Biomedical subjects

H Mano

Publications and source records attributed to H Mano.

At least 73 records · Page 4Linked to original sources

PubMed1996

Comparison of mutagenic activity of bile between Chilean and Japanese female patients having cholelithiasis.

The mutagenic activity of bile was compared between Chilean and Japanese female patients having cholelithiasis by the Ames assay using Salmonella typhimurium tester strain TA98 in the presence of S9 mix with blue rayon adsorption technique. A reason for conducting the present investigation is that Chile and Japan have the highest mortality rates for the gallbladder cancer (GBC) in the world. Of 24 bile samples collected in Chile, 20 (83.3%) samples showed mutagenicity. In the case of Japanese bile, 21 (80.8%) of 26 and 5 (19.2%) of 26 cases were mutagenic in samples from high- and low-risk areas for GBC, respectively. Therefore, both the Chilean and the Japanese samples collected in high-risk areas showed higher mutagenic rates than the Japanese ones in a low-risk area, with a statistical significance (p < 0.001), chi-square test). The average number of revertant colonies were 128 +/- 92 (mean +/- SD), 62 +/- 14 and 66 +/- 13, respectively, when the blue rayon extracts of 200 microliters bile were applied to the Ames test. Thus, Chilean bile had a tendency to show a higher mutagenic activity than Japanese.

Adult↗

PubMed1996

Mutagenicity of 24-hour duplicate of Japanese diet.

In order to elucidate the genotoxicological characteristics of the Japanese diet, the mutagenicity of 24-h duplicate of the diet samples were investigated. The mutagenicity of blue rayon extract was examined in the Ames Salmonella/microsome assay. Thirty-two (91.4%) of 35 samples revealed mutagenicity toward Salmonella typhimurium TA98 in the presence of S9 mix. The mutagenic activities showed significant correlations with the consumption rates of broiled fish (r = 0.517, p = 0.0021) and broiled meat (r = 0.494, p = 0.0036). In other test conditions, 6 (17.1%), 5 (14.3%) and 8 (22.9%) samples were mutagenic to Salmonella typhimurium TA98 without S9 mix, TA100 with S9 mix and TA100 without S9 mix, respectively. Findings in the present study suggest that high consumption of broiled fish and broiled meat are important as the source of mutagens/carcinogens in the Japanese diet. In the present study, however, biological inference of these findings could not be made in relation to the occurrence of cancers, especially of the gastric cancer, which is the most prevalent form of cancer in Japan.

Aged↗

PubMed1996

Mammalian mature osteoclasts as estrogen target cells.

The decrease in estrogen levels that follows the onset of menopause causes rapid bone loss, resulting in osteoporosis. However, the mechanism by which this occurs remains unclear, especially concerning the regulation of osteoclasts, i.e. the bone-resorbing cells. Using a pit assay involving isolated mature osteoclasts from rabbit long bones, we found that estrogen inhibited the bone-resorbing activity in a dose- and time-dependent manner. Furthermore, we clarified by Northern analysis that estrogen down-regulated the mRNA levels of cathepsin K/OC-2 and that putative estrogen receptor (ER) mRNA was expressed in these osteoclasts. Moreover, other sizes of mRNAs that hybridized with ER cDNA probe were found in these cells. Our results suggest that osteoclasts may be indeed target cells for estrogen and that estrogen might regulate a part of bone metabolism through osteoclasts.

Animals↗

PubMed1996

Cloning and characterization of mouse tec promoter.

Tec is a cytoplasmic protein-tyrosine kinase highly expressed in hematopoietic precursor cells. To investigate the mechanism regulating its expression, we cloned and characterized the mouse tec promoter. The promoter region does not contain the authentic TATA or CAAT box but has several consensus binding motifs for GATA and SP1. The nucleotide sequence surrounding the major transcription initiation site determined 5' RACE-PCR appeared to be homologous to the "initiator" consensus sequence which is frequently found in genes lacking TATA and CAAT. The promoter activity of the 5' flanking region was confirmed by using the luciferase assay. The present analysis will provide insights into the mechanism for the expression and regulation of the Tec molecule.

3T3 Cells↗

PubMed1996

Retinoic acid differentially up-regulates the gene expression of retinoic acid receptor alpha and gamma isoforms in embryo and adult rats.

The diverse biological effects of retinoic acid (RA) are exerted by its nuclear receptor-mediated gene expression. One of the two nuclear retinoic acid receptor subfamilies is composed of three subtypes of the all-transretinoic acid receptor (RAR-alpha, RAR-beta, and RAR-gamma). Furthermore, several isoforms are generated from each of three RARs by differential promoter usage and/or alternative splicing. It is thus thought that the developmental stage-specific actions of RA are modulated through the spatio-temporal expression of the subtype and isoforms of RARs. In this study, the auto-regulation of the RAR subtypes (RAR-alpha total, RAR-beta total and RAR-gamma total) and their major isoforms (RAR-alpha 1, alpha 2, RAR-beta 1, beta 2 and RAR-gamma 1, gamma 2) by RA was examined by means of Northern blotting in the 11.5 day embryo and maternal tissues by administering pregnant rats with an excess of all-trans RA. The expression of RAR-beta isoforms as well as the RAR-beta total was auto-regulated by RA in all maternal tissues and embryos examined. The gene expression of RAR-alpha 2, which was not affected by RA in the maternal tissues, was up-regulated in embryos, though there were no significant effects of RA on the levels of RAR-alpha 1 and the alpha total in the maternal tissues and the embryos. Likewise, RA did not affect the levels of RAR-gamma 1 and gamma total. However, unlike RAR-alpha 2, RAR-gamma 2 expression was up-regulated by RA only in the maternal tissues. Thus, these results indicates that two retinoic acid receptor isoforms (RAR-alpha 2 and RAR-gamma 2) are differentially auto-regulated in embryo and adult rats.

Animals↗

PubMed1996

Vitamin K2 inhibits osteoclastic bone resorption by inducing osteoclast apoptosis.

In contrast to vitamin K1(VK1), vitamin K2(VK2) inhibited osteoclastic bone resorption by unfractionated bone cells and isolated osteoclasts. To investigate the mechanism of inhibition of osteoclastic bone resorption by VK2, we examined the effect of this vitamin on osteoclast apoptosis using a DNA-binding fluorescent dye, Hoechst 33258. In unfractionated bone cells and isolated osteoclasts on dentin slices, we first demonstrated that VK2 induced osteoclast apoptosis, but VK1 did not. Moreover, cycloheximide inhibited VK2-induced osteoclast apoptosis. These results suggest the possibility that VK2 inhibits osteoclastic bone resorption by targeting osteoclasts to undergo apoptosis, which leads to cell death.

Animals↗

PubMed1996

Human calcitonin has the same inhibitory effect on osteoclastic bone resorption by human giant cell tumor cells as salmon calcitonin.

Human calcitonin (hCT) has been reported to have a less hypocalcemizing effect on rats and to have a lower binding affinity for the receptor of mouse osteoclasts than salmon CT(sCT). In this study we comparatively examined the effect of hCT and sCT on osteoclastic bone-resorbing activity of unfractionated cells obtained from human giant cell tumor of bone and from rabbit and mouse long bones. We found that hCT had the same inhibitory effect as sCT on the bone-resorbing activity of human and rabbit osteoclastic cells, but a different one on that of mouse cells. These results indicate that the activity of drugs should be assayed using human cells if possible.

Animals↗

PubMed1996

Tec protein-tyrosine kinase is an effector molecule of Lyn protein-tyrosine kinase.

The Tec family is a recently emerging subfamily among nonreceptor type protein-tyrosine kinases (PTKs) consisting of Tec, Txk, Btk, Bmx, and Itk/Tsk/Emt. They have a long amino-terminal unique region containing a pleckstrin homology domain and a Tec-homology domain. We could previously show that, through the Tec-homology domain, Tec is bound to Lyn kinase both in vitro and in vivo. Because Tec is coexpressed with Lyn in many hematopoietic cell types, it has been intriguing to investigate the biological role of the Tec-Lyn association. Here we demonstrate that Lyn can phosphorylate tyrosine residues of the Tec protein, and thereby activate Tec in 3T3 fibroblasts. However, coexpression of Tec has little effect on the phospho-tyrosine-contents of Lyn. By using the in vitro kinase assay and the yeast system, we could prove that the Tec protein is a direct substrate of the Lyn kinase both in vitro and in vivo. From this evidence we conclude that Tec acts downstream of Lyn in intracellular signaling pathways. This is a novel case where one PTK is phosphorylated and regulated by another.

3T3 Cells↗

PubMed1996

Deletion of Src homology 3 domain results in constitutive activation of Tec protein-tyrosine kinase.

Tec protein-tyrosine kinase (PTK) is the prototype of a new subfamily of non-receptor type PTKs, and is abundantly expressed in hematopoietic tissues. We have revealed that Tec is inducibly tyrosine-phosphorylated and activated by stimulation with a wide range of cytokines. To get more insight into the signaling mechanism through Tec, we have generated a constitutively active form of Tec PTK. Deletion of the Src homology (SH) 3 domain gave rise to a hyperphosphorylated and activated Tec kinase (Tec deltaSH3). The activity of Tec deltaSH3 was confirmed in 293 cells, as well as in cytokine-dependent hematopoietic cells (BA/F3). Tec deltaSH3 should be a useful tool to study the in vivo substrates of Tec PTK.

Animals↗

PubMed1996

High expression of the tec gene product in murine testicular germ cells and erythroblasts.

Tec is a novel non-receptor-type protein tyrosine kinase that was originally identified from a murine liver cDNA library. While the function of Tec remains unknown, it was shown recently that two Tec-related kinases are involved directly in the growth and differentiation of bone marrow stem cells. As the localization of Tec protein has not been reported yet, immunohistochemical and immunochemical studies of various murine organs were conducted in the present study to clarify which cells express this kinase protein. An intense immunohistologic reaction was observed in neonatal and adult testicular germ cells, and neonatal and fetal hepatic erythroblasts. In addition, a clear immunostaining was noted in neonatal and adult tubal epithelial cells, hepatocytes, basal cells of the non-glandular stomach, foveolar epithelium of the glandular stomach, sebaceous cells of the skin and fetal cartilage. The immune reaction of germ cells and erythroblasts was observed in the cell membrane, although this protein does not have a transmembrane domain. Supportive western blotting of testis, adult liver, spleen and heart of adult C.B-17 mice with the use of anti-Tec antibody demonstrated a heavy 70 kDa band in the liver and testis, and a much weaker, small band in the heart and spleen. These results suggest that Tec protein has a specific role in testicular germ cells and erythroblasts.

Animals↗

PubMed1996

Bacterially expressed rat retinol-binding protein is functional for retinol and transthyretin bindings.

Retinol-binding protein (RBP) was expressed in Escherichia coli using the cDNA for rat RBP, and characterized. The expressed RBP was fused to maltose-binding protein (MBP) at the N-terminal end (MBP-RBP), and MBP was enzymatically removed from the MBP-RBP with proteinase factor Xa. The binding of retinol and transthyretin (TTR) to the recombinant RBP was monitored by means of gel filtration. The recombinant RBP specifically bound to retinol with an affinity similar to that of purified RBP from rat serum. Furthermore, the retinol-bound recombinant RBP formed hetero-complexes with TTR similar to RBP. Thus, the results showed that the recombinant RBP expressed in E. coli is as functional as serum RBP in terms of retinol and TTR bindings.

ATP-Binding Cassette Transporters↗

PubMed1996

Tec protein tyrosine kinase is involved in the signaling mechanism of granulocyte colony-stimulating factor receptor.

Granulocyte colony-stimulating factor (G-CSF) is a critical cytokine to promote the growth, differentiation, and functional activation of myeloid cells. Despite its important roles, little is still understood how one cytokine can trigger such pleiotropic effects. By using mouse cell lines which can grow or differentiate in response to G-CSF, we investigated whether Tec protein tyrosine kinase is involved in either of the signaling pathways. Interestingly, Tec is shown to be tyrosine phosphorylated and activated by the G-CSF stimulation in both cell growth and differentiation mechanisms. Vav is also shown to be associated with Tec and tyrosine phosphorylated in response to G-CSF. Tec is a good candidate for protein-tyrosine kinases involved in both growth and differentiation mechanisms of myeloid cells.

Animals↗

PubMed1995

Interleukin 3 and erythropoietin induce association of Vav with Tec kinase through Tec homology domain.

Although hematopoietic cytokine receptors lack tyrosine kinase domains, the binding of their ligands to the receptors induce rapid tyrosine phosphorylation of various cellular target proteins. The specific tyrosine kinases which phosphorylate these substrates, however, have not been identified, other than that JAK kinases which phosphorylate STAT proteins and the receptors. We found that the c-vav proto-oncogene product, Vav, is rapidly and transiently tyrosine-phosphorylated in response to erythropoietin and IL3 stimulations and that Tec kinase is also transiently activated by these cytokines. Immunoprecipitation experiments demonstrated that Tec kinase binds to Vav upon these cytokine stimulations and that Grb2 constitutively associates with Vav. In vitro binding assays showed that erythropoietin and IL3 stimulation induce the specific binding of Vav to Tec kinase through Tec homology domains. We therefore concluded that Tec kinase is one of the key enzymes in Epo and IL3 receptor-mediated signaling pathways and that Vav plays an important role in the cytokine receptor-mediated signal transduction.

Animals↗

PubMed1995

Retinoic acid directly stimulates osteoclastic bone resorption and gene expression of cathepsin K/OC-2.

Vitamin A metabolites such as all-trans-retinoic acid (all-trans-RA) affect several steps of metabolic processes in vertebrates. In the last few years, several studies have shown the effect of RA on bone formation and metabolism. However, mechanisms of its action still remain unclear, especially with respect to the regulation of bone cells. Therefore, this study was carried out to clarify how RA regulates the activity of osteoclasts. Using a pit assay involving unfractionated bone cells, including osteoclasts obtained from rabbits, we found that RA stimulated an increase in the bone-resorbing activity in a dose- and time-dependent manner. Furthermore, this effect occurred more rapidly than that of treatments with 1 alpha,25-dihydroxyvitamin D3. However, this effect of RA may be partly related to cross-talk between osteoclasts and other types of cells. Therefore we studied the effect of RA on isolated osteoclasts. We found that all-trans-RA regulates the gene expression of cathepsin K/OC-2, a dominant cysteine proteinase, at the transcriptional level in mature osteoclasts isolated from rabbits. Moreover, retinoic acid-receptor alpha mRNA and retinoid X-receptor beta mRNA were expressed in these mature osteoclasts. Our results indicate that osteoclasts are target cells for RA and that RA might regulate a part of bone formation and metabolism through osteoclasts.

Animals↗

PubMed1995

Expression of p210bcr/abl by metallothionein promoter induced T-cell leukemia in transgenic mice.

The p210bcr/abl chimeric protein is considered to be implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. To investigate its biologic function in vivo, we generated transgenic mice expressing p210bcr/abl driven by the metallothionein enhancer/promoter. Two of six founder mice and the transgenic progeny developed leukemias several months after birth. In the leukemic tissues, the expression of the p210bcr/abl transgene product was detected and the increased tyrosine-phosphorylation of cellular proteins was observed. The expressed p210bcr/abl transgene product was shown to possess an enhanced kinase activity. The leukemic cells showed rearrangements in the T-cell receptor loci, indicating that the leukemic cells were monoclonal and committed to the T-cell lineage. Polymerase chain reaction analysis for tissue distribution of p210bcr/abl expression showed that, in the transgenic line that reproducibly developed leukemias, p210bcr/abl was expressed in the hematopoietic tissues such as thymus and spleen; on the other hand, in the transgenic lines that have not developed leukemias, p210bcr/abl expression was detected only in the nonhematopoietic tissues such as the brain and kidney. These results suggest that the tumorigenicity of the p210bcr/abl chimeric protein is restricted to the hematopoietic tissues in vivo and that an event enhancing p210bcr/abl expression contributed a proliferative advantage to hematopoietic precursor cells and eventually developed T-cell leukemia in transgenic mice.

Animals↗

PubMed1995

Association and activation of Btk and Tec tyrosine kinases by gp130, a signal transducer of the interleukin-6 family of cytokines.

Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor constitute the IL-6 family of cytokines and play important roles in hematopoiesis, immune response, and nervous system. The receptors for the IL-6 family of cytokines share gp130 through which signals are generated, although the cytoplasmic region of gp130 does not contain any catalytic domain. In this study we show that in addition to Jak family tyrosine kinase, the stimulation of gp130 by IL-6 plus soluble IL-6 receptor alpha induced the activation of Btk and Tec tyrosine kinases, whereas IL-3 and granulocyte colony-stimulating factor activated Tec but not Btk in a pro-B cell line. Furthermore, both Btk and Tec kinases were associated with gp130 without the ligand stimulation. Because Btk is a critical tyrosine kinase for B lymphopoiesis and Tec is considered to be involved in hematopoiesis, the results suggest the involvement of gp130-Btk-Tec signal pathway in early lymphohematopoiesis.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1995

Tec protein-tyrosine kinase is involved in interleukin-3 signaling pathway.

Among cytoplasmic protein-tyrosine kinases (PTKs) Tec now forms a novel subfamily with recently identified Tec-related PTKs (Btk and Itk/Tsk). Tec is known to be abundantly expressed in myeloid cells, and multiple forms of Tec protein can be generated via the mechanism of alternative splicing. In this report, we have investigated 5'-terminal diversity of the tec messages to demonstrate a predominant form of the Tec protein in mouse hematopoietic cell lines. Using anti-Tec serum, we could show that stimulation with interleukin-3 (IL-3) can induce tyrosine phosphorylation of Tec both in myeloid and pro-B-cell lines. IL-3 stimulation was also shown to induce kinase activity of Tec. Furthermore, we could demonstrate that Tec is constitutively associated with the Shc protein in vivo. Thus, we conclude that Tec is involved in the signaling pathway of IL-3.

Animals↗

PubMed1995

Increased tyrosine-phosphorylation of 55KDa proteins in beta-actin/Tec transgenic mice.

Protein-tyrosine kinases are considered to play important roles in cell proliferation and differentiation. Tec is a cytoplasmic protein-tyrosine kinase expressed in liver and hematopoietic tissues. To better understand Tec function in vivo, we generated transgenic mice expressing tec driven by the cytomegarovirus enhancer and beta-actin promoter. Among six transgenic lines generated, a particular line, named 2-11, expressed tec transgene product more widely and abundantly than the other lines. In the tissues of 2-11, the kinase activity of Tec was enhanced in accordance with the high expression of tec transgene product. Interestingly, tyrosine-phosphorylation of approximately 55KDa proteins in the tissues was induced. These results suggest that cellular proteins of 55KDa might be potential substrates of Tec in vivo.

Actins↗