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Biomedical subjects

K Chihara

Publications and source records attributed to K Chihara.

At least 127 records · Page 7Linked to original sources
PubMed1997

Regulation of inducible nitric oxide synthase expression in L6 rat skeletal muscle cells.

Cytokine-stimulated expression of inducible type of nitric oxide synthase (iNOS) seems to be regulated by various signal pathways in a cell-specific manner. In this study, we examined how it was regulated in L6 rat skeletal muscle cells. In L6 cells, the combination of interleukin-1 beta and interferon-gamma induced a marked accumulation of nitrite, a stable metabolite of nitric oxide. In parallel with this reaction, iNOS mRNA expression was achieved at a maximum between 3 and 6 h, and iNOS protein was detectable at 6 h and peaked at 24 h after stimulation. Tyrosine kinase inhibitors, herbimycin A, and genistein suppressed cytokine-induced iNOS expression and nitrite production. Forskolin, an adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) activator, and phorbol 12-myristate 13-acetate, a protein kinase C (PKC)-activating phorbol ester, enhanced these cytokine-induced reactions. These results indicate that iNOS expression by cytokines is mediated via a protein tyrosine kinase-dependent pathway and is positively modulated by both PKA- and PKC-dependent pathways in this cell type.

Animals↗
PubMed1997

The action of GH/IGF-I/IGFBP in osteoblasts and osteoclasts.

Bone remodelling is a cyclical phenomenon consisting of osteoclastic bone resorption followed by osteoblastic bone formation. Although recent evidence suggests that GH participates in bone remodelling, the exact mechanism remains unclear. The present series of in vitro studies aimed to clarify how GH affects bone formation and resorption. GH binding sites were found to be present in osteoblastic MC3T3-E1 cells. Bovine GH (bGH) increased DNA synthesis, stimulated alkaline phosphatase activity and enhanced both type I procollagen mRNA expression and collagen synthesis. GH also increased the expression of both IGF-I and IGF-binding protein-5 mRNA as well as the release of IGF-I from these cells. The addition of IGF-I or recombinant IGFBP-5 alone significantly increased ALP activity and type I procollagen mRNA expression. These findings indicate that GH acts directly on osteoblasts to stimulate bone formation and that IGF-I and IGFBP-5 are involved in GH-stimulated bone formation. GH also stimulated pit formation on dentine slices and osteoclast differentiation in stromal cell-containing mouse bone cell cultures, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclasts. The addition of IGF-I or rIGFBP-5 alone exhibited similar effects. These stimulatory effects of GH on pit formation and osteoclast differentiation were significantly blocked in the presence of neutralizing anti-IGF-I antibody. PCR products corresponding in size to the mouse GH receptor were detected in osteoclast precursor cells. GH stimulated osteoclast-like cell formation from these cells in the absence of stromal cells, and these osteoclast-like cells formed pits on dentine slices in the presence of MC3T3-G2/PA-6 stromal cells. These findings indicate that GH stimulates osteoclastic bone resorption through both its direct and indirect action on the maturation of osteoclast precursor cells and through its indirect activation of mature osteoclasts, possibly via stromal cells. In conclusion, GH stimulates osteoclastic bone resorption as well as osteoblastic bone formation in vitro, and locally produced IGF-I and/or IGFBP-5 are involved in the stimulation of bone remodelling by GH.

Animals↗
PubMed1997

Novel compound heterozygous mutations of growth hormone (GH) receptor gene in a patient with GH insensitivity syndrome.

A girl with severe growth retardation had the clinical features of Laron syndrome. Her serum insulin-like growth factor-I level was completely unresponsive to exogenous GH administration. The serum GH-binding protein (GHBP) level was below the detectable limit in the patient, but it was normal in her parents and brother. Her parents and brother were normal in their height. Amplification with PCR and direct sequencing of her GH receptor gene revealed compound heterozygous mutations. The allele from her mother contained a transversion of G to T in exon 7 that could introduce a stop codon in place of a glutamic acid at amino acid 224. Another mutation was found in the allele in her father and also identified in her brother. It was a C deletion at position 981 in exon 10 that could introduce a frame shift, thereby causing the production of 20 novel amino acids (310-329) instead of the wild-type sequence, the premature termination at codon 330, and the subsequent deletion of the C terminal portion of the intracellular domain. RT-PCR of her father's lymphocytes and sequencing of its complementary DNA revealed that only the wild-type GH receptor messenger RNA was expressed in his lymphocytes, though the mechanism remains unclear. These results suggest that neither of the mutant alleles could generate a functional GH receptor, which would be consistent with the patient's severe growth retardation and undetectable serum GHBP.

Adolescent↗
PubMed1997

Dexamethasone stimulates osteoclast-like cell formation by directly acting on hemopoietic blast cells and enhances osteoclast-like cell formation stimulated by parathyroid hormone and prostaglandin E2.

Although an excess of glucocorticoid induces secondary osteoporosis, the mechanism still remains unclear, particularly in regard to glucocorticoid-stimulated bone resorption. We examined the effects of dexamethasone (Dex) on osteoclast-like cell formation and bone-resorbing activity by employing mouse bone and spleen cell cultures and further investigated whether Dex would modulate osteoclast-like cell formation stimulated by several bone-resorbing factors. Dex stimulated osteoclast-like cell formation in stromal cell-containing mouse bone cell cultures in a concentration-dependent manner. Also, Dex significantly stimulated osteoclast-like cell formation from hemopoietic blast cells in spleen cell cultures derived from 5-fluorouracil-pretreated mice. In contrast, Dex (10(-8) M) did not affect the bone-resorbing activity of mature osteoclasts. Pretreatment with 10(-8) M Dex significantly enhanced osteoclast-like cell formation in unfractionated mouse bone cell cultures stimulated by 10(-8) M human (h) parathyroid hormone (PTH) (1-34), 10(-8) M hPTH-related protein (1-34) and 10(-6) M prostaglandin E2, but not by 10(-8) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Moreover, pretreatment with 10(-8) M Dex significantly enhanced osteoclast-like cell formation stimulated by both forskolin and dbcAMP. In contrast, pretreatment with 10(-8) M Dex significantly inhibited osteoclast-like cell formation in mouse spleen cell cultures stimulated by both 10(-8) M hPTH(1-34) and 10(-8) M 1,25(OH)2D3. These findings suggest that Dex stimulates osteoclast-like cell formation, at least in part by directly acting on hemopoietic blast cells. They further suggest that Dex enhances osteoclast-like cell formation stimulated by PTH and prostaglandin E2 through an indirect pathway via cells other than hemopoietic blast cells.

Animals↗
PubMed1997

Serum levels of insulin-like growth factor (IGF) I, IGF-binding protein (IGFBP)-2, and IGFBP-3 in osteoporotic patients with and without spinal fractures.

The present study was performed to investigate the role of insulin-like growth factor I (IGF-I), IGF-binding protein-2 (IGFBP-2), and IGFBP-3 in age-dependent bone loss in postmenopausal Japanese women. One hundred and sixty-five Japanese women aged 43-88 years (mean age, 62) were enrolled in the cross-sectional study. Bone mineral density (BMD) was measured at the lumbar spine, femoral neck, and midradius by dual-energy X-ray absorptiometry or single-photon absorptiometry. Serum levels of IGF-I, IGFBP-2, and IGFBP-3 were measured by radioimmunoassay. BMD at all sites as well as serum levels of IGF-I and IGFBP-3 declined with age, while the serum IGFBP-2 level increased with age. Serum IGFBP-3 and -2 levels were positively and negatively correlated with the serum IGF-I level, respectively. Serum IGF-I and IGFBP-3 levels showed positive correlationship with BMD at any site, particularly at the midradius, while the serum IGFBP-2 level showed negative correlation with BMD. Multiple regression analyses showed age-independent positive correlation between the serum IGF-I level and BMD at all sites as well as age-independent positive correlation between the serum IGFBP-3 level and midradius BMD. The relationship between susceptibility to osteoporotic spinal fracture and serum IGF-I, IGFBP-3, or -2 levels was examined by decade to exclude the influence of aging. Serum levels of IGF-I and IGFBP-3 were significantly lower in subjects with spinal fractures than those without fractures at any decade. No significant difference of serum IGFBP-2 level was observed between subjects with and without fractures. The present findings suggest that IGF-I and IGFBP-3 are important to maintaining bone mass quantitatively as well as qualitatively, and that the determination of serum IGF-I and IGFBP-3 levels could be clinically useful to predict the severity of osteoporosis, particularly the risk of bone fracture associated with osteoporosis.

Absorptiometry, Photon↗
PubMed1997

Clinical and biochemical presentation of primary hyperparathyroidism in Kansai district of Japan.

Although the number of patients with primary hyperparathyroidism (pHPT) has increased since biochemical screening came to be widely employed, few data are available concerning the clinical analysis of Japanese patients. We therefore tried to determine the recent clinical and biochemical state of Japanese patients with pHPT. Clinical and biochemical data were analyzed in a series of 103 pHPT patients who had been hospitalized in Kobe University Hospital during a 17 year period from 1979 to 1995. The data were obtained from the hospital records and additionally from information through questionnaires performed in 1995. Patients were 29 males and 74 females. The average ages at diagnosis were 53.4 +/- 16.0 (SD) year-old and 53.9 +/- 15.2 year-old, respectively. The major clinical symptoms were nephrolithiasis (35.9%), thirst/polyuria (33.0%), easy fatigability (20.4%) and back pain/lumbago (16.5%), but 19.4% of the patients were asymptomatic. Out of 87 cases who received parathyroidectomy, 10 (11.5%) were diagnosed with parathyroid carcinoma. Age- and sex-corrected bone mineral density (BMD) of the radius was significantly lower in the group with back pain/lumbago than in the group without the symptom. Similarly, radial BMD was lower in the group with a high serum alkaline phosphatase value. Patients with nephrolithiasis had a lower urinary calcium/creatinine ratio (UCa/ UCr; 0.305 +/- 0.188 mg/mg) than the patients without nephrolithiasis (0.400 +/- 0.160 mg/mg). This inconsistent result suggests that some important factors except urinary calcium would contribute to urinary stone formation in pHPT. Our data mostly agreed with previous reports on a Caucasian population except for a relatively high prevalence of parathyroid carcinoma, but the negative correlation between nephrolithiasis and urinary calcium was not easily explicable. This finding should be confirmed by analyzing a larger number of cases.

Adolescent↗
PubMed1997

Effect of natural menopause on serum levels of IGF-I and IGF-binding proteins: relationship with bone mineral density and lipid metabolism in perimenopausal women.

The present study was performed to examine the effect of natural menopause on serum levels of IGF-I, IGF-binding protein (IGFBP)-2 and -3 as well as on bone mass and lipid metabolism in perimenopausal women. One hundred and twenty-one healthy Japanese women, who were 45-55 years old, were studied (71 premenopausal and 50 postmenopausal women 1-9 years after menopause). Bone mineral density (BMD) was measured at the middle third of the radius by using dual energy X-ray absorptiometry. Serum levels of IGF-I, but not those of IGFBP-2 or -3, were significantly reduced in the postmenopausal group compared with the premenopausal group. One year after menopause, serum IGF-I levels were significantly lower, and the biochemical markers of bone turnover, such as serum total alkaline phosphatase level and urinary calcium to creatinine ratio, were significantly higher than the premenopausal levels. Serum levels of IGF-I, but not those of IGFBP-2 or -3, were positively correlated with BMD. Serum levels of IGFBP-2, but not those of IGF-I or IGFBP-3, were negatively correlated with body mass index and body weight. Finally, serum levels of IGFBP-3, but not those of IGF-I, were positively correlated with serum levels of total cholesterol and triglyceride. The present findings suggest that a rapid decrease in serum IGF-I levels after menopause might be partly involved in bone loss following gonadal failure and that IGFBP-2 and -3 might be related to the regulation of body mass and lipid metabolism during perimenopause respectively, although the mechanisms remain unknown.

Bone Density↗
PubMed1997

[Recurrent acute transverse myelopathy: an 83-year-old man with antiphospholipid syndrome].

We reported here an 83-year-old man with recurrent acute transverse myelopathy probably due to antiphospholipid syndrome (APS). The patient was admitted to a local hospital on May 8, 1996, because of hypoesthesia and muscle weakness of both legs, accompanied by dysuria. T2-weighted magnetic resonance image (MRI) showed significant swelling and increased intensity of the thoracic spinal cord between Th3 and Th7 without enhancement by contrast medium. After the treatment with steroid, both clinical symptoms and abnormal findings on MR imaging disappeared. On 24 June, however, motor paralysis of both legs and sensory loss below the chest relapsed. The lesion in the thoracic spinal cord between Th4 and Th8 reappeared on T2-weighted MRI. Blood examination disclosed the presence of a lupus anticoagulant. Repeated examinations of CSF revealed the continuous elevation of IgG. Clinical and laboratory findings indicated that immunological mechanisms related with APS might be responsible for the transverse myelopathy in this patient.

Acute Disease↗
PubMed1996

Leptin induces tyrosine phosphorylation of cellular proteins including STAT-1 in human renal adenocarcinoma cells, ACHN.

Several lines of evidence from in vivo animal experiments and human studies suggest that leptin, a peptide secreted from adipose tissue, plays a role in regulating food intake and energy expenditure. However, the signal transduction mechanism of leptin in its target cells remains unknown thus far since leptin-responsive cell lines have not been available yet. We found that leptin caused the tyrosine phosphorylation of several proteins in human renal cell carcinoma cells, ACHN cells, in which STAT-1, but neither STAT-3 nor STAT-5, was involved. An ACHN cell line would serve as a useful tool for analyzing the signal transduction mechanism of leptin.

Adenocarcinoma↗
PubMed1996

G protein-coupled cholecystokinin-B/gastrin receptors are responsible for physiological cell growth of the stomach mucosa in vivo.

Many peptide hormone and neurotransmitter receptors belonging to the seven membrane-spanning G protein-coupled receptor family have been shown to transmit ligand-dependent mitogenic signals in vitro. However, the physiological roles of the mitogenic activity through G protein-coupled receptors in vivo remain to be elucidated. Here we have generated G protein-coupled cholecystokinin (CCK)-B/gastrin receptor deficient-mice by gene targeting. The homozygous mice showed a remarkable atrophy of the gastric mucosa macroscopically, even in the presence of severe hypergastrinemia. The atrophy was due to a decrease in parietal cells and chromogranin A-positive enterochromaffin-like cells expressing the H+,K(+)-ATPase and histidine decarboxylase genes, respectively. Oral administration of a proton pump inhibitor, omeprazole, which induced hypertrophy of the gastric mucosa with hypergastrinemia in wild-type littermates, did not eliminate the gastric atrophy of the homozygotes. These results clearly demonstrated that the G protein-coupled CCK-B/gastrin receptor is essential for the physiological as well as pathological proliferation of gastric mucosal cells in vivo.

Animals↗
PubMed1996

Autocrine loop through cholecystokinin-B/gastrin receptors involved in growth of human leukemia cells.

The cholecystokinin (CCK)-B/gastrin receptor binds two brain-gut hormones, CCK and gastrin, with high affinities. These peptides have a trophic effect on gastrointestinal cells expressing the receptor in vivo as well as in vitro. Recently, this receptor mRNA was reported to be expressed in immunocytes localized in the lamina propria of normal rat stomach mucosa. Here, we studied the receptor expression in human hematopoietic cells in order to determine whether they play a role in cell growth. The CCK-B/gastrin receptor mRNA was detectable in the polymorphonuclear (PMN) cells but not in the mononuclear cells of normal peripheral white blood cells by reverse transcription-polymerase chain reaction. The receptor transcript was, however, expressed in human leukemia cell lines (14 of 18 cell lines tested) derived from not only myeloid, but also T- and B- lymphoid lineages. The CCK-B/gastrin receptors on several leukemia cell lines were shown to be biologically active by demonstrating ligand-dependent cell proliferation in serum-deprived medium. Interestingly, a human CCK-B/gastrin receptor specific antagonist, YM022, but not its stereotype isoform, selectively inhibited the DNA synthesis of THP-1, MOLT-16, MOLT-14, and CCRF-CEM in the absence of exogenous peptide ligands. Further investigation revealed that these leukemia cell lines and normal PMN cells also expressed gastrin mRNA. These results suggest that growth of human leukemia cells is promoted by an autocrine mechanism through the CCK-B/gastrin receptors.

Cell Division↗
PubMed1996

Somatostatin neurons form a distinct peptidergic neuronal group in the rat suprachiasmatic nucleus: a double labeling in situ hybridization study.

Accumulating evidence indicates that somatostatin (SS) is a key substance for the circadian rhythm of rodents. In the present study, we investigated whether SS mRNA coexists with arginine-vasopressin (AVP) mRNA, vasoactive intestinal peptide/peptide histidine isoleucine amide (VIP/PHI) mRNA and glutamate decarboxylase (GAD) mRNA in neurons of the rat suprachiasmatic nucleus (SCN) by double labeling in situ hybridization technique. SS mRNA-positive neurons were scattered in the whole region of rostral SCN, in the intermediate region between dorsomedial and ventrolateral region at the middle level, and in the mid to lateral region at the caudal level. These neurons were located in the close vicinities of the dorsomedial AVP and ventrolateral VIP/PHI mRNA-positive cell clusters. They rarely coexpressed AVP mRNA or VIP/PHI mRNA, but mostly coexpressed GAD mRNA. Thus, SS-synthesizing neurons are GABAergic and form a distinct cell group different from AVP or VIP/PHI cell groups.

Animals↗
PubMed1996

Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).

The small GTPase Rho is implicated in physiological functions associated with actin-myosin filaments such as cytokinesis, cell motility, and smooth muscle contraction. We have recently identified and molecularly cloned Rho-associated serine/threonine kinase (Rho-kinase), which is activated by GTP Rho (Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) EMBO J. 15, 2208-2216). Here we found that Rho-kinase stoichiometrically phosphorylated myosin light chain (MLC). Peptide mapping and phosphoamino acid analyses revealed that the primary phosphorylation site of MLC by Rho-kinase was Ser-19, which is the site phosphorylated by MLC kinase. Rho-kinase phosphorylated recombinant MLC, whereas it failed to phosphorylate recombinant MLC, which contained Ala substituted for both Thr-18 and Ser-19. We also found that the phosphorylation of MLC by Rho-kinase resulted in the facilitation of the actin activation of myosin ATPase. Thus, it is likely that once Rho is activated, then it can interact with Rho-kinase and activate it. The activated Rho-kinase subsequently phosphorylates MLC. This may partly account for the mechanism by which Rho regulates cytokinesis, cell motility, or smooth muscle contraction.

Animals↗
PubMed1996

Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase)

The small guanosine triphosphatase Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacted with the myosin-binding subunit (MBS) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP.RhoA, phosphorylated MBS and consequently inactivated myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increased phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase.

3T3 Cells↗
PubMed1996

Introduction of basic fibroblast growth factor gene into mouse renal cell carcinoma cell line enhances its metastatic potential.

To clarify the role of basic fibroblast growth factor (FGF-2) in the malignant progression of renal cell carcinoma, we transfected the FGF-2 gene, which lacks the typical signal sequence, into RenCa, a mouse renal cell carcinoma cell line that does not express FGF-2 mRNA. In an in vitro tumor cell invasion assay, the FGF-2-transfected cell lines (RenCa/F) exhibited 3- to 4-fold higher invasive potential than either the parental RenCa (RenCa/P) or the vector-only transfected cell line (RenCa/C). Zymography showed a marked increase in matrix metalloproteinase 2 (MMP-2) production in the culture supernatants of RenCa/F. Furthermore, when injected i.v. or into the renal subcapsule in syngeneic mice, RenCa/F formed more than 10 times as many metastatic nodules in the lung as did RenCa/P and RenCa/C. Metastases to the liver and mesenteric lymph nodes were observed only after the injection of RenCa/F into the renal subcapsule. In contrast, there was no significant difference in either cell proliferation in vitro or tumor growth in vivo among RenCa sublines. These results suggest that if it is overexpressed, endogenous native FGF-2 plays an important role in the invasion and metastasis of renal cell carcinoma, probably through the production of MMP-2.

Animals↗
PubMed1996

Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway.

3T3 Cells↗