Biomedical subjects

K Chihara

Publications and source records attributed to K Chihara.

At least 109 records · Page 6Linked to original sources

PubMed1998

Carboxyl-terminal parathyroid hormone fragments stimulate type-1 procollagen and insulin-like growth factor-binding protein-5 mRNA expression in osteoblastic UMR-106 cells.

The effects of carboxyl-terminal (C-) PTH fragments, (35-84), (53-84) and (69-84), on the proliferation and function of osteoblastic UMR-106 cells were compared with those of amino-terminal (N-) (1-34) and intact (I-) (1-84) PTH. I-PTH as well as N-PTH at 10(-8)M significantly inhibited [3H] thymidine incorporation and stimulated alkaline phosphatase activity in UMR-106 cells. No C-PTH fragments affected them. In contrast, the expression of type-1 procollagen mRNA in these cells was stimulated by all C-PTH fragments, inhibited by N-PTH and not affected by I-PTH. All C-PTH fragments except (69-84) as well as N-PTH and I-PTH stimulated IGFBP-5 mRNA expression. The present study suggests that the C-portion of the PTH molecule exercises biological activities in mRNA expression of type-1 procollagen as well as IGFBP-5 in osteoblasts, and that it might be involved in the anabolic action of PTH on bone in vivo.

Cell Line↗

PubMed1998

Age-dependent changes in body composition in postmenopausal Japanese women: relationship to growth hormone secretion as well as serum levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3.

The present study was performed to investigate the age-dependent changes in body composition and the possible role of growth hormone (GH), insulin-like growth factor (IGF)-I and IGF-binding protein-3 (IGFBP-3) in these changes in postmenopausal Japanese women. A total of 161 Japanese women aged 45-88 years (mean 62) were enrolled in the cross-sectional study. Body composition (bone mineral content (BMC), lean body mass (LBM) and fat) was measured by dual-energy X-ray absorptiometry, and the percentage of BMC, LBM and fat was calculated by dividing each absolute value of body composition by total body mass. Urinary GH concentration divided by creatinine in nocturnal urine samples collected just after waking was used as an index of endogenous GH secretion. Serum levels of IGF-I and IGFBP-3 were measured by RIA. Urinary GH levels as well as serum levels of IGF-I and IGFBP-3 declined with age. BMC, %BMC and LBM also declined with age, while fat mass and %fat did not obviously change with age. Urinary GH levels as well as serum levels of IGF-I and IGFBP-3 correlated positively with BMC, even if age was taken into account. On the other hand, urinary GH correlated negatively with fat and %fat. In contrast, serum levels of IGF-I and IGFBP-3 correlated positively with fat and %fat. LBM did not correlate with either urinary GH or serum IGFBP-3 levels but exhibited a weakly positive correlation with serum IGF-I level. The present study suggests that the GH-IGF-I-IGFBP-3 axis positively regulates bone mass, and that GH and IGF-I-IGFBP-3 inversely regulate fat mass, i.e. GH negatively and IGF-I-IGFBP-3 positively regulates it.

Aged↗

PubMed1998

Fatal liver cirrhosis and esophageal variceal hemorrhage in a patient with type IIIa glycogen storage disease.

A 45-year-old woman with type IIIa glycogen storage disease (GSD IIIa) died of variceal hemorrhage secondary to liver cirrhosis. The postmortem examination disclosed increased intracellular glycogen in the liver as well as in the heart and skeletal muscle. Although most liver injuries in GSD IIIa have been considered to be non-progressive in adulthood, liver cirrhosis can be a cause of death in some patients.

Diagnosis, Differential↗

PubMed1998

Clinical significance and molecular mechanisms of bioinactive growth hormone (review).

About 80% of short children are not deficient in endogenous growth hormone (GH) and termed idiopathic short stature (ISS). The causes of impaired growth in children with ISS are various. Short stature and low insulin-like growth factor-I (IGF-I) concentration despite normal to high GH concentration suggest impaired GH effect. The prototypical GH insensitivity syndrome was described and characterized by the absent or defective GH receptors. Growth retardation resulting from biologically inactive GH was also described, but the molecular basis of biologically inactive GH has remained unclear. Recently, two unique point mutations in the GH-1 gene in the children with short stature whose GH were supposed as bioinactive were reported. Mutant GH R77C not only failed to stimulate tyrosine phosphorylation by itself, but it also inhibited the activity of wild-type GH. This mutant GH exerted an antagonistic effect. Another mutant D112G was only bio-inactive. This case was a typical Kowarski syndrome. The molecular heterogeneity of mutant GH reflected clinical phenotype of bioinactive GH syndrome.

Child↗

PubMed1998

[Multicentric Castleman's disease with lymphoid interstitial pneumonia and polyneuropathy].

A 51-year-old woman was admitted to a local hospital because of pneumonia, and received a diagnosis of cervical lymphadenopathy, anemia, and hyperimmunoglobulinemia with suspected multicentric Castleman's disease (MCD). At the age of 53 she was transferred to our hospital because of numbness and muscle weakness. Polyclonal hyperimmunoglobulinemia was observed, and biopsy specimens of lymph node tissue revealed infiltration of plasma cells in the interfollicular areas, thus leading to a diagnosis of MCD. A chest computed tomography scan revealed diffuse nodular shadows in all lung fields, and a transbronchial lung biopsy disclosed infiltration of plasma cells in the alveolar septa. Together, these findings indicated lymphoid interstitial pneumonia (LIP). Polyneuropathy was diagnosed by neurological examination and nerve conduction studies. Treatment with prednisolone and cyclophosphamide was ineffective against LIP probably because the patient had coexising pulmonary fibrosis. Since pulmonary complications of MCD determine its prognosis, their early detection and treatment are essential.

Castleman Disease↗

PubMed1998

[Disseminated carcinomatosis of the bone marrow occurring 11 years after subtotal gastrectomy for gastric cancer].

A 44-year-old man was admitted to our hospital because of purpura, increased serum alkaline phosphatase, and thrombocytopenia. He had undergone subtotal gastrectomy for gastric cancer 11 years earlier. A biopsy specimen of the bone marrow revealed metastatic mucin-forming, moderately differentiated adenocarcinoma. Because the primary tumor was not detected in any other organ, the gastric cancer the patient was treated for 11 years earlier was suspected as the primary tumor. Microangiopathic hemolytic anemia and disseminated intravascular coagulation developed during the clinical course, and the patient deteriorated despite treatment with anticoagulants. Finally, he died of pulmonary carcinomatous lymphangitis. Autopsy revealed a small number of adenocarcinomatous cells in the lymphoduct of the remaining stomach in spite of its mucosa being intact. We concluded that the bone marrow was infiltrated by cancer cells which originated in the stomach 11 years before. It is unclear why adenocarcinoma cells remained dormant for as long as 11 years in the gastric lymphoduct and bone marrow.

Adenocarcinoma↗

PubMed1997

Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase.

The small GTPase Rho is implicated in cytoskeletal rearrangements including stress fiber and focal adhesion formation and in the transcriptional activation of c-fos serum response element. In vitro, Rho-kinase, which is activated by Rho, phosphorylates not only myosin light chain (MLC) (thereby activating myosin ATPase) but also myosin phosphatase, thus inactivating myosin phosphatase. Rho-kinase is involved in the formation of stress fibers and focal adhesions in fibroblasts. Here we show that the expression of constitutively active Rho-kinase increased the level of MLC phosphorylation. The activity of Rho-kinase was necessary for maintaining the vinculin-containing focal adhesions, whereas organized actin stress fibers were not necessary for this. The microinjection of constitutively active Rho-kinase into fibroblasts induced the formation of focal adhesions to some extent under the conditions where organized actin stress fibers were disrupted. The expression of constitutively active Rho-kinase also stimulated the transcriptional activity of c-fos serum response element. These results suggest that Rho-kinase has distinct roles in divergent pathways downstream of Rho, which include MLC phosphorylation leading to stress fiber formation, focal adhesion formation, and gene expression.

3T3 Cells↗

PubMed1997

Cloning of mouse diastrophic dysplasia sulfate transporter gene induced during osteoblast differentiation by bone morphogenetic protein-2.

Although intensive studies have been directed at understanding osteoblastic differentiation, the molecular mechanisms are still unclear. In this study, we describe a cDNA that encodes a sulfate transporter that was cloned as a gene induced in osteoblast precursor cells in association with osteoblastic differentiation. Based on the fact that bone morphogenetic protein-2 (BMP-2) induces osteoblastic phenotypes in immature mouse fibroblastic C3H10T1/2 cells, we performed a subtraction hybridization between BMP-2-treated and untreated cells, and have isolated one clone (designated as st-ob for sulfate transporter in osteoblast) induced by BMP-2 that is constantly expressed in osteoblastic cells. The deduced amino acid sequence and proposed structure of st-ob are mostly identical to those of the human diastrophic dysplasia sulfate transporter gene product (DTDST). St-ob mRNA was abundantly expressed in the thymus, testis, calvaria and osteoblastic MC3T3-E1 cells, whereas its expression was faint in C3H10T1/2 cells. Expression of st-ob in C3H10T1/2 cells was increased by transforming growth factor-beta1 (TGF-beta1), retinoic acid and dexamethasone as well as BMP-2. Furthermore, BMP-2 increased sulfate incorporation in C3H10T1/2 cells about twice as high as the baseline level. Osteoblasts actively take up sulfate to synthesize proteoglycans, which are one of the major components of the extracellular matrix of bone and cartilage. The present study demonstrates that st-ob induced during osteoblastic differentiation is an important phenotype of osteoblasts for characterizing their function.

Animals↗

PubMed1997

Cloning and characterization of the 5'-flanking region of the mouse diastrophic dysplasia sulfate transporter gene.

Dyastrophic dysplasia sulfate transporter (DTDST) plays an important role in proteoglycan synthesis in the extracellular matrix of bone and cartilage. Recently, we found that the mouse DTDST gene was induced in pluripotent C3H10T1/2 cells during differentiation by bone morphogenetic protein-2 (BMP-2). To clarify the transcriptional regulation of the DTDST gene, we have cloned the 5'-flanking region of the mouse DTDST gene by the PCR based gene walking method. Sequence analysis revealed the presence of the TATA box followed by GC rich sequences containing two Sp-1 binding sites and a CBFA1 binding site. Transient transfection assays demonstrated that the basal transcriptional activity in osteoblastic MC3T3-E1 cells was mainly present between -309 and -275 bp upstream of the transcription start site (Segment -309/-275) which contained the consensus sequence for the xenobiotic-responsible element (XRE). Nuclear proteins from MC3T3-E1 cells and C3H10T1/2 cells could bind to this short segment in vitro. BMP-2 increased the promoter activity as well as the nuclear protein binding to the sequence in C3H10T1/2 cells. The present data suggest that the DTDST gene expression in osteoblasts and differentiating precursor cells to osteoblast/chondrocyte lineage would be mainly regulated by undetermined XRE binding transcription factors.

Animals↗

PubMed1997

Biologically inactive growth hormone caused by an amino acid substitution.

Short stature caused by biologically inactive growth hormone (GH) is characterized by lack of GH action despite high immunoassayable GH levels in serum and marked catch-up growth to exogenous GH administration. We found a heterozygous single-base substitution (A-->G) in exon 4 of the GH-1 gene of a girl with short stature, clinically suspected to indicate the presence of bioinactive GH and resulting in the substitution of glycine for aspartic acid at codon 112. We confirmed the presence of mutant GH in the serum using isoelectric focusing analysis. The locus of mutation D112G was found within site 2 of the GH molecule in binding with GH receptor (GHR)/GH binding protein (GHBP). The expressed recombinant mutant GH tended to form a 1:1 instead of the 1:2 GH-GHBP complex normally produced by wild-type GH. The formation of a 1:2 GH-GHBP complex is compatible with the dimerization of GHRs by GH, a crucial step in GH signal transduction. Mutant GH was less potent than wild-type GH not only in phosphorylation of tyrosine residues in GHR, janus kinase 2 (JAK2), and signal transducers and activators of transcription 5 (STAT5) in IM-9 cells, but also in metabolic responses of BaF/GM cells, a stable clone transfected with cDNA of the chimera of the extracellular domain of human GHR, the transmembrane and the cytoplasmic domain of the human thrombopoietin receptor. These results indicate that the D112G mutation in the GH-1 gene causes production of bioinactive GH, which prevents dimerization of GHR and is therefore responsible for the patient's short stature.

Child, Preschool↗

PubMed1997

Expression of a kinase-defective Eph-like receptor in the normal human brain.

We have identified a human Eph-family protein, HEP, gene located in human chromosomal region 7q33-->q35. The deduced amino acid sequence shared primary structural properties of Eph-family receptor tyrosine kinases. However, six invariant amino acids such as a lysine in the ATP-binding site and an aspartic acid in the phosphotransfer site of a conserved catalytic domain were substituted with other amino acid residues in HEP. Thus, no intrinsic tyrosine kinase activity was detectable in the catalytic domain expressed in CHO-K1 cell transfectants. Although most kinase-defective mutants of growth factor receptors have been reported as pathogenic receptors, its transcript was abundantly expressed in normal human adult tissues. A 135-kDa HEP protein was expressed in the human brain as much as in CHO-K1 cells transfected with a HEP cDNA expression vector. HEP is the first description of a kinase-defective Eph-family protein expressed abundantly in normal human tissues.

Adenosine Triphosphate↗

PubMed1997

Stimulatory effects of parathyroid hormone and 1,25-dihydroxyvitamin D3 on insulin-like growth factor-binding protein-5 mRNA expression in osteoblastic UMR-106 cells: the difference between transient and continuous treatments.

The transient treatment with parathyroid hormone (PTH) for 12 h, followed by its removal for 36 h, stimulated insulin-like growth factor-binding protein (IGFBP)-5 mRNA expression more strongly than the continuous treatment for 48 h in osteoblastic UMR-106 cells. The transient but not continuous treatment with A23187 also stimulated it. In contrast, 1,25-dihydroxyvitamin D3 stimulated it, irrespective of the treatment design. IGFBP-5 stimulated type-1 procollagen mRNA expression. The present study first indicated that the transient treatment with PTH more effectively stimulated IGFBP-5 mRNA expression than its continuous treatment partly via an increase in intracellular calcium and suggested that IGFBP-5 might be involved in the anabolic action of PTH in bone.

Animals↗

PubMed1997

Leptin induces mitogen-activated protein kinase-dependent proliferation of C3H10T1/2 cells.

Leptin, secreted by adipocytes, regulates satiety and energy expenditure. Several forms of leptin receptors produced by alternative mRNA splicing are found in many tissues, including the hypothalamus, liver, lung, kidney, hematopoietic cells, and gonads, suggesting that leptin exerts effects in these tissues. In accordance with the distribution of leptin receptors, there is accumulating evidence that leptin plays various roles in reproduction, hematopoiesis, and the immune systems in addition to the regulation of food intake and energy expenditure. In the present study, we examined the in vitro effects of leptin on proliferation of a mouse embryonic cell line, C3H10T1/2, and its mechanism of action. Leptin caused a dose- and time-dependent increase in mitogen-activated protein kinase (MAPK) activity that was accompanied by an increase in C3H10T1/2 cell number. The MAPK kinase-1-specific inhibitor PD98059 completely blocked the increases in both MAPK activity and cell proliferation caused by leptin. These findings indicate that leptin stimulates the proliferation of C3H10T1/2 cells via the MAPK cascade.

Animals↗

PubMed1997

Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase.

The small guanosine triphosphatase (GTPase) Rho is implicated in the formation of stress fibers and focal adhesions in fibroblasts stimulated by extracellular signals such as lysophosphatidic acid (LPA). Rho-kinase is activated by Rho and may mediate some biological effects of Rho. Microinjection of the catalytic domain of Rho-kinase into serum-starved Swiss 3T3 cells induced the formation of stress fibers and focal adhesions, whereas microinjection of the inactive catalytic domain, the Rho-binding domain, or the pleckstrin-homology domain inhibited the LPA-induced formation of stress fibers and focal adhesions. Thus, Rho-kinase appears to mediate signals from Rho and to induce the formation of stress fibers and focal adhesions.

3T3 Cells↗

PubMed1997

Insulin-like growth factor-I mediates osteoclast-like cell formation stimulated by parathyroid hormone.

There have been several lines of evidence that parathyroid hormone (PTH) stimulates production of insulinlike growth factor I (IGF-I) in bone and that IGF-I stimulates osteoclast formation. Thus, the present study was performed to clarify the possible role of IGF-I in PTH-stimulated osteoclastlike cell formation and the role of PTH-responsive dual signal transduction systems (cyclic [c] AMP-dependent protein kinase [PKA] and calcium/protein kinase C [PKC]) in its mechanism. Treatment with anti-IGF-I antibody (1-10 micrograms/ml) partially but significantly blocked hPTH-(1-34)-stimulated osteoclastlike cell formation in unfractionated mouse bone cell cultures, although it did not affect osteoclastlike cell formation stimulated by 1,25-dihydroxyvitamin D3. Rp-cAMP5 (10(-4) M), a direct PKA inhibitor, as well as two types of PKC inhibitors, H-7 (10 microM) and staurosporine (3 nM), and dantrolene (10(-5) M), an inhibitor of calcium mobilization from intracellular calcium stores, all significantly blocked PTH-stimulated osteoclastlike cell formation. Anti-IGF-I antibody (3 micrograms/ml) significantly blocked osteoclastlike cell formation stimulated by 10(-4) M dbcAMP, 10(-4) M Sp-cAMPS, a direct PKA activator, and 10(-5) M forskolin in mouse bone cell cultures. Dibutyryl cAMP, forskolin, and hPTH-(1-34) significantly stimulated mRNA expression of both IGF-I and IGF-binding protein 5 (IGFBP-5) in these cultures, but neither 10(-7) M PMA, a PKC activator, nor 10(-7) M A23187 did. Moreover, anti-IGF-I antibody significantly blocked osteoclastlike cell formation stimulated by the conditioned medium from MC3T3-E1 cells pretreated with 10(-8) PTH-(1-34), which induced IGF-I and IGFBP-5 mRNA expression in these cells. In conclusion, the present study indicates that IGF-I mediates osteoclastlike cell formation stimulated by PTH and that the PKA pathway is involved in its mechanism. However, IGF-I does not seem to be the sole effector molecule to be active in this system.

Animals↗

PubMed1997

Lesion-induced neuronal nitric oxide synthase in Purkinje cells of the rat cerebellar cortex: histochemical and in situ hybridization study.

Lesion-induced induction of neuronal nitric oxide synthase (nNOS) was examined in the rat cerebellum. The stab-lesioned cerebellar cortex was examined with NADPH-diaphorase (NADPH-d) histochemistry and in situ hybridization using nNOS cRNA probe at 1, 3, 7, 14, 35 days post-lesion. NADPH-d- and nNOS mRNA-positive Purkinje cells appeared adjacent to the lesion by 3 days after the lesion. The area of distribution expanded and the number of positive cells increased at 7 days after the lesion, and at 14 days post-lesion, shrunken NADPH-d-positive Purkinje cells with irregular surface appeared. NADPH-d activity and nNOS mRNA signal could not be detected in Purkinje cells after 35 days post-lesion. Combined NADPH-d histochemistry and in situ hybridization using glutamic acid decarboxylase (GAD) cRNA probe revealed that nNOS-expressing Purkinje cells showed fewer GAD mRNA signals than those in normal Purkinje cells. The atrophic contour and the lower expression of GAD mRNA signals in NADPH-d positive Purkinje cells suggest that nNOS is expressed under a degenerating process.

Animals↗

PubMed1997

Adrenocorticotrophin-independent macronodular adrenal hyperplasia in a patient with lysine vasopressin responsiveness but insensitivity to gastric inhibitory polypeptide.

The aetiology of ACTH-independent macronodular adrenal hyperplasia (AIMAH) is uncertain. We examined a 55 year old man with Cushing's syndrome due to AIMAH, whose cortisol levels increased after stimulation with lysine-8-vasopressin (LVP) in vitro as well as in vivo. Abdominal MRI revealed nodular enlargement of both adrenal glands. No adenoma was evident on pituitary MRI. 131I-adosterol scintigraphy exhibited marked uptake into both adrenal glands. Although baseline plasma cortisol levels were within normal limits, urinary free cortisol excretion was 3-fold higher than the upper limit of the normal range. Plasma ACTH levels were undetectable. Oral dexamethasone failed to suppress plasma cortisol levels irrespective of dose, and administration of corticotrophin releasing hormone failed to increase plasma ACTH and cortisol levels. LVP injection failed to increase plasma ACTH levels, but elicited an increase in plasma cortisol levels. The direct stimulatory effect of LVP on cortisol secretion was confirmed in vitro in cultured adrenocortical cells from macronodules obtained at surgery. Food intake, gastric inhibitory polypeptide (GIP), or octreotide administration, which were reported to regulate cortisol release in patients with AIMAH, failed to affect plasma cortisol levels. In conclusion, plasma cortisol responsiveness to LVP, GIP, and octreotide is heterogeneous in patients with AIMAH.

Adrenal Glands↗

PubMed1997

Characterization of GH3 cells overexpressing basic fibroblast growth factor (FGF-2).

Basic fibroblast growth factor (FGF-2) is not only a potent mitogen for various cells but also a multifunctional factor with angiogenic and chemotactic activity, and the capacity to induce the synthesis of various proteinases and to modulate endocrine function. To clarify the role played by FGF-2 in the progression of pituitary tumor, we fused rat FGF-2 cDNA to the promoter SR alpha, consisting of the early promoter of SV40 and HTLV(I)-LTR, and we cotransfected GH3 cells with pSV2-neo by an electroporation method. After selection by G418, we obtained 7 neomycin-resistant clones. Southern blot analysis of genomic DNA revealed the presence of transfected rat FGF-2 cDNA in 4 of the 7 clones. To measure FGF-2 molecules, we established a new immuno-fluorometric assay system, using 3 monoclonal antibodies against different portions of human FGF-2. This assay had a minimum sensitivity of 10 pg/ml and cross-reacted neither with acidic fibroblast growth factor (FGF-1) nor insulin-like growth factor 1 (IGF-1), even at a concentration of 100 ng/ml. Although FGF-2 was undetectable in the culture medium of any of the clones, the cell homogenate contained a significant amount of FGF-2 (7.2 ng/mg protein) in 1 of the 4 FGF-2-transfected clones (GH3FGF(+)), whereas FGF-2 was not detected (< 5.2 pg/mg protein) in the cell homogenates of either the parent GH3 cells or the control cells transfected with pSV2-neo alone (GH3FGF(-)), GH3FGF(+) grew as adherent cells and formed epithelial sheets with a growth rate similar to that of control cells. The amount of prolactin(PRL) released by TRH was greater in GH3FGF(+) than that in GH3 or GH3FGF(-). On the other hand, the sensitivity to SRIF was increased in GH3FGF(+) compared with that in other clones. The findings of these in vitro studies indicate that FGF-2, if it is expressed in pituitary tumor cells, plays little if any role in cell growth but may modulate certain cell functions such as responsiveness to hormones.

Adenoma↗