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Biomedical subjects

T Akiyama

Publications and source records attributed to T Akiyama.

At least 19 recordsLinked to original sources
PubMed1996

Expression of activin A in human keratinocytes at early stages of cultivation.

Activins are members of the TGF-beta superfamily and are classified into 3 types: activin A, which consists of a homodimer of betaA, activin B, which consists of a homodimer of betaB, and activin AB, which consists of a heterodimer of betaAbetaB. We studied the expression of activin mRNAs by RT-PCR in normal human epidermis, cultured keratinocytes, and DJM-1 cells (a squamous cell carcinoma line). We could detect only activin A mRNA (betaA) in normal human epidermis. In cultured keratinocytes and DJM-1 cells, activin betaA mRNA was observed at 4 h but not at 96 h after plating. Activin A activity was detected in the conditioned medium of DJM-1 cells within 48 h. In addition, although follistatin mRNA was not observed in human epidermis in situ, it was transiently expressed in cultured cells at 4 h after plating. These findings suggest that the expression of these molecules in keratinocytes is associated with cell proliferation. In an in vitro tissue injury model, activin A was observed at the wound edge, where cell migration and proliferation may be activated. In DJM-1 cells cultured for 92 h, betaA mRNA was observed 4 h after injury treatment. These findings suggest that activin A acts as a potent inducer of proliferation in vitro, at least in keratinocytes.

Activins↗
PubMed1996

Midkine as a novel target gene for the Wilms' tumor suppressor gene (WT1).

Midkine (MK) is a heparin-binding growth factor which is strongly expressed during the midgestation period of mouse embryogenesis. Wilms' tumor is an embryonal kidney malignancy in infants, and WT1 has been identified as its tumor suppressor gene. The high expression level of MK in all Wilms' tumor specimens so far examined and the presence of two WT1 elements (5'-GCGGGGGCG-3') in the human MK promoter region led us to examine the possible role of the WT1 gene product in the regulation of MK gene expression. A gel shift assay verified the complex formation between the WT1 gene product and WT1 consensus sequence of MK gene. DNase1 footprint analysis also demonstrated that the downstream WT1 element was protected from DNase1 cleavage by the addition of the WT1 protein. The human MK promoter fused with the chloramphenicol acetyltransferase gene (phMK2.3kCAT) was co-transfected with an effector plasmid containing the WT1 gene into several cell lines. Transient transfection assays showed suppression of the MK promoter by WT1 co-transfection in recipient cells; deletion of the WT1 binding site abolished the suppression. The evidence reported in this study indicates that MK gene is a newly identified WT1 target gene.

3T3 Cells↗
PubMed1996

Microscopic DNA flexibility analysis. Probing the base composition and ion dependence of minor groove compression with an artificial dna bending agent.

We have used an artificial DNA bending agent to monitor the local flexibility of the DNA helix as a function of Mg2+ cation concentration, sequence, and temperature. A DNA bending agent was constructed from a pair of triple helix-forming oligonucleotides connected by a flexible polymeric linker, which, when the linker is short enough, causes a bend in a minor groove region separating the two sites of triple helix formation. The unique aspect of this system is that, since the bent region is not in direct contact with the linker or the triple helix-forming oligonucleotides, the free energy reflecting the bendability of the minor helix groove can be estimated from a comparison of binding affinity between the bent and unbent triple helices. A binding competition experiment and association and dissociation kinetic assays executed at 37 degrees C in the presence of 10 mM Mg2+ have revealed an extremely small difference in binding affinity between bent (50 degrees ) and straight triple helices, suggesting that DNA flexibility with respect to minor groove compression is extremely high and virtually independent of the sequence of the distorted duplex. This unexpectedly small difference in binding affinity was detected over the temperature range from 25 to 65 degrees C, and over a Mg2+ concentration range from 0.3 to 10 mM. Thus, these findings provide evidence that DNA bendability for minor groove compression is inherently high and independent of DNA sequence, temperature, or a 30-fold variation of Mg2+ ion concentration.

Binding, Competitive↗
PubMed1996

The design of an agent to bend DNA.

An artificial DNA bending agent has been designed to assess helix flexibility over regions as small as a protein binding site. Bending was obtained by linking a pair of 15-base-long triple helix forming oligonucleotides (TFOs) by an adjustable polymeric linker. By design, DNA bending was introduced into the double helix within a 10-bp spacer region positioned between the two sites of 15-base triple helix formation. The existence of this bend has been confirmed by circular permutation and phase-sensitive electrophoresis, and the directionality of the bend has been determined as a compression of the minor helix groove. The magnitude of the resulting duplex bend was found to be dependent on the length of the polymeric linker in a fashion consistent with a simple geometric model. Data suggested that a 50-70 degrees bend was achieved by binding of the TFO chimera with the shortest linker span (18 rotatable bonds). Equilibrium analysis showed that, relative to a chimera which did not bend the duplex, the stability of the triple helix possessing a 50-70 degrees bend was reduced by less than 1 kcal/mol of that of the unbent complex. Based upon this similarity, it is proposed that duplex DNA may be much more flexible with respect to minor groove compression than previously assumed. It is shown that this unusual flexibility is consistent with recent quantitation of protein-induced minor groove bending.

Base Sequence↗
PubMed1996

Suppression of fibroblast cell growth by overexpression of LIM-kinase 1.

LIM-kinase 1 (LIMK1) is a serine/threonine kinase containing two LIM motifs at the N-terminus. The functional role of LIMK1 has remained unknown. In this study, we examined the role of LIMK1 in cell growth of fibroblasts. Induced expression of LIMK1 in NIH3T3 cells led to growth retardation. Transfection of LIMK1 sense cDNA into NIH3T3 and H-ras-transformed FYJ10 fibroblasts significantly suppressed colony formation of these cells. In contrast, transfection with LIMK1 antisense cDNA strongly stimulated colony formation of the NIH3T3 cells. These findings suggest that LIMK1 functions as a negative regulator of fibroblast cell growth, and may play a role in tumor suppression.

3T3 Cells↗
PubMed1996

Elevation of either axoplasmic norepinephrine or sodium level induced release of norepinephrine from cardiac sympathetic nerve terminals.

Using dialysis technique, prominent accumulation of norepinephrine (NE) in the myocardial interstitial space was observed under two different conditions: (1) local administration of a monoamine oxidase inhibitor (pargyline, 1, 10 mM), (2) local administration of a Na, K-ATPase inhibitor (ouabain, 10, 100 microM). Further, pretreatment with a vesicle uptake inhibitor (reserpine, 10 microM) augmented these responses. Either condition independently caused endogenous NE release and a brisk increase in dialysate NE levels might occur as a consequence of non-exocytotic release of NE.

Animals↗
PubMed1996

Relation of circadian ventricular ectopic activity to cardiac mortality. CAST Investigators.

The relation between the circadian occurrence of ventricular premature depolarizations (VPD) and sudden arrhythmic death was examined in a subset of patients entered into the Cardiac Arrhythmia Suppression Trial (CAST). Ambulatory electrocardiographic recordings with hourly measurement of VPD frequency were available in 357 patients. Forty percent of the patients (142 of 357) demonstrated circadian variation in VPD frequency between 6:00 A.M. and 9:59 A.M. that was significantly higher (p < 0.05) than what could randomly be expected from an overall 24-hour average for that patient. The only baseline characteristics in patients with circadian VPDs were age (p < 0.04), history of cardiac arrest (p < 0.01), presence of higher frequency of VPDs (p < 0.002), more frequent episodes of ventricular tachycardia (p < 0.04), and more frequent episodes of slow runs (p < 0.04). There was no difference in mortality in patients with or without circadian VPD variation; drug treatment did not effect mortality. These data indicate that the presence of circadian VPDs is not a predictor of sudden arrhythmic death in patients with a high frequency of VPDs.

Case-Control Studies↗
PubMed1996

Constitutive expression of the Wilms tumor suppressor gene WT1 in F9 embryonal carcinoma cells induces apoptotic cell death in response to retinoic acid.

The product of the Wilms tumor suppressor gene, WT1, is thought to be a tissue specific transcription factor regulating cell growth and differentiation. To elucidate the function of WT1 in cellular differentiation, we examined the changes in the level of WT1 expression during retinoic acid induced-differentiation of embryonal carcinoma F9 cells into parietal endoderm cells. We found that, in response to retinoic acid addition, the expression of WT1 increased significantly after 12--24 h of incubation, then decreased and finally disappeared after 4 days, by which time most of the cells had differentiated into primitive endoderm cells. To examine the significance of these changes in WT1 expression, we established cell lines constitutively expressing one of the WT1 splicing variants. These cell lines showed a phenotype very similar to parental F9 cells in the absence of retinoic acid. However, in the presence of retinoic acid, they failed to differentiate into primitive endoderm cells and underwent apoptotic death 36 h after the addition of retinoic acid. These results suggest that downregulation of WT1 expression is necessary for normal differentiation of F9 cells into parietal endoderm cells.

3T3 Cells↗
PubMed1996

Expression of truncated midkine in human colorectal cancers.

Midkine (MK) is a growth differentiation factor originally found as the product of a retinoic acid-responsive gene. The expression of MK was examined in 35 surgically resected specimens of primary colorectal cancer using the reverse transcription-polymerase chain reaction (RT-PCR). All of the cancerous tissues expressed MK. In 5/25 cancerous tissues a truncated form of MK, which was recently found in various human tumor cell lines, was detected in addition to the full-size MK. In contrast, the truncated from of MK could not be detected in non-cancerous tissues, whereas the wild-type form was detected in 8/10 non-cancerous tissues. These results suggest that the expression of the truncated form of MK may be associated with tumorigenesis.

Carrier Proteins↗
PubMed1996

Upregulation of the APC gene product during neuronal differentiation of rat pheochromocytoma PC12 cells.

The adenomatous polyposis coli (APC) gene, the mutation of which is responsible for familial adenomatous polyposis and sporadic colorectal tumors, is highly expressed in the central nervous system. To elucidate the contribution of the APC protein to neuronal differentiation, changes in APC expression were examined during nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma PC12 cells. The expression of APC gradually increased throughout the time course, in particular it increased markedly after 7 days of exposure to NGF. However, forced expression of APC did not induce neuronal differentiation of PC12 cells. These results suggest that the APC protein itself does not have the potential to induce neuronal differentiation, but rather is upregulated secondary to the differentiation of PC12 cells.

Adenomatous Polyposis Coli Protein↗
PubMed1996

The tumor suppressor protein APC colocalizes with beta-catenin in the colon epithelial cells.

The APC gene is mutated in familial adenomatous polyposis and sporadic colorectal tumors. The product of this gene is a 300 kDa cytoplasmic protein associated with catenin. In the present study, we examined the subcellular localization of the APC protein and beta-catenin in the mouse colon by double-labeling immunocytochemistry. While the APC protein was localized in the lateral and apical cytoplasm and in microvilli of the epithelial cells, beta-catenin was present exclusively in the lateral cytoplasm. Double-labeling-immunoelectron microscopy demonstrated precise colocalization of the APC protein and beta-catenin along the lateral plasma membrane. These results suggest that the APC protein functions in cooperation with beta-catenin in the lateral cytoplasm but has other functions independent of beta-catenin in the apical cytoplasm and in microvilli.

Adenomatous Polyposis Coli Protein↗
PubMed1996

Binding of APC to the human homolog of the Drosophila discs large tumor suppressor protein.

The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors, and its product binds to the adherens junction protein beta-catenin. Overexpression of APC blocks cell cycle progression. The APC-beta-catenin complex was shown to bind to DLG, the human homolog of the Drosophila discs large tumor suppressor protein. This interaction required the carboxyl-terminal region of APC and the DLG homology repeat region of DLG. APC colocalized with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function.

Adenomatous Polyposis Coli Protein↗
PubMed1996

MCC, a cytoplasmic protein that blocks cell cycle progression from the G0/G1 to S phase.

The MCC gene was isolated from the human chromosome 5q21 by positional cloning and was found to be mutated in several colorectal tumors. In this study, we prepared specific antibodies and detected the MCC gene product as a cytoplasmic 100-kDa phosphoprotein in mouse NIH3T3 cells. Immunoelectron microscopic analysis showed that the MCC protein is associated with the plasma membrane and membrane organelles in mouse intestinal epithelial cells and neuronal cells. The amount of the MCC protein remained constant during the cell cycle progression of NIH3T3 cells, while its phosphorylation state changed markedly in a cell cycle-dependent manner, being weakly phosphorylated in the G0/G1 and highly phosphorylated during the G1 to S transition. Overexpression of the MCC protein blocked the serum-induced cell cycle transition from the G1 to S phase, whereas a mutant MCC, initially identified in a colorectal tumor, did not exhibit this activity. These results suggest that the MCC protein may play a role in the signaling pathway negatively regulating cell cycle progression.

3T3 Cells↗
PubMed1996

Persistent activation of CDK4 during neuronal differentiation of rat pheochromocytoma PC12 cells.

It has been shown that suppression of cyclin-dependent kinase 4 (CDK4) expression or its kinase activity is a critical event for the differentiation of hematopoietic and myoblastic cells. To investigate the role of CDK4 during NGF-mediated neuronal differentiation of PC12 cells, we examined the changes in the expression and kinase activity of CDK4. Unexpectedly, both the expression of CDK4 and its kinase activity remained high throughout 10 days of exposure to NGF. Furthermore, constitutive overexpression of the human CDK4 did not inhibit the differentiation. These results suggest that suppression of CDK4 is not required for the NGF-induced neuronal differentiation of PC12 cells.

Animals↗
PubMed1996

Growth inhibition of human leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis.

We have previously reported expression of WT1 in acute leukemia. To elucidate its biological significance, we examined the effect of the suppression of the WT1 expression by WT1 antisense oligomers on the growth of the leukemic cells expressing WT1. When 20 different WT1 antisense (AS) oligomers covering from the 5' cap sites of the WT1 gene to the 3' end were examined for the inhibitory effect on the growth of K562 cells expressing WT1, four WT1 AS oligomers inhibited the cell growth, whereas WT1 sense and random sequence oligomers had no effect on the cell growth of K562. Moreover, WT1 AS oligomers significantly inhibited the growth of the clonogenic cells of fresh leukemic cells in six of 14 patients with acute myeloid leukemia, in one of two patients with chronic myelogenous leukemia (CML) chronic phase, and in one of one patient with CML blastic crisis. However, these oligomers did not inhibit normal colony-forming unit-granulocyte-macrophage. Western blot analysis clearly demonstrated the significant reduction in the WT1 protein levels in the K562 and fresh leukemic cells that were treated with the WT1 AS oligomers, confirming that the inhibitory effect of the WT1 AS oligomers on the cell growth operates via the reduction in the WT1 protein levels. These results show that WT1 plays an important role in leukemogenesis.

Base Sequence↗
PubMed1996

The tumor suppressor gene product APC is hyperphosphorylated during the M phase.

The APC gene is mutated in familial adenomatous polyposis and sporadic colorectal tumors. The product of this gene is a 300 kDa cytoplasmic protein and its overexpression results in the block of cell cycle progression from the G0/G1 to the S phase. In the present study, we studied the expression and phosphorylation of the APC protein through the cell cycle. The APC protein was found to be constantly expressed and phosphorylated at serine and threonine residues. Moreover, the APC protein immunoprecipitated from cells arrested in the M phase by nocodazole treatment migrated in SDS-PAGE more slowly than those from the G1 and S phases. Phosphatase treatment abolished this M phase-specific retarded migration, suggesting that APC is transiently hyperphosphorylated in the M phase.

Adenomatous Polyposis Coli Protein↗
PubMed1996

Onset study of English-speaking temporary residents in Japan.

This is a study of the onset time of psychiatric episodes of 103 English-speaking temporary residents in Japan. The onset time is defined as the interval between the time of arrival in Japan and the request for treatment. The best-fit model was composed of 13 factors and had a highly significant effect on onset time. The existence of a past history of psychiatric disorder was linked with a high vulnerability. Higher proficiency in the language and experience of having been married indicated a lower vulnerability. Vulnerability defined by onset time may share a certain common ground with liability defined by prevalence.

Adolescent↗