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Biomedical subjects

J Miyoshi

Publications and source records attributed to J Miyoshi.

At least 19 recordsLinked to original sources
PubMed2003

Cot/Tpl2 is essential for RANKL induction by lipid A in osteoblasts.

Lipopolysaccharide (LPS) is a pathogenic factor that increases bone resorption in periodontal diseases. LPS treatment of osteoblasts was shown to induce the receptor activator of NF-kappa B ligand (RANKL), an essential secretory or membrane-bound factor for osteoclast function, in a manner dependent on extracellular signal-regulated kinase (ERK) activation. However, the mechanisms regulating this process remained unknown. Here, we show that RANKL mRNA induction and ERK activation, when treated with synthetic lipid A (an active center of LPS), were markedly reduced in mouse osteoblasts lacking Cot/Tpl2, which was recently recognized as an essential kinase for the induction of TNF-alpha by LPS in macrophages. In contrast, c-Jun N-terminal kinase (JNK), p38 kinase, Raf-1, and NF-kappa B were normally activated in cot/tpl2-/- osteoblasts. These findings indicate that Cot/Tpl2 is essential for LPS-induced ERK activation and RANKL induction in osteoblasts.

Animals↗
PubMed2001

Role of Rab3 GDP/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction.

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca(2+)-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP-/- mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction approximately 10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Action Potentials↗
PubMed2001

Reduced expression of focal adhesion kinase in liver metastases compared with matched primary human colorectal adenocarcinomas.

The focal adhesion kinase (FAK) is implicated in integrin-mediated signal transduction pathways used in cell adhesion, cell motility, apoptosis, and anchorage-independent growth. Because cancer invasion and metastasis are thought to be associated with alterations in cellular adhesive and motile properties, we studied the expression of four focal adhesion proteins including FAK in matched samples of human normal colorectal mucosa (N), primary colorectal adenocarcinomas (T) and liver metastases (M) from 10 patients by Western blot analysis. This gave us the advantage of directly comparing levels of focal adhesion protein expression within the same genetic background. Average FAK expression level was significantly higher in T than in N and it was significantly lower in M than in T. Average paxillin expression level was also significantly higher in T than in N, but it was not significantly different between T and M. Similar results were obtained by immunohistochemical analyses of FAK and paxillin expression. Average vinculin and talin expression levels showed no significant differences among these three samples (N, T, and M). These data demonstrate that the FAK expression level increases in primary tumors compared with normal mucosa and decreases in liver metastases to the level of normal mucosa in the majority of human colorectal adenocarcinomas. Up- and down-regulation of FAK protein expression observed in this study may have a profound effect on the signal transduction.

Adenocarcinoma↗
PubMed2000

Role of rab GDP dissociation inhibitor alpha in regulating plasticity of hippocampal neurotransmission.

Rab GDP dissociation inhibitor alpha (Rab GDIalpha) is a regulator of the Rab small G proteins implicated in neurotransmission, and mutations of Rab GDIalpha cause human X-linked mental retardation associated with epileptic seizures. In Rab GDIalpha-deficient mice, synaptic potentials in the CA1 region of the hippocampus displayed larger enhancement during repetitive stimulation, which was apparently opposite to the phenotype of Rab3A-deficient mice. Furthermore, the Rab GDIalpha-deficient mice showed hypersensitivity to bicuculline, an inducer of epileptic seizures. These results suggest that Rab GDIalpha plays a specialized role in Rab3A recycling to suppress hyperexcitability via modulation of presynaptic forms of plasticity.

Animals↗
PubMed2000

Targeted deletion of the H-ras gene decreases tumor formation in mouse skin carcinogenesis.

To clarify the role of the H-Ras in vivo, we generated H-ras null mutant mice by gene targeting. In spite of the importance of the Ras in cell proliferation and differentiation, H-ras null mutant mice grew normally and were fertile. The oldest H-ras mutant mice grew to be more than 30 months old. We used the H-ras deficient mice to study the importance of the H-ras and other ras genes in the development of skin tumors induced by initiation with 7, 12-dimethylbenz(a)anthracene (DMBA) followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). We showed that H-ras null mutant mice develop approximately six times less papillomas compared with wild-type littermates after 20 weeks of TPA treatment. While all papillomas examined (17 out of 17) in wild-type mice have mutations of H-ras at codon 61, 13 (62%) out of 21 papillomas in H-ras null mutant mice have mutations of K-ras gene at codon 12, 13, or 61 and another eight (38%) papillomas have no mutations in these codons of K-ras or N-ras genes. This suggests that the activation of H-ras gene is critical in the wild-type mice, but the activation of K-ras gene can replace the H-ras activation in the initiation step of skin tumor development in the H-ras deficient mice. Oncogene (2000).

Animals↗
PubMed2000

Increased expression of S100A6 at the invading fronts of the primary lesion and liver metastasis in patients with colorectal adenocarcinoma.

Two members of the S100 gene family, S100A6 and S100A4 have been suggested to be associated with cancer invasion and metastasis. To study their involvement in the malignancy of human colorectal adenocarcinoma, we examined the protein expression levels of S100A6 and S100A4 in the primary colorectal adenocarcinoma (T) and paired adjacent normal colorectal mucosa (N) from 12 cases, quantitatively by Western blot analysis. In 11 of 12 and seven of 12 cases, S100A6 and S100A4 expression levels were higher in T than in N, respectively. Average S100A6 level in T was significantly higher than in N (about x 2.3;P = 0.001), whereas average S100A4 level in T was not. When S100A6 expression levels in three sets of matched samples of primary colorectal adenocarcinoma (T) and liver metastasis (M) were examined, S100A6 levels were higher in M than in T in two of three cases. Immunohistochemical analysis using monoclonal anti-S100A6 antibody showed that 23 of 42 (55%) primary colorectal adenocarcinoma and 15 of 16 (94%) liver metastasis specimens were positively stained. S100A6 immunostaining of primary colorectal adenocarcinomas was significantly more intense in the invading fronts with structural atypia than in central portions with glandular structure (P< 0.0001), whereas Ki-67 staining (a growth marker) was similar in these two portions. Interestingly, S100A6 and Ki-67 immunostaining patterns in liver metastases were also the same as in primary lesions. These results suggest that S100A6 is involved in the invasive process of human colorectal adenocarcinomas and that S100A6 expression levels decrease when carcinoma cells form glandular structure again at the central portions of metastatic nodules.

Adenocarcinoma↗
PubMed2000

Involvement of a small GTP-binding protein (G protein) regulator, small G protein GDP dissociation stimulator, in antiapoptotic cell survival signaling.

Small GTP-binding protein GDP dissociation stimulator (Smg GDS) regulates GDP/GTP exchange reaction of Ki-Ras and the Rho and Rap1 family members and inhibits their binding to membranes. In fibroblasts, Smg GDS shows mitogenic and transforming activities in cooperation with Ki-Ras. However, the physiological function of Smg GDS remains unknown. Here we show that mice lacking Smg GDS died of heart failure shortly after birth, not resulting from developmental heart defects but from enhanced apoptosis of cardiomyocytes triggered by cardiovascular overload. Furthermore, neonatal thymocytes and developing neuronal cells underwent apoptotic cell death. Smg GDS-/- thymocytes were susceptible to apoptotic inducers, such as etoposide and UV irradiation. Smg GDS-/- thymocytes were protected from etoposide-induced cell death by ex vivo transduction of the Smg GDS cDNA. These phenotypes partly coincide with those observed in Ki-Ras-deficient mice, suggesting that Smg GDS is involved in antiapoptotic cell survival signaling through Ki-Ras.

Animals↗
PubMed2000

Increased expression of S100A6 (Calcyclin), a calcium-binding protein of the S100 family, in human colorectal adenocarcinomas.

The expression of S100A6 (also known as Calcyclin/2A9/ 5B10/PRA) in surgically resected human colorectal adenocarcinomas was examined to investigate whether S100A6 plays a role in the malignancy of human tumor cells. Western blot analysis using the lysates from colorectal adenocarcinomas and adjacent normal mucosa from 10 patients revealed that the average S100A6 level of adenocarcinomas was significantly higher (about 2.4-fold) than that of normal mucosa. Immunohistochemical analysis using formalin-fixed paraffin-embedded surgical specimens and monoclonal anti-S100A6 antibody (mAbA6) demonstrated that 2(5%) of 42 normal mucosa and 6 (46%) of 13 adenoma specimens were mAbA6-positive and showed granular staining localized at the supranuclear regions of epithelial cells, whereas 23 (55%) of 42 adenocarcinomas and 13 (100%) of 13 carcinoma cells that metastasized to the liver were mAbA6-positive and showed diffuse cytoplasmic staining. A significant correlation between S100A6 expression and Dukes' tumor stage or lymphatic permeation but not with other clinicopathological factors was shown. S100A6 was stained more intensely in peripheral portions than in central portions of adenocarcinomas, whereas Ki-67 (a growth marker) was stained equally in these two portions. These results suggest that S100A6 may be involved in the progression and invasive process of human colorectal adenocarcinomas.

Adenocarcinoma↗
PubMed1999

Progressive impairment of kidneys and reproductive organs in mice lacking Rho GDIalpha.

The Rho small G protein family members regulate various actin cytoskeleton-dependent cell functions. The Rho GDI (GDP dissociation inhibitor) family, consisting of Rho GDIalpha, -beta, and -gamma, is a regulator that keeps the Rho family members in the cytosol as the GDP-bound inactive form and translocates the GDP-bound form from the membranes to the cytosol after the GTP-bound form accomplishes their functions. Rho GDIalpha is ubiquitously expressed in mouse tissues and shows GDI activity on all the Rho family members in vitro. We have generated mice lacking Rho GDIalpha by homologous recombination to clarify its in vivo function. Rho GDIalpha -/- mice showed several abnormal phenotypes. Firstly, Rho GDIalpha -/- mice were initially viable but developed massive proteinuria mimicking nephrotic syndrome, leading to death due to renal failure within a year. Histologically, degeneration of tubular epithelial cells and dilatation of distal and collecting tubules were readily detected in the kidneys. Secondly, Rho GDIalpha -/- male mice were infertile and showed impaired spermatogenesis with vacuolar degeneration of seminiferous tubules in their testes. Thirdly, Rho GDIalpha -/- embryos derived from Rho GDIalpha -/- female mice were defective in the postimplantation development. In addition, these morphological and functional abnormalities showed age-dependent progression. These results suggest that the signaling pathways of the Rho family members regulated by Rho GDIalpha play important roles in maintaining the structure and physiological function of at least kidneys and reproductive systems in adult mice.

Age Factors↗
PubMed1999

Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

Actins↗
PubMed1999

Unsuspected extra-adrenal pheochromocytoma simulating ovarian tumor.

We report on a case of an extraadrenal pheochromocytoma simulating an ovarian tumor. Before intervention, the patient exhibited no symptoms suggestive of pheochromocytoma. Nevertheless, during surgery she experienced marked blood pressure fluctuations, and an unsuspected extraadrenal pheochromocytoma was diagnosed. Thus, although rare, when preparing to remove a pelvic mass, the gynecologist should consider the possibility of an extraadrenal pheochromocytoma.

Blood Pressure↗
PubMed1997

K-ras is essential for the development of the mouse embryo.

ras genes encode members of the small GTP-binding proteins. Ras protein in highly conserved in various species from yeast to humans and plays a key role in signal transduction. Ras is related to cell proliferation and differentiation. While, in addition, mutations in the ras genes are implicated in a variety of tumors. However, the physiological functions and specific roles of each ras gene, H-ras, K-ras and N-ras, are still not fully understood. To clarify the role of the K-Ras in vivo, we generated K-ras mutant mice by gene targeting. In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term. At embryonic day 15.5, their ventricular walls are extremely thin. Besides, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord. Our results thus indicate K-Ras to be essential for normal development in mice and residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice.

Animals↗
PubMed1997

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor.

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.

Amino Acid Sequence↗
PubMed1995

Participation of rhop21 in serum-dependent invasion by rat ascites hepatoma cells.

Rat ascites hepatoma cells (MM1 cells) penetrate through a cultured mesothelial cell monolayer (MCL) in the presence of fetal calf serum (FCS), but scarcely do so in its absence. Inactivation of rhop21 of MM1 cells by ADP-ribosyltransferase C3 resulted in the suppression of this serum effect on the penetration, suggesting that the serum effect was mediated by rhop21. To ascertain this assumption MM1 cells were transfected with an activated (Val14) human rhoA cDNA (Neo/RhoA 1-7). The transfectants penetrated MCL extensively even in the absence of FCS and became largely independent of serum for the penetration. These results suggest that serum-induced invasion by MM1 cells is mainly mediated by rhop21.

ADP Ribose Transferases↗
PubMed1994

Function-dependent cooperation between oncogene activation and nonrandom chromosome change during tumorigenic conversion of Syrian hamster cells.

We determined the tumorigenicity of SHOK cells, a Syrian hamster cell line, morphologically transformed by the introduction of either c-Ha-ras, c-Ki-ras, N-ras, v-mos, v-fgr, or v-src genes. When 1 x 10(6) cells were injected subcutaneously into nude mice, SHOK cells containing one of those oncogenes gave rise to tumors within 3 weeks, while the parental SHOK cells, or SHOK cells containing the neo gene, did not show tumorigenicity even 200 days after injection. From karyotype analysis, the addition of chromosome 3 was consistently found in tumor cells derived from SHOK cells containing the c-Ha-ras, c-Ki-ras, or v-mos genes, and the acquisition of chromosome 9 was a common change observed in tumor cells derived from SHOK cells morphologically transformed by the v-fgr or v-src gene. No consistent chromosomal change was observed in tumor cells containing the N-ras gene. The results obtained from Southern blot analysis showed that amplification of introduced oncogenes was detected in the case of c-Ha-ras, c-Ki-ras, N-ras, v-fgr, and v-src genes. These results indicate that nonrandom chromosome changes, along with the amplification of introduced oncogenes, are required for the tumorigenic conversion. Our findings propose the possibility that secondary chromosomal changes associated with the tumorigenic conversion of morphologically transformed cells occur dependent on the function of the primary activated oncogene introduced.

Animals↗
PubMed1993

Syrian hamster embryo cell lines useful for detecting transforming genes in mouse tumours: detection of transforming genes in X-ray-related mouse tumours.

The Syrian hamster embryo cell lines, SHOK and MC-1, were used as recipient cells for DNA transfection assay to detect transforming genes in experimental mouse tumours. A mouse repeat sequence was utilised to check whether each transformed focus included mouse genomic DNA in the Hamster background. We investigated five mouse tumours that are related to X-ray radiation, and detected activated c-K-ras, c-mos, and c-cot oncogenes which induced foci of hamster cells. These results show that SHOK and MC-1 cells have unique properties for detecting transforming genes in experimental mouse tumours.

3T3 Cells↗