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T Habu

Publications and source records attributed to T Habu.

At least 19 recordsLinked to original sources
PubMed2000

A novel cis-acting element regulates HES-1 gene expression in P19 embryonal carcinoma cells treated with retinoic acid.

The regulatory mechanisms of mammalian hairy and Enhancer of split homologue-1 (HES-1) genes were examined in mouse P19 embryonic carcinoma cells (P19 cells). Undifferentiated P19 stem cells expressed a basal level of the HES-1 gene, whereas the expression of this gene was increased upon induction of the cells to a neural cell lineage using retinoic acid (RA). Reporter co-transfection analysis identified an activating region within the upstream promoter region of HES-1 from nucleotides -201 to -172. This activating region, called activating region X (ARX), shows a high GC content and contains both an AP-2 binding motif and a CCAAT box. An electrophoretic mobility shift assay using nuclear proteins extracted from P19 cells showed that ARX forms a specific DNA-protein complex. Importantly, ARX-dependent transcription, as well as ARX-binding activity, was significantly increased in P19 cells treated with RA. These results indicate that ARX transduces signals that up-regulate HES-1 gene expression in response to RA-treatment. Thus, a novel cis-acting element involved in HES-1 gene regulation that plays a role in RA-induced neural differentiation of P19 cells has been identified.

Animals↗
PubMed2000

Osmotic membrane stretch increases cytosolic Ca(2+) and inhibits bone resorption activity in rat osteoclasts.

Although the importance of mechanical stress on bone metabolism is well known, the intracellular mechanisms involved are not well understood. To evaluate the role of mechanical stress on osteoclastic function, we investigated the effects of membrane stretch induced by osmotic cell swelling on cytosolic Ca(2+) and bone resorption activity in freshly isolated rat osteoclasts. The intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 microspectrofluorimetry. Exposure to hypotonic solution (211-151 mOsm) caused cell swelling and reversibly increased [Ca(2+)](i) in the osteoclasts. This [Ca(2+)](i) increase was abolished by the omission of extracellular Ca(2+), but was not affected by the depletion of intracellular Ca(2+) stores. Gd(3+) and La(3+) inhibited the swelling-induced [Ca(2+)](i) increase, while nifedipine and Bay K 8644 did not. Neither protein kinase A inhibitors (Rp-cAMP, H-89) nor protein kinase C inhibitors (staurosporine, chelerythrine) affected the [Ca(2+)](i) increase. Membrane depolarization was not essential for the [Ca(2+)](i) increase either. To assess the effects of membrane stretch on the bone resorption activity of osteoclasts, we investigated actin ring formation, the intracellular structure responsible for bone resorption in osteoclasts. Hypotonic stimulation acutely disrupted actin ring formation in an extracellular Ca(2+)-dependent manner, and this disruption was prevented by Gd(3+). Moreover, Ca(2+) ionophore (ionomycin) also induced disruption of the actin rings. These results indicate that mechanical stress inhibits osteoclastic bone resorption activity, possibly via the elevation of [Ca(2+)](i) through stretch-activated, non-selective cation channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells.

We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I-III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of Mr 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells.

Amino Acid Sequence↗
PubMed1999

The association of ATR protein with mouse meiotic chromosome cores.

The ATR (ataxia telangiectasia- and RAD3-related) protein is present on meiotic prophase chromosome cores and paired cores (synaptonemal complexes, SCs). Its striking characteristic is that the protein forms dense aggregates on the cores and SCs of the last chromosomes to pair at the zygotene-pachytene transition. It would appear that the ATR protein either signals delays in pairing or it is directly involved in the completion of the pairing phase. Atm-deficient spermatocytes, which are defective in the chromosome pairing phase, accumulate large amounts of ATR. The behaviour of ATR at meiotic prophase sets it apart from the distribution of the RAD51/DMC1 recombinase complex and our electron microscope observations confirm that they do not co-localize. We failed to detect ATM in association with cores/SCs and we have reported elsewhere that RAD1 protein does not co-localize with DMC1 foci. The expectation that putative DNA-damage checkpoint proteins. ATR, ATM and RAD1, are associated with RAD51/DMC1 recombination sites where DNA breaks are expected to be present, is therefore not supported by our observations.

Adenosine Triphosphatases↗
PubMed1998

The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis.

The mouse Dmc1 gene is an E. coli RecA homolog that is specifically expressed in meiosis. The DMC1 protein was detected in leptotene-to-zygotene spermatocytes, when homolog pairing likely initiates. Targeted gene disruption in the male mouse showed an arrest of meiosis of germ cells at the early zygotene stage, followed by apoptosis. In female mice lacking the Dmc1 gene, normal differentiation of oogenesis was aborted in embryos, and germ cells disappeared in the adult ovary. Meiotic chromosome analysis of Dmc1-deficient mouse spermatocytes revealed random spread of univalent axial elements without correct pairing between homologs. In rare cases, however, we observed complex pairing among nonhomologs. Thus, the mouse Dmc1 gene is required for homologous synapsis of chromosomes in meiosis.

Adenosine Triphosphatases↗
PubMed1996

Cell cycle-dependent expression of the mouse Rad51 gene in proliferating cells.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouse Rad51 mRNA was examined in synchronized mouse m5S cells. The Rad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression of Rad51 mRNA. By immunohistochemical analyses, in mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouse Rad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes.

Animals↗
PubMed1996

The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis.

Genetic recombination in meiosis plays an important role in generating diversity of genetic information. In yeast an Escherichia coli RecA-like gene, DMC1, is expressed in meiotic prophase and its product co-localizes with Rad51 protein on zygotene chromosomes. We have cloned the mouse and human homologs of the yeast DMC1 gene. The predicted human and mouse DMC1 proteins showed 54.1% sequence identity with yeast Dmc1 protein. The domain II region, highly conserved in the E.coli RecA-like protein family, was also found in the mammalian DMC1 proteins, including the two ATP binding motifs and DNA binding sites with the region. In situ hybridization analysis revealed expression of the mouse Dmc1 gene in testicular germ cells in meiosis; RT-PCR showed expression in embryonal ovaries. These findings suggest that DMC1 plays an important role in meiotic homologous recombination. From both the man and mouse we have isolated an alternative spliced form of Dmc1 cDNA (Dmc1-d), which is deleted for a region between the two motifs involved in nucleotide binding. Since the alternatively spliced Dmc1-d transcript was detected in both male and female germ cells, the encoded protein DMC1-D may have a novel role in mammalian genetic recombination in meiosis.

Adenosine Triphosphatases↗
PubMed1996

Effect of light-exposure duration on the amount of leachable monomers from light-activated reline material.

Leaching of monomers from light-activated direct intraoral reline material (Lebaron LC) was determined by means of high-pressure liquid chromatography analysis. This study evaluated the effects of exposure duration and thickness to determine appropriate curing conditions that reduce the levels of unreacted monomeric components. Prolonged duration of exposure (30 minutes) reduced the amount of leached monomer. However, the results of this study indicated that the amount of leached monomeric components increased with an increasing reline material thickness. The results suggest that the light-activated reline material should be cured for sufficient prolonged exposure duration.

Chromatography, High Pressure Liquid↗
PubMed1995

Regulation of the mouse histone H2A.X gene promoter by the transcription factor E2F and CCAAT binding protein.

We have molecularly cloned the genomic gene encoding the mouse histone variant H2A.X and characterized the promoter. The promoter region of the H2A.X gene was characterized by chloramphenicol acetyltransferase analysis using Balb/c 3T3 cells. Maximal promoter activity was found in the construct containing up to -282 base pairs H2A.X upstream region. Within this region, we found two sequences regulating the promoter activation; one was an E2F site and another was a CCAAT box. These sequences were also required for the DNA/protein binding activities. Thus, these activities corresponded to the promoter activities, implying that the promoter activity H2A.X gene was controlled by both the transcription factor E2F and H1TF2 through the E2F and CCAAT element. The CCAAT box binding activity was constitutive when cell cycle was progressed by release from G1 arrest, but transiently transfected chloramphenicol acetyltransferase activity slightly increased when cells entered S phase. Similarly, the level of the smallest form of E2F (free E2F) became higher when cells reentered the cell cycle, indicating that the free E2F was one capable of inducing the promoter activation. Thus, the free E2F and CCAAT DNA binding activity correlated with regulation of the promoter activity.

3T3 Cells↗
PubMed1990

Protein secretion by rat submandibular glands in response to isoproterenol, alpha-methylnoradrenaline and clonidine during aging.

Changes in salivary volumes and the three types of proteins secreted by the submandibular salivary gland (SMG) of male rats at 3.5, 5.5, 8, 12, 13, 14, 15, 19, 21 and 24 months of age in response to the beta 1-, alpha 1- and alpha 2-adrenoceptor agonists, isoproterenol (IPR), alpha-methylnoradrenaline (alpha-mNA) and clonidine (Clonid), were studied and compared by measuring the weight and by isoelectric focusing electrophoresis with the Phast System on both the gradient pH 3.5-5 and 3.5-9 gels with silver staining. A protein (protein A, tentatively termed in this study) purified by FPLC from saliva elicited by IPR was also analyzed by SDS-polyacrylamide gel electrophoresis, the immuno-thermoblotting method, carbohydrate determination and neuraminidase treatment. Unexpected findings were observed that salivary volumes, but not the protein concentration, were substantially increased by Clonid-, but not IPR-, stimulation with ages up to 24 months of age and that the three types of proteins elicited by each agonist were different during aging. The gamma-type of proteins elicited by Clonid was not greatly changed during aging, whereas several proteins at about neutral pI in the alpha-type, elicited by alpha-mNA, at 5.5 to 21 months of age and a protein A in the beta-type, elicited by IPR, at 13 to 24 months of age were greatly increased. This protein A without any carbohydrate and sialic acid, located only in the acinar cells, but not in any duct system, had a molecular weight of 16,000 and a pI of 4.05. We conclude that the secretory function of the SMG in the aged animals is in general little changed.

Aging↗
PubMed1990

Changes in protein secretion by rat submandibular glands in response to isoproterenol, alpha-methylnoradrenaline, and clonidine during post-natal development.

We studied developmental changes in salivary volumes and proteins secreted by the submandibular glands of male rats at weekly intervals from two to ten weeks of age in response to the beta 1-, alpha 1-, and alpha 2-adrenoceptor agonists, isoproterenol (IPR), alpha-methylnoradrenaline (alpha-mNA), and clonidine (Clonid). The types of proteins in saliva samples were determined and compared by isoelectric-focusing electrophoresis with the Phast system in both the gradient pH -3.5-to-5 and pH-3.5-to-9 gels by means of silver staining. Salivary volume and protein concentration in saliva samples elicited by IPR and alpha-mNA were positively related to the weight of the submandibular glands up to six or seven weeks of age, whereas in saliva elicited by Clonid, no relation was found in the protein concentration [corrected]. The isoelectric-focusing electrophoretic patterns of proteins secreted by the glands in response to three stimuli were different from each other during post-natal development. Within one stimulation, differences were also observed at two and three weeks of age for Clonid, and from seven weeks of age for the three stimuli, respectively. The alpha-type proteins, but not the beta-type proteins, were very similar to those in extracts from glands of rats at seven weeks of age. Almost all of the alpha-type proteins, but not the beta-type proteins, reacted with antibodies to two proteases. We conclude that functional maturation precedes morphological maturation in the submandibular glands of rats.

Age Factors↗
PubMed1990

[Supplemental studies on self-curing resins. 2. On polymerizing temperature of reline materials].

The objective of this study was to find the best way to keep down the peak temperature of self-curing reline materials. The experiments were done by using three kinds of self-curing reline materials (Rebaron, REBASE and KOOLiner) and three kinds of the reline materials of different thickness (1.5mm, 2mm and 3mm) under three different intraoral conditions. When these reline materials cured under rinsing the mouth with cold water (18 degrees C), the data analysis of these reline materials showed a considerable reduction in the peak curing temperatures clinical acceptance. The results obtained were as follows: 1. Both extraoral and interoral curing tests showed that the thicker the material is the higher the peak temperature becomes. 2. Highly significant differences in time to the peak temperature were found among the three kinds of reline materials. 3. Dangerously high temperature was found in polymerizing of the thickest material, 3mm.

Denture Liners↗
PubMed1988

Age changes in secretory function of male and female rat parotid glands in response to methoxamine and pilocarpine.

Saliva secreted in response to methoxamine and pilocarpine was collected from the cannulated ducts of both parotid glands of male and female rats at weekly age intervals from three to 10 weeks, and at 3.5, 8, and 15 months of age. It was analyzed for the concentrations of protein, potassium, calcium, inorganic phosphate, and for amylase activity. The type of protein were determined electrophoretically, and an amino acid analysis of the total protein was also carried out. The wet weights of the glands increased substantially up to eight weeks of age, then reached almost plateau values, and finally tended to decrease at 15 months of age in both sexes. The salivary volumes secreted in response to methoxamine and pilocarpine were positively correlated with the parotid gland weights in both sexes. The concentrations of protein, potassium, and inorganic phosphate were inversely related to the salivary flow rates only at relatively low rates of flow. The amylase activity was positively correlated with the concentration of protein, independent of the nature of the stimulus, age, and sex. With methoxamine as a stimulus, the amylase activity was positively correlated with the concentration of calcium, independent of age and sex. The types of protein and amino acid concentrations were independent of the nature of the stimulus, age, and sex up to 15 months of age. However, in parotid saliva of several rats at 8 and 15 months of age, unusual proteins were observed electrophoretically, independent of the nature of the stimulus and sex.

Aging↗