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Biomedical subjects

Kazuo Chihara

Publications and source records attributed to Kazuo Chihara.

At least 91 records · Page 5Linked to original sources
PubMed2003

Morphologic, flow cytometric and cytogenetic evaluation of bone marrow involvement in B-cell lymphoma.

BACKGROUND AND OBJECTIVES: Flow cytometric immunophenotypic analysis (FC) and cytogenetic analysis are essential techniques for the diagnosis and classification of many hematologic disorders. The roles of these analyses in B-cell lymphoma to detect bone marrow (BM) involvement in clinical staging and to evaluate the effectiveness of treatments have not yet been determined. The aim of this study was to evaluate the usefulness of FC and cytogenetic analysis in the assessment of BM involvement in B-cell lymphoma. DESIGN AND METHODS: We retrospectively analyzed the usefulness of three-color FC and cytogenetic analysis in detecting BM involvement by examining 104 BM specimens from patients with B-cell lymphoma. RESULTS: By morphologic evaluation of BM biopsy (BMB), the BM was involved in 11 specimens (10.6%), not involved in 92 specimens (88.5%) and involvement could not be determined in 1 specimen (0.9%). FC identified a monoclonal B-cell population in 24 samples (23.1%). FC detected BM involvement in all but one BMB positive sample, and showed negative results in a BMB undetermined sample. Conclusively, FC found a monoclonal B-cell population in 14 of 92 BMB negative samples (15.2%). In particular, FC detected smaller amounts of BM involvement than did morphologic evaluation. Cytogenetic analysis revealed clonal abnormalities in only 9 of 104 samples (8.7%). However, 2 of these 9 samples were from patients with aggressive lymphoma with complex structural chromosomal abnormalities detectable only by cytogenetic analysis. INTERPRETATION AND CONCLUSIONS: Although morphologic evaluation of adequate amounts of BMB specimens remains essential for the evaluation of BM involvement, three-color FC is more sensitive in detecting BM disease than morphologic or cytogenetic analysis. Cytogenetic analysis seems to have low sensitivity and specificity, but this method may improve the detection of BM involvement in a small number of aggressive lymphomas that have many mitotic cells.

B-Lymphocytes↗
PubMed2003

Expression of mPOU protein in the human pituitary adenomas.

OBJECTIVE: mPOU is a POU protein classified as class VI. It is present in the pituitary gland as well as the brain, heart muscle, skeletal muscle, lung, and lymphocytes. In our previous investigation, mPOU bound to the Pit-1-binding DNA elements of the rat PRL gene, and promoted transcription of the GH and PRL genes. In this study, we immunohistologically investigated the expression of mPOU in pituitary adenomas. METHODS: 17 patients with pituitary adenoma underwent tumor excision by transsphenoidal approach at our hospital (PRL: 5, GH: 4, FSH: 1, non-functioning: 7). The expression in the tissue sections was investigated using immunostaining (ABC method). RESULTS: In all GH-producing and PRL-producing adenomas, mPOU protein was specifically expressed, particularly in the nuclei. DISCUSSION: Pit-1 has been considered to be a factor determining the expression of GH and PRL genes, but mPOU may also be involved in the expression.

Adenoma↗
PubMed2003

[Not Available].

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Journal Article↗
PubMed2003

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Journal Article↗
PubMed2002

The extracellular calcium Ca2+o-sensing receptor is expressed in myeloma cells and modulates cell proliferation.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca(2+)(o)) homeostasis by enabling parathyroid, kidney, and other cells to directly "sense" changes in Ca(2+)(o). In multiple myeloma-associated bone disease, myeloma cells could raise the level of Ca(2+)(o) within their immediate vicinity in the bone marrow microenvironment, through their known capacity to cause bone destruction by stimulating osteoclastic bone resorption. Thus if myeloma cells expressed the CaR, they might sense these locally elevated levels of Ca(2+)(o), which could, in turn, potentially modify their function(s) in ways that could contribute to myeloma bone disease or other aspects of the pathophysiology of this disabling hematological malignancy. In this study, we examined the expression of the CaR in three myeloma cell lines, human U266, IM-9, and RPMI8226 cells. CaR protein was present in all three cell lines as assessed by immunocytochemistry and Western blot analysis using a monoclonal antibody specific for the CaR. Moreover, the use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in the three cell lines. Exposure to known polycationic agonists of the CaR, including high Ca(2+)(o) (2.5mM), neomycin, and gadolinium (Gd(3+)) as well as a specific CaR activator, NPS R467, augmented cell proliferation in all three cell lines. RT-PCR revealed that U266 cells, but not IM-9 cells or RPMI8226 cells, expressed interleukin-6 (IL-6), the expression of which was not enhanced by treatments with CaR agonists. Therefore, taken together, our data first document the fact that the myeloma cell lines, U266, IM-9, and RPMI8226, all express CaR protein and mRNA. Moreover, the CaR expressed on myeloma cells could sense the locally high levels of Ca(2+)(o) in the vicinity of sites of osteoclastic bone resorption and stimulate their proliferation in an IL-6-independent manner. These processes may result in promoting further growth of the tumor and aggravating the associated bone disease.

Animals↗
PubMed2002

Alternative splicing variants of the human DBL (MCF-2) proto-oncogene.

The DBL (MCF-2) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates the Rho family of GTPases. In this communication we describe the isolation of three novel splicing variants of Dbl. The prototype Dbl gene (designated var.1 here) contains 25 exons, while splicing variant 2 (var.2) lacks exons 23 and 24. Var.3 contains additional 3 exons from 5(')-UTR in place of exon 1, while var.4, var.2, and var.3 contain a 48bp insertion between exons 10 and 11, resulting in the insertion of 16 amino acids. We found that var.1 was expressed only in brain, whereas var.3 was expressed in heart, kidney, spleen, liver, and testis, and var.4 in brain, heart, kidney, testis, placenta, stomach, and peripheral blood. The Dbl protein was detectable in brain, heart, kidney, intestine, muscle, lung, and testis. An assay for GEF activity revealed that the var.2 exhibits decreased GEF activity towards Cdc42, var.3 exhibits a weak but significant activity toward Rac1 and Cdc42, var.4 exhibits significant activity toward RhoA and Cdc42, while var.1 exhibits no activity toward RhoA, Rac1, or Cdc42. In summary, we describe 4 splicing variants of the human DBL proto-oncogene that show different tissue distributions and GEF specificities.

Alternative Splicing↗
PubMed2002

Novel function of the transactivation domain of a pituitary-specific transcription factor, Pit-1.

Pit-1 stimulates the expression of growth hormone, prolactin, and thyrotropin beta subunit genes. Consequently, abnormality of the Pit-1 gene results in combined pituitary hormone deficiency (CPHD). In this study, we analyzed the function of Pit-1 with a mutation (proline to leucine at codon 24) in the transactivation domain, P24L, which has a normal POU domain important for binding to DNA, because this mutation had been reported in a patient with CPHD. We found that codon 24 proline in the transactivation domain as well as the POU domain of Pit-1 was crucial to recruit coactivator CREB-binding protein (CBP) in the cultured cells. P24L completely lost the responsiveness to cAMP to stimulate the expression of the Pit-1-targeted genes. Furthermore, CBP and Pit-1, but not P24L, markedly enhanced the expression of the Pit-1-targeted gene to cAMP, and adenovirus E1a that binds to CBP and abrogates its function blocked the induction by cAMP of Pit-1-stimulated gene transcription in the pituitary-derived GH3 cells. These results suggest that CBP and proline at codon 24 in the transactivation domain of Pit-1 are important for the cAMP-induced activation of Pit-1-targeted genes. However, P24L maintained basal transcriptional activity, suggesting that CBP is unlikely to be an essential coactivator for Pit-1.

Animals↗
PubMed2002

Activations of ERK1/2 and JNK by transforming growth factor beta negatively regulate Smad3-induced alkaline phosphatase activity and mineralization in mouse osteoblastic cells.

Transforming growth factor (TGF) beta inhibits alkaline phosphatase (ALP) activity and mineralization in mouse osteoblastic MC3T3-E1 cells, whereas local administration of TGF-beta stimulates bone formation in vivo. We recently demonstrated that Smad3, a TGF-beta signaling molecule, promotes ALP activity and mineralization in MC3T3-E1 cells. Moreover, the target disruption of Smad3 in mouse is reported to cause a decrease in bone mineral density. These findings indicate that Smad3 plays an important role in the regulation of bone formation. However, why the effects of TGF-beta and Smad3 on ALP activity and mineralization are different remains unknown. The purpose of the present study is to clarify the role of mitogen-activated protein kinase (MAPK) in TGF-beta and Smad3 pathways in osteoblast. TGF-beta activated extracellular signal-regulated kinases/p42/p44 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in mouse osteoblastic MC3T3-E1 cells. The expression of dominant negative type Smad3, Smad3DeltaC, affected neither TGF-beta-activated MAPKs nor TGF-beta-inhibited ALP activity. Specific inhibitors of ERK1/2 activation (PD98059 and U0126), as well as JNK inhibitors (curcumin and dicumarol) antagonized the inhibitory effects of TGF-beta on ALP activity and mineralization, whereas the specific inhibitor of p38 MAPK (SB203580) did not affect them. PD98059 and curcumin enhanced Smad3-induced ALP activity and mineralization, whereas SB203580 inhibited them. In the luciferase reporter assay using 3TP-lux with the specific Smad3-responsive element, PD98059, and curcumin enhanced TGF-beta- and Smad3-induced transcriptional activity in MC3T3-E1 cells. On the other hand, TGF-beta-induced production of type I collagen was antagonized by curcumin but not by PD98059. The present study indicated that TGF-beta-responsive ERK1/2 and JNK cascades negatively regulate Smad3-induced transcriptional activity as well as ALP activity and mineralization in osteoblasts.

Alkaline Phosphatase↗
PubMed2002

IGF-I mediates the stimulatory effect of high phosphate concentration on osteoblastic cell proliferation.

Although high concentrations of inorganic phosphate (Pi) are known to have a distinct anabolic effect on bone structure and metabolism, the precise mechanism by which phosphate possesses anabolic effect on bone formation has not been elucidated. The present study was performed to examine the effects of an increase in extracellular Pi concentration ([Pi](e)) on the proliferation of osteoblastic MC3T3-E1 cells. Increase in [Pi](e)(2-4 mM) dose-dependently stimulated DNA synthesis. Indomethacin, an inhibitor of prostaglandin synthesis, did not affect high [Pi](e)-induced DNA synthesis. DNA synthesis first increased affer a 3 h exposure to 4 mM [Pi](e) and its stimulatory effect was observed in a time-dependent manner up to 24 h. On the other hand, DNA synthesis was significantly but partially blocked by cycloheximide, suggesting that this stimulatory effect of high [Pi](e) was at least in part dependent on new protein synthesis. There is recent evidence that MG3T3-E1 cells constitutively produce and secrete insulin-like growth factor-I (IGF-I) and possess IGF-I receptors. IGF-I antiserum (1:10,000 to 1:100) significantly but partially blocked the stimulatory effect of [Pi](e) (4 mM) on DNA synthesis in a concentration-dependent manner. A neutralizing IGF-I antibody as well as IGF-I receptor antibody also significantly but partially blocked DNA synthesis stimulated by high [Pi](e) in a concentration-dependent manner, indicating that IGF-I at least in part mediated the high [Pi](e)-induced effect. Actually, high [Pi](e) significantly increased the secretion of immunoreactive IGF-I into the medium as well as the expression of IGF-I mRNA. Present findings indicate that an increase in [Pi](e) stimulated DNA synthesis partly via an increase in IGF-I action.

3T3 Cells↗
PubMed2002

A novel heterozygous T51I mutation of growth hormone receptor is not associated with short stature.

A novel missense mutation of the growth hormone receptor (GHR) gene was identified in a Japanese short boy with GH insensitivity. The analysis of the GHR gene revealed a novel heterozygous T51I mutation in exon 4. To clarify the effect of this mutation on GH signaling, the mutant GHR was expressed in CHO cells and its functional properties were investigated. The signal transducer and activator of transcription 5 (STAT5)-mediated transcriptional activation in mutant GHR (GHR-T51I)-expressing cells was significantly reduced as compared with wild-type GHR (GHR-wt)-expressing cells. The CHO cells co-expressing the GHR-T51I and GHR-wt, however, revealed no functional differences as compared with cells expressing GHR-wt alone, indicating that the T51I mutation does not exert the dominant negative effect. In addition, the mother and the elder brother of the proband, whose heights were normal, possessed the same heterozygous mutation. These results suggest that the heterozygous T51I mutation of GHR does not inhibit the signal transduction of GH and is not responsible for GH insensitivity.

Amino Acid Sequence↗
PubMed2002

Expression of parathyroid hormone-related protein (PTHrP) in multiple myeloma.

Multiple myeloma is a plasma cell neoplasia often associated with multiple skeletal lesions and hypercalcemia. Several cytokines, including interleukin (IL)-1, IL-6 and tumor necrosis factor-beta (TNF-beta), derived from myeloma cells are thought to accelerate osteoclastic bone resorption and cause hypercalcemia through a paracrine mechanism. We report on a case of a 69-year-old man with multiple myeloma associated with hypercalcemia and advanced osteolytic lesions. After bisphosphonate treatment and MP (melphalan and prednisolone) therapy, the patient's serum calcium level was successfully but transiently recovered to the normal range. Biochemical analysis showed a remarkable increase in serum parathyroid hormone-related protein (PTHrP; 3.7 pmol/L) and IL-6 (22.0 pg/mL). On the other hand, parathyroid hormone and 1alpha,25(OH)2 vitamin D3 were suppressed. By immunohistochemistry and in situ hybridization on aspiration-biopsied bone marrow clot sections, PTHrP mRNA and protein were detected in the cytoplasm of myeloma cells. The rate of PTHrP-positive myeloma cells was estimated to be at least one-third. Since PTHrP can, as an endocrine factor, systemically act on bone and kidney, hypercalcemia in this case might have been caused through both local osteolytic hypercalcemia and humoral hypercalcemia of malignancy mechanisms.

Aged↗
PubMed2002

Familial hypocalciuric hypercalcemia caused by an R648stop mutation in the calcium-sensing receptor gene.

In this study, we report an 84-year-old female proband in a Japanese family with familial hypocalciuric hypercalcemia (FHH) caused by an R648stop mutation in the extracellular calcium-sensing receptor (CaR) gene. At the age of 71 years, she presented with hypercalcemia (11.4 mg/dl), hypocalciuria (Cca/Ccr = 0.003), hypermagnesemia (2.9 mg/dl), and a high-serum parathyroid hormone (PTH) level (midregion PTH, 3225 [160-520] pg/ml). At the age of 74 years, a family screening was carried out and revealed a total of 9 hypercalcemic individuals (all intact PTH values <62 pg/dl) among 17 family members tested, thus, being diagnosed as FHH. Two and one-half of three clearly enlarged parathyroid glands were resected, because persistently high PTH levels (intact PTH, 292 pg/ml; midregion PTH, 5225 pg/ml) and the presence of a markedly enlarged parathyroid gland by several imaging modalities (ultrasonography, computed tomography [CT], magnetic resonance imaging [MRI], and subtraction scintigraphy) suggested coexistent primary hyperparathyroidism (pHPT); however, hypercalcemia persisted postoperatively. Histological and immunohistochemical examination revealed that the resected parathyroid glands showed lipohyperplasia as well as normally expressed Ki67, vitamin D receptor (VDR), and the CaR. Sequence analysis disclosed that the proband and all affected family members had a heterozygous nonsense (R648stop) mutation in the CaR gene. This mutation is located in the first intracellular loop; thus, it would be predicted to produce a truncated CaR having only one transmembrane domain (TMD) and lacking its remaining TMDs, intracellular loops, and C-terminal tail. Western analysis of biotinylated HEK293 cells transiently transfected with this mutant receptor showed cell surface expression of the truncated protein at a level comparable with that of the wild-type CaR. The mutant receptor, however, exhibited no increase in intracellular free calcium concentration (Ca2+i) when exposed to high extracellular calcium concentrations (Ca2+o). The proband's clinical course was complicated because of associated renal tubular acidosis (RTA) and nephrotic syndrome. However, it was unclear whether their association affected the development of elevated serum PTH and parathyroid gland enlargement. This report is the first to show that an R648stop CaR mutation yields a truncated receptor that is expressed on the cell surface but is devoid of biological activity, resulting in FHH.

Base Sequence↗
PubMed2002

Smad3 promotes alkaline phosphatase activity and mineralization of osteoblastic MC3T3-E1 cells.

Transforming growth factor (TGF) beta is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-beta signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-beta on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-beta inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L-azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.

Alkaline Phosphatase↗
PubMed2002

Plasma lipids and osteoporosis in postmenopausal women.

Many clinical studies have shown that osteoporosis is associated with atherosclerosis and cardiovascular death. Although both high plasma levels of low density lipoprotein cholesterol (LDL-C) and low plasma levels of high density lipoprotein cholesterol (HDL-C) are known to be risk factors for atherosclerosis, it is unclear whether such lipid derangements are also associated with the pathogenesis of osteoporosis. In this study, we evaluated the relationships between plasma levels of total C, LDL-C, HDL-C, or triglyceride (TG) versus bone mineral density (BMD) at the lumbar spine, femoral neck, radius, or total body as well as the presence of vertebral fractures in 214 Japanese postmenopausal women (age range, 47-86 years, mean 62.7). Multiple regression analysis was performed between BMD at each skeletal site versus each lipid level adjusted for age, years after menopause, body mass index (BMI), and %fat. Plasma LDL-C levels were significantly and inversely correlated with the absolute values of both one-third radial (1/3R) and distal radial (UDR) BMD (p<0.01), and tended to be inversely correlated with the absolute values of L-BMD (p=0.051). In contrast, plasma HDL-C levels were significantly and positively correlated with the absolute values of L, 1/3R and UDR BMD (p<0.05). On the other hand, plasma TG levels were significantly lower in women with vertebral fractures than in those without fractures (97.0+/-36.5 vs. 126.4+/-65.8 mg/dl, mean+/-SD, p<0.05). When multivariate logistic regression analysis was performed with the presence of vertebral fractures as a dependent variable and each lipid level adjusted for age, years after menopause, BMI, and %fat as independent variables, TG alone was selected as an index affecting the presence of vertebral fractures (odds ratio: 0.51, 95% confidential interval: 0.29-0.89 per SD increase, p<0.05). Our study showed that plasma LDL-C and HDL-C levels were inversely and positively correlated with both R- and L-BMD values, respectively, while low plasma TG levels were associated with the presence of vertebral fractures in postmenopausal women. Thus, plasma lipids might be related to bone mass and bone fragility, and might be the common factor underlying both osteoporosis and atherosclerosis.

Aged↗
PubMed2002

Time-dependent effects of vitamin K2 (menatetrenone) on bone metabolism in postmenopausal women.

Vitamin K is known to mediate carboxylation of glutamyl residues of osteocalcin. We evaluated the effects of vitamin K2 (menatetrenone) treatment (45 mg/day) for 48 weeks on the markers of bone formation and resorption, bone mineral density (BMD), and the incidence of vertebral fractures in 34 Japanese postmenopausal women (aged 48-82 years). Serum levels of alkaline phosphatase (ALP) increased gradually and became significant at 48 weeks after menatetrenone treatment, while urinary excretion of deoxypyridinoline (DPD) decreased transiently but significantly at 4 weeks. Serum levels of both intact osteocalcin (OC) and carboxylated OC (Gla-OC) increased rapidly and significantly within 4 weeks and sustained their high values up to 48 weeks after the treatment, while those of undercarboxylated OC (Glu-OC) decreased reciprocally. These results can be interpreted to suggest that Glu-OC was converted to Gla-OC in vivo. On the other hand, lumbar BMD values showed no significant change and only one subject with a previous vertebral fracture had one newly occurring vertebral fracture. These results indicate that menatetrenone treatment of postmenopausal women constantly elevates bone formation markers as well as converts Glu-OC to Gla-OC. Thus, vitamin K2 treatment may promote bone formation, at least as measured biochemically in these subjects.

Aged↗
PubMed2002

Adult growth hormone deficiency in Japan: results of investigation by questionnaire.

Adult GH deficiency (AGHD) has been established as a syndrome associated with various metabolic disturbances such as hyperlipidemia, impaired glucose tolerance and protein catabolism, in addition to changes in body composition such as increased visceral fat, decreased muscle mass and bone density. We investigated the clinical findings, complications and prognosis of AGHD in Japan. The questionnaire was sent to various expert facilities of endocrinology and metabolism to gather cross-sectional information as well as longitudinal follow-up data on adult patients with hypopituitarism. We received answers on 422 subjects, of which number the GH stimulation test was performed in only 63% of them. An age- and sex-matched group of 259 adults with hypopituitarism (125 male and 134 female subjects) was finally selected for this investigation. Of them 185 subjects (81 male and 104 females) were diagnosed as AGHD with plasma peak GH levels less than 3 ng/ml after GH stimulation test. Male adult patients with GHD had significantly lower ratio of smoking and drinking in their life style compared with those without GHD. Male adult patients with GHD revealed significantly higher BMI on physical examination, and significantly higher plasma ALT, AST, total cholesterol, and LDL cholesterol in blood chemistry compared with those without GHD (P < 0.05). Though patients with ischemic heart disease were more frequent in female patients than male patients, the rate of frequency was not different between female adult patients with and without GHD. Clinical characteristics found in especially male adult patients with GHD in Japan were consistent with findings reported so far in foreign countries. However, consequent complications such as atherosclerosis seemed less severe than expected. Moreover, GH stimulation test for the diagnosis of AGHD as well as clinical test to perform when AGHD was suspected is still less frequently carried out. Therefore, the clinical outcome of AGHD in our country requires further investigation.

Adult↗