesions but also cell-cell adhesions in Ras-transformed Rat1 cells. In addition, the coexpression of constitutively activated Rho-kinase, a downstream effector of Rho, restored the assembly of stress fibers and focal adhesions. Treatment of Ratl cells with lysophosphatidic acid, which is known to activate the Rho-Rho-kinase pathway, enhanced the stress fiber formation, whereas it failed to induce the stress fiber formation in the cells expressing RasV12. These results suggest that the Rho-Rho-kinase pathway may be inactivated in the cells expressing RasV12, and this may contribute to oncogenic Ras-induced transformation.

Actins↗
PubMed1998

Proliferative reaction of myelogenous leukemia cells with cytokines G-CSF, GM-CSF, M-CSF, SCF and TPO.

In recent years, many cytokines have been defined and some of them used clinically. In hematological malignancies, cytokines, including granulocyte colony-stimulating factor (G-CSF), have been widely used for leukopenia after chemotherapy. However, in acute myelogenous leukemia (AML), some leukemic cells may be induced to proliferate by these cytokines and they must be used with care. In this study, we have investigated cell reactivity and proliferation with G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CMF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF) and thrombopoietin (TPO) in cases of AML. We have also investigated the reactivity of some myeloid leukemia cell lines to TPO. G-CSF, GM-CSF, M-CSF, SCF and TPO caused proliferation of leukemic cells in 25%, 58.3%, 8.3%, 21.1% and 0% of cases, respectively. Because of this result, the use of G-CSF in AML should be regarded as potentially hazardous. TPO did not cause proliferation of leukemic cells in any case of AML, or in cell lines except MO7E, which is a megakaryocytic cell line. This result suggests that TPO might cause proliferation of some megakaryocytic leukemia cells. We cannot conclude that TPO does not cause proliferation of other AML cells, as the number of cases was small and it has been reported elsewhere that leukemia cells may proliferate when exposed to TPO in 50% of AML cases. Reactivity of AM L cells to TPO is an important factor when deciding the indications of TPO in AML and myelodysplastic syndrome.

Cell Division↗
PubMed1998

Myosin II activation promotes neurite retraction during the action of Rho and Rho-kinase.

BACKGROUND: The Rho small GTPase regulates myosin II activity through the phosphorylation of the myosin light chain (MLC) by activating Rho-kinase, which is a target of Rho. Several lines of evidence point to an important role of Rho in the action of lysophosphatidic acid (LPA) and thrombin in provoking neurite retraction in N1E-115 neuroblastoma cells. RESULTS: Here we examined whether Rho-kinase and myosin II are involved in neurite retraction in N1E-115 cells. We showed that the expression of constitutively active forms of Rho-kinase induced neurite retraction in NIE-115 cells and MLC phosphorylation in NIH 3T3 cells, whereas the expression of dominant negative forms of Rho-kinase inhibited the LPA-induced neurite retraction in N1E-115 cells and the serum-induced MLC phosphorylation in NIH 3T3 cells. The expression of mutant MLCT18D,S19D (substitution of Thr and Ser by Asp), which is known to lead to the activation of myosin ATPase and a conformational change of myosin II when reconstituted with myosin heavy chains in vitro, also promoted neurite retraction. CONCLUSION: These results indicate that Rho-kinase is involved in the LPA-induced neurite retraction downstream of Rho, and that myosin II activation promotes neurite retraction downstream of Rho and Rho-kinase.

3T3 Cells↗
PubMed1998

Identification of an enhancer sequence in 5'-flanking region of 1A exon of mouse liver/bone/kidney-type alkaline phosphatase gene.

The 5'-flanking region of the 1A exon of the mouse liver/bone/kidney-type alkaline phosphatase (L/B/K ALP) gene was analyzed for the promoter activity. Luciferase assays revealed that a segment containing a 9 bp inverted repeat (nt -1212 to -1179, designated as IRS for inverted repeat segment) had an enhancer activity. Though an E-box at nt -234 did not have a prominent promoter activity, the effect of IRS required its presence. Both transcriptional activity and IRS binding to the nuclear proteins were similarly observed even in cells that did not express ALP. Bone morphogenetic protein-2 (BMP-2) induced the ALP expression in pluripotent C3H10T1/2 cells, but it did not increase the transcriptional activity of the promoter region. Though they do not determine cell specific or BMP-2 inducible ALP expression, IRS and the E-box at position -241 seem to be important for the basic transcriptional activity of ALP gene.

3T3 Cells↗
PubMed1998

Short stature caused by a natural growth hormone antagonist.

Severe short stature in a male child due to a single mutation in the GH-1 gene was first reported in 1996 by Takahashi et al. [N Engl J Med 1996;334:432-436]. This missense mutation was predicted to convert codon 77 from arginine (R) to cysteine (C). The child's chronological age was 4 years and 11 months, and his bone age 2 years and 6 months, i.e., equal to only 51% of his chronological age. Body proportions were normal except for the prominent forehead and saddle nose. Pituitary size was normal on magnetic resonance imaging examinations. Serum IGF-1, IGFBP-3 and GHBP were all decreased or at the lower limit of the normal range. Nocturnal urinary growth hormone (GH) excretion was high. Isoelectric focusing analysis revealed the presence of an abnormal GH peak in addition to the normal one. The R77C mutant GH possessed a 6 times greater affinity to GHBP than the wild-type GH, and inhibited tyrosine phosphorylation in IM-9 cells 10 times more potently than the wild-type GH, showing an antagonistic or a dominant negative action. In agreement with the antagonistic property of the mutant GH exhibited, the child did not show any increase in serum IGF-1 levels after exogenous hGH administration. It should be noted that the child in this study is not a typical case of Kowarski syndrome in which endogenous GH is found to be simply bioinactive, as in the patient we recently described elsewhere. Therefore, this patient's condition should be categorized as a new syndrome of short stature caused by a natural GH antagonist.

Age Determination by Skeleton↗
PubMed1998

Growth hormone (GH) insensitivity syndrome with high serum GH-binding protein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain.

Most of the GH receptor (GHR) gene abnormalities causing GH insensitivity syndrome (GHIS) are located in the region coding the extracellular domain, and serum GH-binding protein (GHBP) levels, determined by ligand-mediated immunofunctional assay, are low in most of the patients with GHIS. We present here a heterozygous point mutation of the donor splice site in intron 9 of the GHR gene in two Japanese siblings with GHIS, whose serum GHBP levels were high. The same mutation was found in their mother as well. The analysis of ribonucleic acid from the peripheral leukocytes revealed complete skipping of exon 9 from one allele, but not the other, in the GHR complementary DNA and appearance of a premature stop codon in exon 10. The translated protein was truncated with deletion of 98% of the intracellular domain of the GHR, including boxes 1 and 2, which are critical for GH signal transduction and GHR internalization, respectively. Recently, it was shown that the truncated GHR lacking the intracellular domain was physiologically present in a minute amount, served as a negative regulator for GH signaling, and possessed increased capacity to generate GHBP. Therefore, the mutation found in our patients caused the pathogenetic production of the truncated GHR with a dominant negative effect on GH signaling, which is probably responsible for their short stature and high serum GHBP levels.

Adolescent↗
PubMed1998

Estrogen via the estrogen receptor blocks cAMP-mediated parathyroid hormone (PTH)-stimulated osteoclast formation.

Several lines of evidence indicate that estrogen inhibits parathyroid hormone (PTH)-induced bone resorption in vivo and in vitro. However, its precise mechanism remains unknown. The present study was performed to investigate whether osteoclast precursor cells possess the receptors for PTH/PTH-related protein (PTHrP) and/or estrogen and to clarify the mechanism by which estrogen affects PTH-induced osteoclast-like cell (Ocl) formation. The polymerase chain reaction (PCR) product corresponding in size to the mouse PTH/PTHrP receptor cDNA was detected in mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as in osteoblastic MC3T3-E1 cells. The nucleotide sequence of the PTH/PTHrP receptor PCR product of hemopoietic blast cells was found to be 95.4% identical to that of PTH/PTHrP receptor cDNA of rat osteoblastic ROS cells. The PCR product corresponding in size to the mouse estrogen receptor cDNA was detected in mouse hemopoietic blast cells supported by GM-CSF as well as in MC3T3-E1 cells. The nucleotide sequence of the estrogen receptor PCR product of hemopoietic blast cells was completely identical to that of mouse estrogen receptor cDNA. 17Beta-estradiol (17beta-E2) but not 17alpha-E2 dose dependently antagonized Ocl formation stimulated by human (h) PTH(1-34) at a minimal effective concentration of 10(-10) M in the hemopoietic blast cell culture. 17Beta-E2 also significantly inhibited Ocl formation stimulated by 10(-8) M hPTHrP(1-34), while it did not affect 1,25-dihydroxyvitamin D3-induced Ocl formation. However, 10(-8) M 17beta-E2 significantly inhibited Ocl formation stimulated by dibutyryladenosine cAMP (10(-4) M) and Sp-cAMPS (10(-4) M), an activator of cAMP-dependent protein kinase (PKA) as well as forskolin (10(-5) M). In contrast, 17beta-E2 did not affect Ocl formation by either phorbol myristate acetate (10(-7) M), an activator of protein kinase C (PKC), or A23187 (10(-7) M), a calcium ionophore. The pretreatment with 17beta-E2 significantly inhibited Ocl formation induced by the combined treatment with PTH and PKC inhibitors (H7 or staurosporine), while it did not affect Ocl formation stimulated by the combined treatment with PTH and Rp-cAMPS, a PKA inhibitor. The present data indicate that estrogen inhibits PTH-stimulated Ocl formation by directly acting on hemopoietic blast cells, possibly through blocking a PKA pathway but not a calcium/PKC pathway.

Animals↗
PubMed1998

Vitamin D receptor genotype is associated with cortical bone loss in Japanese patients with primary hyperparathyroidism.

Vitamin D receptor (VDR) genotype was determined in 66 Japanese patients with primary hyperparathyroidism (pHPT). In contrast to previous report showing an association between VDR genotype bb and the development of pHPT in a Swedish population, we did not find any differences in the frequencies of the VDR genotypes between pHPT and the control. The bone mineral density in the radius was significantly lower (Z score, -1.723 +/- 1.819) in bb genotype than in BB/Bb genotype (+0.255 +/- 2.344) in pHPT patients. Based on the fact that PTH requires vitamin D3 to take effect on calcium mobilization from bone, it is possible that VDR polymorphism may influence the PTH action on bone.

Adult↗