Biomedical subjects

H Mano

Publications and source records attributed to H Mano.

At least 55 records · Page 3Linked to original sources

PubMed1998

Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells.

Bone metabolism consists of osteoblast-mediated bone formation coupled to osteoclastic resorption of bone. Osteoclastic bone resorption plays an important role in normal skeletal development and the maintenance of its integrity throughout life. Although osteoclastic activity is thought to be under the control of feedback regulation by extracellular cations, the molecular mechanism of detecting extracellular cations within the bone microenvironment remains to be clarified. In the present study we showed by reverse transcription-polymerase chain reaction and Northern blot analysis that cultured mature osteoclasts express the calcium-sensing receptor (CaSR) mRNA. The nucleotide sequence of rabbit osteoclast CaSR was approximately 90% identical to that of CaSR cDNA from human, bovine, and rat parathyroid glands. Moreover, the activity of osteoclastic bone resorption, as determined by pit formation, was regulated by extracellular calcium ion as well as its agonists that are known to act through the CaSR. We conclude that CaSR, homologous to that identified in parathyroid glands, is present in mature osteoclasts and calcium ion released from bone may directly regulate osteoclastic bone resorption.

Animals↗

PubMed1998

Tec and Jak2 kinases cooperate to mediate cytokine-driven activation of c-fos transcription.

Although transcriptional activation of the c-fos proto-oncogene plays an intrinsic role in the mechanism of blood cell growth, it is still obscure how protein-tyrosine kinases (PTKs) regulate the cytokine-driven c-fos activation pathway. We present here that Tec PTK is tyrosine-phosphorylated and activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in a human GM-CSF-dependent cell line. Moreover, we could show that introduction of Tec into mouse BA/F3-hGMRalphabeta cells can profoundly activate the c-fos promoter in response to GM-CSF or to interleukin-3 (IL-3). In contrast, introduction of a kinase-deleted Tec could suppress cytokine-driven c-fos activation, indicating that Tec is directly involved in the regulation of c-fos transcription. Interestingly, strong activation by Tec of the c-fos promoter was blocked by the co-expression of dominant negative Jak2. The molecular interaction between Tec and Jak2 was then investigated both in mammalian and insect cell systems, revealing that they can not only bind to each other, but either of the two can phosphorylate the other. Thus, Tec and Jak2 can "cross-talk" in a complexed way to mediate cytokine-driven c-fos activation.

Amino Acid Sequence↗

PubMed1998

Expression and activation of the nonreceptor tyrosine kinase Tec in human B cells.

The tyrosine kinase Tec belongs to a new group of structurally related nonreceptor tyrosine kinases that also includes Btk and Itk. Previous studies have suggested that these kinases have lineage-specific roles, with Tec being involved mainly in the regulation of cytokine-mediated myeloid cell growth and differentiation. In this study, we investigated expression and activation of Tec in human B-lymphoid cell lines representing different stages of B-cell maturation, including pro-B (RS4;11, 380, REH), pre-B (NALM6), and mature B (Ramos, and one Epstein-Barr virus [EBV]-transformed lymphoblastoid line) cells. Like Btk, Tec protein was expressed in all B-cell lines tested. Tec was also highly expressed in two EBV-transformed lymphoblastoid lines derived from patients with X-linked agammaglobulinemia (XLA) lacking Btk expression, as well as in tonsillar lymphoid cells. In surface immunoglobulin-positive B cells (Ramos), ligation of the B-cell antigen receptor (BCR) with anti-IgM antibodies caused marked tyrosine phosphorylation of Tec and increased Tec tyrosine kinase activity. Likewise, cross-linking of CD19 with a monoclonal antibody in BCR-negative pro-B (RS4;11, 380) and pre-B (NALM6) cells induced Tec tyrosine phosphorylation and increased Tec autophosphorylation, as well as Btk activation. Tyrosine phosphorylation of Tec, but not of Btk, was detectable in RS4;11 cells after CD38 ligation, suggesting that these kinases are regulated differently. We conclude that Tec is expressed and can be stimulated throughout human B-cell differentiation, implying that this tyrosine kinase plays a role in B-cell development and activation.

ADP-ribosyl Cyclase↗

PubMed1998

Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblasts.

We investigated the feasibility of an inducible apoptosis system to regulate cells genetically engineered for ectopic cytokine production. In a previous study, cDNA encoding the ligand-binding domain of the rat estrogen receptor was fused to the sequence for murine Fas transmembrane and cytoplasmic regions, and expression of the fusion protein (MfasER) in L929 fibroblasts resulted in estrogen-dependent apoptosis. We applied this MfasER/estrogen strategy to apoptosis-mediated regulation of cytokine production, using the human granulocyte colony-stimulating factor (G-CSF) as a model. Upon estrogen treatment, the G-CSF producers expressing MfasER showed an apoptotic phenotype and died in several hours, with termination of G-CSF production. This estrogen-induced apoptosis was not influenced by whether the target cells were proliferating or resting, unlike a conventional suicide system involving the herpes simplex virus 1 thymidine kinase (HSVtk). That is, estrogen induced prompt and extensive apoptosis in the resting cells which expressed MfasER, while ganciclovir treatment induced only partial reduction of the resting cells which expressed HSVtk. These results imply the feasibility of apoptosis-mediated regulation of cytokine production by genetically modified cells for supplement gene therapy.

Animals↗

PubMed1998

Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase.

BACKGROUND: Tec is a member of the recently emerging subfamily among nonreceptor protein-tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine-mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid screening. RESULTS: One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand-dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine-phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine-driven and Tec-driven activation of the c-fos promoter. CONCLUSION: Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.

Amino Acid Sequence↗

PubMed1997

Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells.

To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)-dependent murine cell line, Ba/F3, the cells showed IL-3-independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Delta(5-195)GCRER, whose product lacks the extracellular G-CSF-binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Delta(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

Animals↗

PubMed1997

SOCS-1/JAB/SSI-1 can bind to and suppress Tec protein-tyrosine kinase.

Tec is the prototype of a recently emerging subfamily among nonreceptor type protein-tyrosine kinases and is known to become tyrosine-phosphorylated and activated by a wide range of cytokine stimulations in hematopoietic cells. Although Tec was recently shown to be involved in the cytokine-driven activation mechanism of c-fos transcription, it is yet obscure how Tec relays the signals from cell surface receptors to the nucleus. To identify signaling molecules acting downstream of Tec, we have looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid system. Here we report the identification and characterization of a novel protein, TIP3, which has been simultaneously identified by other groups as SOCS-1, JAB, or SSI-1. TIP3 carries one Src homology 2 domain with a sequence similarity to that of CIS. In 293 cells, TIP3 associates with Tec and suppresses its kinase activity. Interestingly, TIP3 can also down-regulate the activity of Jak2 but not that of Lyn. We propose that SOCS-1/JAB/SSI-1/TIP3 is a novel type of negative regulator to a subset of protein-tyrosine kinases.

Amino Acid Sequence↗

PubMed1997

Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts.

Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 beta-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor- mediated mechanism.

Animals↗

PubMed1997

Concanavalin A directly stimulates bone-resorbing activity of osteoclasts and their gene expression of cathepsin K/OC-2.

Concanavalin A (Con A), a plant lectin that recognizes cell-surface glycoproteins, up-regulates osteoclastic bone-resorbing activity of cultured isolated rabbit pure osteoclasts as well as unfractionated bone cells. The effect of Con A was blocked by alpha-methyl mannopyranoside. Several Con A-binding proteins were detected in the plasma membranes from osteoclasts. Furthermore, Con A increased the levels of transcripts of cathepsin K/OC-2, one of the proteases responsible for osteoclastic bone-resorbing activity. These results indicate that Con A directly enhances the function of osteoclasts by the association with surface glycoproteins of osteoclasts.

Animals↗

PubMed1997

Stimulation of osteoblast proliferation by the cartilage-derived growth promoting factors chondromodulin-I and -II.

We previously reported the isolation of the cartilage-derived growth promoting factors chondromodulin-I (ChM-I) and chondromodulin-II (ChM-II) from fetal bovine epiphyseal cartilage. Both of these factors stimulate the growth and matrix formation of chondrocytes in vitro. In the present study, we found that ChM-I and ChM-II stimulated the proliferation of clonal mouse osteoblastic MC3T3-E1 cells as well as primary mouse osteoblasts in culture. Unlike other known growth factors, these factors did not support the proliferation of fibroblasts. Concomitantly with growth stimulation of osteoblasts, there was a reduction of alkaline phosphatase (ALP) activity in the cells, the expression of the differentiated phenotype. These results suggest that epiphyseal cartilage may play a functional role in longitudinal bone growth by production of these unique growth-promoting factors.

Alkaline Phosphatase↗

PubMed1997

A selective immunoaffinity chromatography for determination of plasma 1 alpha, 25-dihydroxyvitamin D3: application of specific antibodies raised against a 1 alpha, 25-dihydroxyvitamin D3-bovine serum albumin conjugate linked through the 11 alpha-position.

A selective and simple immunoaffinity chromatography of 1 alpha, 25-dihydroxyvitamin D3 has been developed and found to be a useful pretreatment tool for determining the metabolite in human plasma. A reasonably designed, haptenic derivative, 11 alpha-hemiglutaryloxy-1 alpha, 25-dihydroxyvitamin D3, was linked to bovine serum albumin, and rabbits were immunized repeatedly with the conjugate. The resulting polyclonal antibodies were specific to 1 alpha, 25-dihydroxyvitamin D3, recognizing both the A-ring and the side-chain structures. The antibodies were then immobilized on agarose gel to produce an immunosorbent which was stable and repeatedly usable. A plasma extract prepared with a Chem Elut column was applied to an affinity column containing the immunosorbent. After adequate washing, the adsorbed 1 alpha, 25-dihydroxyvitamin D3 was eluted selectively with a satisfactory recovery rate. This immunoaffinity chromatography enabled a simple radioreceptor assay for human plasma 1 alpha, 25-dihydroxyvitamin D3 which does not require any preparative high-performance liquid chromatography. The mean (+/-SD) values for 30 normal subjects and 8 patients with chronic renal failure were 36.0 (10.2) and 13.1 (2.1) pg/ml, respectively. The present method also gave reliable assay values for the plasma specimens from 1 alpha-hydroxyvitamin D3-administered volunteers, which have conventionally been difficult to measure unless complicated pretreatment is used.

Animals↗

PubMed1997

Prostaglandin F2alpha stimulates tyrosine phosphorylation and mitogen-activated protein kinase in osteoblastic MC3T3-E1 cells via protein kinase C activation.

PGF2alpha stimulates the proliferation of clonal osteoblastic MC3T3-E1 cells via PGF2alpha receptor linked to phospholipase C activation. To elucidate intracellular events elicited by this receptor, we examined the effects of PGF2alpha on tyrosine phosphorylation and mitogen-activated protein kinase (MAPK) activity in MC3T3-E1 cells. PGF2alpha rapidly raised the level of phosphotyrosine of cellular proteins with Mr values of 62, 68, 72, 76, 82, 125, and 150 kDa. This PGF2alpha-induced tyrosine phosphorylation of proteins (except for pp62) was blocked by down-regulating protein kinase C (PKC) by 12-O-tetradecanoylphorbol 13-acetate pretreatment and by GF 109203X, a potent specific PKC inhibitor. The addition of PGF2alpha also transiently activated MAPK in the same range of concentrations that stimulated tyrosine phosphorylation. In addition, PGF2alpha augmented the MAPK kinase kinase activity of Raf-1, whereas basal activity of MAPK/extracellular signal-regulated protein kinase kinase was less than that of Raf-1 and was little affected by PGF2alpha. Like the tyrosine phosphorylation, these activations of Raf-1 and MAPK activities were reduced by inhibition and down-regulation of PKC. Genistein, a potent inhibitor of tyrosine kinases, did not block the Raf-1 induced by PGF2alpha, indicating a tyrosine kinase-independent pathway for Raf-1 activation. However, the tyrosine kinase inhibitor partially inhibited the MAPK activity, suggesting an involvement of another Raf-1-independent kinase cascade for activation of MAPK by PGF2alpha. Fluprostenol, a specific agonist of PGF2alpha receptor, mimicked the actions of PGF2alpha consistent with a PGF2alpha receptor pathway. Thus, the action of PGF2alpha on osteoblastic MC3T3-E1 cells appears to involve a single receptor that uses diverse interacting signal transduction systems.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1997

[Techniques for purification of rabbit osteoclasts and analysis of their functions].

In the process of bone remodeling or modeling, the balance between bone formation and bone resorption maintains normality of function and structures of bone. Major bone-resorbing cells, osteoclasts, are terminally differentiated from hematopoietic stem cells and multinucleate cells. However, direct effects of osteotropic factors on osteoclast function have been unclear. Attempts to obtain isolated mammalian osteoclasts of high purity have been unsuccessful so far. Initially we succeeded in isolating osteoclasts of high purity using tissue culture dishes because of their high affinity for the tissue culture dishes, but the shortcoming of this method is that it was impossible to detach osteoclasts from the dishes. Therefore, we could not estimate bone-resorbing activity of mature osteoclasts on mineralizing substratum without any influence of bone-cells other than osteoclasts. Recently we developed a method to avoid the defect in the above-described method by the use of collagen gel. Our new method may shed new light on our understanding of the cellular and molecular mechanisms of osteoclasts.

Acid Phosphatase↗

PubMed1997

Down-regulation of gap junctional intercellular communication between osteoblastic MC3T3-E1 cells by basic fibroblast growth factor and a phorbol ester (12-O-tetradecanoylphorbol-13-acetate).

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (< 1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (< 5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (> 8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.

3T3 Cells↗

PubMed1997

[Acute myeloid leukemia with monosomy 7 accompanied by central diabetes insipidus].

A 27-year-old female was diagnosed as having atypical aplastic anemia in 1979 because of hypercellular bone marrow with abnormal erythroblasts and megakaryocytes. Afterward the diagnosis was corrected to myelodysplastic syndrome (RA) due to the reevaluation of the bone marrow smears. In March, 1995, thirst and polyurea occurred. In April, 1995, bone marrow aspiration biopsy showed the proliferation of atypical blasts (28%), and two months later, the number of the blasts increased (30%) and leukemic progression was noticed. Only 0.5 percent of the blasts showed weak peroxidase activity, and most of the blasts had CD13, CD33 and several adhesion molecules as CD11a, CD11b, CD44, CD54 and CD56. Karyotype of the bone marrow cells was 45, XX, -7. Her polyurea was caused by central diabetes insipidus. She was also complicated by pleuritis, colon ulcer, sinusitis and hypothalamic dysfunction. The etiology of these signs was due to the leukemic cell infiltration. She died despite of receiving multi-drug chemotherapy.

Adult↗

PubMed1997

[Stimulating conditions of cold air caloric test].

This study is to clarify the effects of stimulating conditions on caloric response evoked by cold air stimulation. The five parameters of caloric nystagmus (latency, maximum slow phase velocity [Max. spv], Max. spv arrival time, frequency and duration) were examined. The different stimulating air temperature(0 degree C, 5 degrees C, 10 degrees C, 15 degrees C, 20 degrees C), flow rate (3.0, 4.0, 5.0, 6.0 l/m) and period(30, 45, 60, 90 sec) were used to evoke caloric nystagmus in normal subjects. 1. Effect to temperature When the intensity of air irrigation temperature increased, Max. spv, frequency and duration also tended to increase. Out of the five parameters, the duration of caloric nystagmus was the most relevant to the intensity of irrigation temperature. 2. Effect of stimulating period When the period of caloric stimuli increased, the Max. spv and its duration also increased. But, when the stimulating period was 90 seconds, the slow phase velocity reached to the maximum responses 60 seconds after starting the stimulation. Therefore, it is not necessary to stimulate for more than 60 seconds. 3. Caloric response evoked by 60 sec, 5.0 l/m at 5 degrees C air irrigation showed the same response as the caloric nystagmus provoked by 20 ml of water at 20 degrees C for 20 sec.

Adult↗

PubMed1997

Tec protein-tyrosine kinase is involved in the thrombopoietin/c-Mpl signaling pathway.

Thrombopoietin (TPO) is the ligand of the proto-oncogene product, c-Mpl, and supports the growth and maturation of megakaryocytic cells. Although much attention has been paid to the in vivo role of the TPO/c-Mpl system in the context of maintenance of circulating platelets, still little is understood about how activation of c-Mpl leads to mitogenesis and differentiation inside cells. Tec protein-tyrosine kinase (PTK) is the prototype of a recently emerging subfamily of nonreceptor type PTKs. Tec has been demonstrated to be activated by a wide range of cytokine stimulations. In this paper we show that TPO stimulation also rapidly enhances tyrosine-phosphorylation and activity of the Tec kinase in a human TPO-dependent cell line. In addition, the Vav protein, a blood cell-specific signaling molecule, is shown to be tyrosine-phosphorylated in response to TPO and to be constitutively associated with the Tec protein. From this evidence, we conclude that Tec is involved in the intracellular signaling system of TPO/c-Mpl.

Cell Cycle Proteins↗

PubMed1996

Autocrine mechanism of epidermal growth factor in choriocarcinoma cell proliferation.

We examined four choriocarcinoma cell lines, NaUCC-1, NaUCC-3, NaUCC-4 and BeWo, for the presence of epidermal growth factor (EGF) by enzyme immunoassay and reverse transcription and polymerase chain reaction, and for EGF receptor (EGFR) by 125I-EGF binding assay. Specific EGF binding and EGF proteins were detected in these four choriocarcinoma cell lines. On the cell lines examined, NaUCC-4 had the greatest EGF binding capacity (18 x 10(5) sites/cell) and the highest amount of immunoreactive EGF (142 pg/ml). These results prompted us to assess the significance of EGF/EGFR autocrine mechanism in NaUCC-4 cells. Low doses of exogenous EGF stimulated 3H-thymidine incorporation, and monoclonal antibodies against EGF or EGFR dose-dependently inhibited 3H-thymidine incorporation. On the other hand, these antibodies did not significantly affect hCG production. These results suggested that EGF might function in an autocrine manner to stimulate proliferation rather than differentiation of NaUCC-4 choriocarcinoma cells.

Adult↗