Biomedical subjects

S Fukumoto

Publications and source records attributed to S Fukumoto.

At least 73 records · Page 4Linked to original sources

PubMed1999

Molecular cloning of a novel alpha2,3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids.

A novel member of the human CMP-NeuAc:beta-galactoside alpha2, 3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of alpha2, 3-sialyltransferase toward Galbeta1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity, i.e. it utilized Galbeta1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.

Amino Acid Sequence↗

PubMed1999

Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides.

Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.

Amino Acid Sequence↗

PubMed1999

Differential expression and regulation of chemokines JE, KC, and IP-10 gene in primary cultured murine hepatocytes.

Chemokines are a superfamily of structurally related chemoattractant cytokines. JE (monocyte chemoattractant protein-1) and IP-10 (interferon-inducible protein-10) have been detected in the diseased liver. However the in vitro expression is unclear. In this report, we revealed that JE, KC (melanoma growth-stimulating activity gene), and IP-10 mRNAs are not expressed in the normal liver but spontaneously and time-dependently expressed in the primary hepatocytes. The serum-independent gene expression of both JE and KC lasted over 72 h, but that of IP-10 became undetectable 24 h after isolation with collagenase perfusion method. The induction of the genes' expression was not due to LPS contamination but nevertheless was associated with isolation procedure. Actinomycin D blocked their expression. The increase of their transcripts resulted from greater increase in gene transcription and lower mRNA stability. Consistent with c-jun, their mRNA expressions were simultaneously superinduced by cycloheximide (1 microg/ml), suggesting that de novo protein synthesis is involved their transcriptions. Inhibition by pyrrolidine dithiocarbamate (PDTC), a NF-kappaB/c-rel inhibitor, and EMSA imply that NF-kappaB/c-rel is important in their expressions. Of particular interest is that dexamethasone upregulated the spontaneous expression of KC, but suppressed that of JE and IP-10. LPS upregulated the mRNA levels of JE and KC but did not affect that of IP-10. IFN-gamma induced the expression of IP-10; however unlike in macrophages, it did not selectively inhibit that of JE and KC. Our data demonstrated the existence and differential gene expression of JE, KC, and IP-10 in primary cultured hepatocytes, and these are considered to be a reflex of the alteration of hepatocyte cellular physiology during and after isolation.

Animals↗

PubMed1999

Diagnostic utility of magnetic resonance imaging skeletal survey in a patient with oncogenic osteomalacia.

Oncogenic osteomalacia is a rare paraneoplastic syndrome characterized by hypophosphatemic osteomalacia due to renal phosphate wasting. The same biochemical features are found in patients with X-linked hypophosphatemic rickets/osteomalacia and sporadic hypophosphatemic osteomalacia with unknown etiology. Oncogenic osteomalacia is cured by resection of the responsible tumor. In contrast, patients with other types of hypophosphatemic rickets/osteomalacia need long-term treatment with large doses of active vitamin D3. Therefore, detection of the responsible tumor for oncogenic osteomalacia has great clinical importance. However, there is no standard method for detecting the tumor for oncogenic osteomalacia, and the responsible tumor is often very difficult to be found. We describe a patient with adult-onset osteomalacia due to renal phosphate wasting. Although oncogenic osteomalacia was suspected, cranial, chest, and abdominal computed tomography scanning, urological and otolaryngological examinations, and detailed palpation for soft tissue mass failed to detect the responsible tumor. However, magnetic resonance imaging skeletal survey revealed a tumor in the right femoral bone. Resection of the tumor resulted in normalization of serum phosphate and renal phosphate handling. Because the most frequent causes for oncogenic osteomalacia are tumors in bone or soft tissue, magnetic resonance imaging skeletal survey is a very powerful method for detecting the responsible tumor. Vigorous search for tumors with this method in patients with hypophosphatemic osteomalacia would be helpful not only for proper management of patients, but also for clarifying the identity of sporadic hypophosphatemic osteomalacia.

Bone Neoplasms↗

PubMed1999

Occurrence and significance of decahydroquinolines from dendrobatid poison frogs and a myrmicine ant: use of 1H and 13C NMR in their conformational analysis

Structures for 2,5-disubstituted decahydroquinolines (DHQs) are reported for the two diastereomeric pairs cis-275B (14) and cis-275B' (15) and 5-epi-trans-269AB (18) and trans-269AB (19), all isolated from skin extracts of dendrobatid frogs, and for 5-epi-cis-275B' (16) and 5-epi-trans-275B (17) found in the extracts of virgin queens of a myrmicine ant [Solenopsis (Diplorhoptrum) azteca]. Detection of such DHQs in an ant, their first reported occurrence, strengthens a dietary hypothesis for the origin of the approximately 30 DHQs that have been detected in extracts of frog skin. NMR data on the two conformers of cis-decahydroquinoline permit assignment of ring conformations and stereochemistry to cis-DHQs of the "N-endo" type or the "N-exo" type. These conformations are also assigned on whether H-8a is equatorial or axial as determined with E-COSY or 1D-HOHAHA spectra.

Journal Article↗

PubMed1999

An adult patient with severe hypercalcaemia and hypocalciuria due to a novel homozygous inactivating mutation of calcium-sensing receptor.

Inactivating mutations in the calcium-sensing receptor (CaSR) cause familial hypocalciuric hypercalcaemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). Earlier investigations showed patients with FHH are heterozygous, and NSHPT are homozygous for inactivating mutations. However, one adult patient with severe hypercalcaemia and hypocalciuria has been reported to have a homozygous inactivating mutation in CaSR (Pro39Ala). This suggested that mutant CaSR in this patient had some residual activity and hypercalcaemia was not so severe as to be fatal. However, the function of this mutant CaSR was not evaluated. In the present study, we describe a novel homozygous mutation in an adult patient with severe hypercalcaemia and hypocalciuria, and evaluate the function of the mutant CaSRs. The DNA sequence of CaSR gene was determined by direct sequencing of the polymerase chain reaction product. The function of mutant CaSR was analysed by creating mutant cDNAs by in vitro mutagenesis, transfection of mutant cDNAs into HEK293 cells and measuring intracellular ionized Ca in response to changes in extracellular Ca. A 26-year-old Japanese woman showed marked hypercalcaemia with an elevated parathyroid hormone (PTH) level. Her consanguineous parents had asymptomatic hypercalcaemia with relative hypocalciuria. The proband had a homozygous mutation at codon 27 of CaSR gene (CAA-->CGA, Gln27Arg). Her parents were heterozygous for this mutation. EC50 for Ca of this mutant CaSR (GIn27Arg) was 4.9 mM. EC50 of another mutant CaSR (Pro39Ala) whose homozygous mutation was discovered in an adult patient was 4.4 mM. These EC50s were significantly higher than that of wild-type CaSR (3.7} 0.1 mM), but were the lowest among the reported EC50s for inactivating mutations of CaSR. These results indicate that serum Ca and PTH levels are determined by residual function of mutant CaSR in patients with homozygous mutation in CaSR, and that patients having homozygous mutant CaSRs with mild dysfunction do not suffer from fatal hypercalcaemia in infancy and can survive into adulthood.

Adult↗

PubMed1999

Prevention of the death of the rat axotomized hypoglossal nerve and promotion of its regeneration by bovine brain gangliosides.

We have examined the time course of the neuronal death and regeneration of rat axotomized hypoglossal nerve with various conditions of the nerve resection, and established a useful system to measure neurotrophic activities of bioactive substances. In this system, neuronal death can be evaluated by counting surviving neurons in the nucleus of hypoglossal neuron at the brain stem, and the degree of the regeneration can be measured by counting horseradish peroxidase-positive cells at the same region after injection of horseradish peroxidase into tongue. Using this system, the effects of brain gangliosides on rat hypoglossal nerve regeneration following 5 mm transection were examined. The addition of a ganglioside mixture from bovine brain as well as the autograft strongly prevented the death of neurons and promoted the regeneration of the lesioned nerve at 10 weeks after the operation. Further analyses on the dose effects and injection sites of gangliosides were performed. Although the mechanisms of the neurotrophic effects of the gangliosides are unknown, the therapeutic application of gangliosides for neuronal degeneration is a promising approach.

Animals↗

PubMed1999

Thiazolidinediones inhibit osteoclast-like cell formation and bone resorption in vitro.

Osteoblasts and adipocytes are derived from common bone marrow stromal cells that play crucial roles in the generation of osteoclasts. Activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) induces adipogenic differentiation of stromal cells; however, whether this would affect osteoblast/osteoclast differentiation is unknown. Thus, we examined the effects of the thiazolidinedione (TZD) class of antidiabetic agents that activate PPARgamma on osteoblast/osteoclast differentiation using mouse whole bone marrow cell culture. As reported, all TZDs we tested (troglitazone, pioglitazone, and BRL 49653) markedly increased the number of Oil Red O-positive adipocytes and the expression of adipsin and PPARgamma 2. 1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] did not affect adipogenic differentiation induced by TZDs. TZDs did not affect alkaline phosphatase activity, an early marker of osteoblastic differentiation, despite their marked adipogenic effects. TZDs decreased the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cells induced by 1,25-(OH)2D3 or PTH. Troglitazone dose dependently inhibited basal and 1,25-(OH)2D3- and PTH-induced bone resorption as assessed by pit formation assay. Interleukin-11 blocked the induction by troglitazone of adipogenesis, but had no effect on the inhibition of osteoclast-like cell formation. These results indicate that TZDs are potent inhibitors of bone resorption in vitro. Inhibitory effects of TZDs on osteoclastic bone resorption was not osteotropic factor specific and did not appear to be related to their adipogenic effects. Thus, TZDs may suppress bone resorption in diabetic patients and prevent bone loss.

Acid Phosphatase↗

PubMed1999

Stimulation of osteoclast formation by 1,25-dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic cells: studies using VDR knockout mice.

Previous studies have shown that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays important roles in the formation of osteoclasts through its actions on osteoblastic cells. We have generated mice lacking vitamin D receptor (VDR) by gene targeting (VDR-/-). These mice had tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and exhibited similar levels of parameters for bone resorption to those in wild type mice. The present studies were undertaken to clarify whether effects of 1,25(OH)2D on osteoclast formation require VDR in osteoblasts, and to examine mechanisms of the formation of osteoclasts without VDR-mediated actions using VDR-/- mice. When wild-type calvarial osteoblasts and spleen cells were co-cultured with 1,25(OH)2D, TRAP-positive osteoclasts were formed regardless of the genotypes of spleen cells. In contrast, when osteoblasts from VDR-/- mice were co-cultured, no osteoclasts could be formed even with wild-type spleen cells. Parathyroid hormone and interleukin-1alpha stimulated osteoclast formation by co-cultures from VDR-/- mice, and the generated osteoclasts showed resorbing activity. These results demonstrate that VDR-mediated actions of 1,25(OH)2D in osteoblasts are essential for osteoclast formation by 1,25(OH)2D, and that functionally intact osteoclasts can be formed without 1,25(OH)2D actions under stimulations by other agents. It is suggested that osteoclastic bone resorption can be maintained without 1,25(OH)2D actions by other stimulatory agents.

Acid Phosphatase↗

PubMed1999

Extracellular matrix-associated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro.

Osteoblastic differentiation is an essential part of bone formation that compensates resorbed bone matrix to maintain its structural integrity. Cells in an osteoblast lineage develop differentiated phenotypes during a long-term culture in vitro. However, intrinsic mechanisms whereby these cells differentiate into mature osteoblasts are yet unclear. Bone morphogenetic proteins (BMPs) stimulate osteoblastic differentiation and bone formation. We demonstrate that mouse osteoblastic MC3T3-E 1 cells constitutively expressed messenger RNAs (mRNAs) for BMP-2 and BMP-4 and accumulated BMPs in collagen-rich extracellular matrices. BMPs associated with the extracellular matrices were involved in the induction of osteoblastic differentiation of nonosteogenic mesenchymal cells as well as cells in the osteoblast lineage. MC3T3-E1 cells constitutively expressed type IA and type II BMP receptors. When a kinase-deficient type IA BMP receptor was stably transfected to MC3T3-E 1 cells to obliterate BMP-2/4 signaling, these cells not only failed to respond to exogenous BMP-2 but lost their capability of differentiation into osteoblasts that form mineralized nodules. These observations strongly suggest that endogenous BMP-2/4 accumulated in extracellular matrices are essential for the osteoblastic differentiation of cells in the osteoblast lineage. Therefore, the regulatory mechanism of BMP-2/4 actions in osteoblastic cells is a principal issue to be elucidated for better understanding of pathogenesis of bone losing diseases such as osteoporosis.

Animals↗

PubMed1999

Heterozygous gsp mutation renders ion channels of human somatotroph adenoma cells unresponsive to growth hormone-releasing hormone.

Ionic mechanisms play an important role in the regulation of hormone secretion. The GHRH-induced GH release by human GH-secreting cells is transmitted through protein kinase A (PKA), which activates nonselective cation current (NSCC) and induces membrane depolarization, intracellular Ca2+ increase, and GH secretion. To evaluate whether ionic mechanisms have pathophysiological significance in GH oversecretion of GH-secreting pituitary adenomas, we examined four adenomas with constitutively active Gs alpha mutation (gsp mutation) and compared with three gsp-negative adenomas. In primary-cultured cells of gsp-positive adenomas, GHRH did not increase the NSCC under voltage-clamp experiments. Detailed examination showed that NSCC was maximally activated at the basal level and application of GHRH did not increase the current in these adenomas. Furthermore, by using single-cell RT-PCR method, we demonstrated for the first time at the single cell level that gsp mutation is heterozygous in GH-secreting pituitary adenomas. These indicate that heterozygous gsp mutation fully activates NSCC at the basal level, which may account for the GH oversecretion in gsp-positive GH-secreting pituitary adenomas.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

A novel activating mutation in calcium-sensing receptor gene associated with a family of autosomal dominant hypocalcemia.

Autosomal dominant hypocalcemia (ADH), caused by activating mutations of the calcium-sensing receptor (CaSR), is characterized by hypocalcemia with an inappropriately low concentration of PTH. Among 11 missense mutations of CaSR reported to date in patients with ADH or sporadic hypocalcemia, functional properties of 8 mutant CaSRs were characterized. Here, we describe a novel mutation of CaSR and its functional property in a family with ADH. The 41-yr-old male proband had asymptomatic hypocalcemia with a history of recurrent nephrolithiasis. His father had asymptomatic hypocalcemia, but his mother was normocalcemic. PCR-single strand conformation polymorphism and sequencing revealed that both the proband and the father had a novel heterozygous mutation in CaSR gene that causes lysine to asparagine substitution at codon 47 (K47N), which is in the extracellular domain of CaSR, like 6 of 11 known activating mutations. Using HEK293 cells transfected with wild-type or K47N CaSR complementary DNA, the intracellular Ca2+ concentration was assessed in response to changes in the extracellular Ca2+ concentration. The EC50 of the mutant CaSR for the extracellular Ca2+ concentration was 2.2 mmol/L and was significantly lower than that of wild-type (3.7 mmol/L). These results confirm that this mutation is responsible for ADH in this family. The fact that several inactivating mutations in familial hypocalciuric hypercalcemia occur in amino acid around K47 suggests the importance of the N-terminal portion of the receptor in extracellular Ca sensing.

Adult↗

PubMed1999

No enzyme activity of 25-hydroxyvitamin D3 1alpha-hydroxylase gene product in pseudovitamin D deficiency rickets, including that with mild clinical manifestation.

Pseudovitamin D deficiency rickets (PDDR) is an autosomal recessive disorder caused by defect in the activation of vitamin D. We recently isolated 25-hydroxyvitamin D3 1alpha-hydroxylase gene and identified four homozygous inactivating missense mutations in this gene by analysis of four typical cases of PDDR. This disease shows some phenotypic variation, and it has been suspected that patients with mild phenotypes have mutations that do not totally abolish the enzyme activity. To investigate the molecular defects associated with the phenotypic variation, we analyzed six additional unrelated PDDR patients: one with mild and five with typical clinical manifestation. By sequence analysis, all six patients were proven to have mutations in both alleles. The mutations varied, and we identified four novel missense mutations, a nonsense mutation, and a splicing mutation for the first time. The patient with mild clinical symptoms was compound heterozygous for T321R and a splicing mutation. The splice site mutation caused intron retention. Enzyme activity of the T321R mutant was analyzed by overexpressing the mutant 1alpha-hydroxylase in Escherichia coli cells to detect the subtle residual enzyme activity. No residual enzyme activity was detected in T321R mutant or in the other mutants. These results indicate that all of the patients, including those of mild phenotype, are caused by 1alpha-hydroxylase gene mutations that totally abolish the enzyme activity.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1999

Short-term treatment with troglitazone decreases bone turnover in patients with type 2 diabetes mellitus.

Poorly controlled type 2 or non-insulin dependent diabetes mellitus (NIDDM) patients exhibit high bone turnover, which decelerate with treatment according to the degree of improvement in glycemic control. In adults, higher bone turnover results in rapid bone loss. Therefore, deceleration of bone turnover is beneficial for bone. Troglitazone (Tro), a new anti-diabetic drug, is a thiazolidinedione (TZD) which promotes adipocyte differentiation by activating peroxisome proliferator activated receptor gamma (PPARgamma). Because, in the bone marrow, adipocytes and osteoblasts originate in common mesenchymal stem cells that are also essential for osteoclastogenesis, TZDs may directly affect bone metabolism. Thus, we examined the effects of Tro on metabolic bone markers in type 2 DM patients. Tro (400 mg/day) was administered to 33 type 2 DM patients for four weeks. The day before and four weeks after starting Tro, serum and urine samples were collected after overnight fasting. Metabolic bone markers and glycemic indices were assessed. As bone resorption markers, urinary free and total deoxypyridinoline as well as urinary collagen type I C-terminal telopeptide were measured; as bone formation markers, serum bone type and total alkaline phosphatase (BALP and ALP) levels along with osteocalcin (OC) were used. No significant changes in fasting plasma glucose or HbA1c levels were observed in our short-term treatment with Tro. All the bone resorption markers, BALP and ALP were significantly decreased. OC was not significantly changed. The discrepant changes of OC from all the other metabolic bone markers suggest limitation of the use of OC as a reliable bone formation marker in diabetics. Our results that Tro decreased metabolic bone markers before significantly improving glucose metabolism suggest that it has direct effects on bone and decreased bone turnover. TZDs may spare bone mass in NIDDM subjects through its dual effects on glucose and bone metabolism.

Aged↗

PubMed1999

Pituitary metastasis from carcinoma of the urinary bladder mimicking pituitary apoplexy--case report.

A 70-year-old male presented with pituitary metastasis from transitional cell carcinoma of the urinary bladder manifesting as sudden headache, transient unconsciousness, and visual disturbance mimicking apoplexy of pituitary adenoma. Computed tomography showed a suprasellar tumor with intratumoral and intraventricular hemorrhage. Magnetic resonance imaging demonstrated an intra- and suprasellar mass lesion mimicking pituitary adenoma. Diabetes insipidus developed soon after. The tumor was subtotally removed. Histological examination revealed transitional cell carcinoma. An intratumoral hemorrhage may be associated with a pituitary metastasis if the patient presents with symptoms such as pituitary apoplexy.

Aged↗

PubMed1999

[Echinococcus].

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Animals↗

PubMed1999

Transposition of mesial and distal aspects of maxillary first molars: case report.

Congenital absence of one or more teeth, hypodontia, is the most common developmental anomaly and is often accompanied by the presence of other tooth anomalies. In this case two Japanese sisters have several congenitally missing primary and permanent teeth and morphological abnormalities of maxillary first molars. One sister has transposition of mesial and distal aspects of a maxillary first molar, whose cusps display a normal shape. Another sister has maxillary first molars, which look like maxillary second molars. Mesio-distally shift of teeth is a very rare anomaly making this particular case important to analyze the teeth formation and development.

Adolescent↗

PubMed1998

Complex gangliosides are essential in spermatogenesis of mice: possible roles in the transport of testosterone.

Mice, homozygous for disrupted ganglioside GM2/GD2 synthase (EC 2.4. 1.94) gene and lacking all complex gangliosides, do not display any major neurologic abnormalities. Further examination of these mutant mice, however, revealed that the males were sterile and aspermatogenic. In the seminiferous tubules of the mutant mice, a number of multinuclear giant cells and vacuolated Sertoli cells were observed. The levels of testosterone in the serum of these mice were very low, although testosterone production equaled that produced in wild-type mice. Testosterone was found to be accumulated in interstitial Leydig cells, and intratesticularly injected testosterone was poorly drained in seminiferous fluid in the mutant mice. These results suggested that complex gangliosides are essential in the transport of testosterone to the seminiferous tubules and bloodstream from Leydig cells. Our results provide insights into roles of gangliosides in vivo.

Animals↗