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Biomedical subjects

H Wada

Publications and source records attributed to H Wada.

At least 127 records · Page 7Linked to original sources
PubMed2000

Conservation and elaboration of Hox gene regulation during evolution of the vertebrate head.

The comparison of Hox genes between vertebrates and their closest invertebrate relatives (amphioxus and ascidia) highlights two derived features of Hox genes in vertebrates: duplication of the Hox gene cluster, and an elaboration of Hox expression patterns and roles compared with non-vertebrate chordates. We have investigated how new expression domains and their associated developmental functions evolved, by testing the cis-regulatory activity of genomic DNA fragments from the cephalochordate amphioxus Hox cluster in transgenic mouse and chick embryos. Here we present evidence for the conservation of cis-regulatory mechanisms controlling gene expression in the neural tube for half a billion years of evolution, including a dependence on retinoic acid signalling. We also identify amphioxus Hox gene regulatory elements that drive spatially localized expression in vertebrate neural crest cells, in derivatives of neurogenic placodes and in branchial arches, despite the fact that cephalochordates lack both neural crest and neurogenic placodes. This implies an elaboration of cis-regulatory elements in the Hox gene cluster of vertebrate ancestors during the evolution of craniofacial patterning.

Animals↗
PubMed2000

Activation of the beta-catenin gene by interstitial deletions involving exon 3 as an early event in colorectal tumorigenesis.

beta-Catenin has been identified as an oncogene in several tumors including colorectal cancers. beta-Catenin gene is activated by interstitial deletions involving exon 3 in colorectal carcinomas of Japanese population, in contrast to amino acid substitutions detected among Caucasian population. The aim of this study was to examine the type and frequency of beta-catenin gene mutation during early stages of colorectal tumorigenesis. We screened 100 colorectal adenomas for somatic mutations in the beta-catenin gene by single-strand conformation polymorphism method, as well as polymerase chain reaction amplification. In cases with mutations, sequencing analyses and immunohistochemical staining were also performed. Somatic interstitial deletions of 272-413 bp, each of which included all parts of exon 3, were detected in three tumors. However, no adenoma carried missense mutations. We confirmed accumulation of aberrant beta-catenin protein in cytoplasm and nuclei of adenoma cells by immunohistochemical analysis. Our results suggested that activation of the beta-catenin gene by interstitial deletions involving exon 3 might be less frequent compared with frequent alterations of adenomatous polyposis coli (APC) gene, but could be an early event in colorectal tumorigenesis equivalent to APC gene alterations in the Japanese population.

Adenoma↗
PubMed2000

Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes.

To investigate the role of phosphatidylglycerol (PG) in photosynthesis, we constructed a mutant defective in the CDP-diacylglycerol synthase gene from a cyanobacterium, Synechocystis sp. PCC6803. The mutant, designated as SNC1, required PG supplementation for growth. Growth was repressed in PG-free medium concomitantly with the decrease in cellular content of PG. These results indicate that PG is essential, and that SNC1 is defective in PG synthesis. Decrease in PG content was accompanied by a reduction in the cellular content of chlorophyll, but with little effect on the contents of phycobilisome pigments, which showed that levels of chlorophyll-protein complexes decreased without alteration of those of phycobilisomes. Regardless of the decrease in the PG content, CO(2)-dependent photosynthesis by SNC1 was similar to that by the wild type on a chlorophyll basis, but consequently became lower on a cell basis. Simultaneously, the ratio of oxygen evolution of photosystem II (PSII) measured with p-benzoquinone to that of CO(2)-dependent photosynthesis, which ranged between 1.3 and 1.7 in the wild type. However, it was decreased in SNC1 from 1.3 to 0.4 during the early growth phase where chlorophyll content and CO(2)-dependent photosynthesis were little affected, and then finally to 0.1, suggesting that PSII first lost its ability to reduce p-benzoquinone and then decreased in its level and actual activity. These results indicate that PG contributes to the accumulation of chlorophyll-protein complexes in thylakoid membranes, and also to normal functioning of PSII.

Amino Acid Sequence↗
PubMed2000

A p300 protein as a coactivator of GATA-6 in the transcription of the smooth muscle-myosin heavy chain gene.

The mechanisms that regulate smooth muscle development and differentiation are poorly understood. Although recent studies have suggested the possible role of a zinc finger transcription factor, GATA-6, in the differentiation of vascular smooth muscle cells (VSMCs), the downstream gene targeted by GATA-6 is unknown. The expression of smooth muscle-myosin heavy chain (Sm-MHC) provides a highly specific marker for the differentiated phenotype of VSMCs as well as the smooth muscle cell lineage. Here, we show that GATA-6 bound to a GATA-like motif (-810/-805) within the rat Sm-MHC promoter in a sequence-specific manner and activated this promoter through this site. In addition, we show that the transcriptional coactivator p300 associated with GATA-6 during the transcription of the Sm-MHC gene. A p300/GATA-6 complex in VSMCs was up-regulated by induction of the quiescent phenotype. A wild-type E1A, which interferes with endogenous p300, but not a mutant E1A defective for p300 binding, markedly down-regulated the expression of endogenous Sm-MHC in quiescent-phenotype VSMCs. These studies provide the first identification of a functionally important GATA-6 binding site within a smooth muscle-specific promoter and suggest a role for p300 in the maintenance of the differentiated phenotype in VSMCs as a coactivator of GATA-6.

Adenovirus E1A Proteins↗
PubMed2000

Characterization of novel and identified genes in guinea pig organ of corti.

A number of proteins are expressed in the organ of Corti and are considered to be responsible for hearing. However, most of them have not been identified. Therefore, to achieve a better understanding of the genetic factors influencing these traits, the first step is to characterize the genes expressed in the organ of Corti. In the present study, a cDNA library was constructed from the guinea pig organ of Corti. After sequencing isolated clones, 196 expressed sequence tags (ESTs) were identified with FASTA analysis: 65 ESTs showed significant sequence homology to previously identified genes in guinea pig, human or other species, and 131 ESTs showed no significant matches to sequences already present in the DNA database DDBJ/GenBank/EMBL. A variety of matching sequences, some of which were known to be cochlea-specific, were found through FASTA analysis of the 65 clones. RT-PCR with a panel of 10 different tissue mRNA revealed the restricted expression of 13 unknown clones. The results of our analysis allowed the establishment of a list of genes expressed in the guinea pig organ of Corti.

Animals↗
PubMed2000

A dominant-negative UBC12 mutant sequesters NEDD8 and inhibits NEDD8 conjugation in vivo.

NEDD8, a novel ubiquitin-like protein, has been shown to conjugate to proteins in a manner analogous to ubiquitination and sentrinization. Recently, human UBC12 was identified as a putative NEDD8 conjugation enzyme (E2). While investigating the in vivo function of UBC12, we found that the point mutant, UBC12(C111S), showed a dominant-negative effect on NEDD8 conjugation. This mutant, with a single Cys-to-Ser substitution at the conserved Cys residue in the E2 family, could specifically inhibit NEDD8 conjugation. We observed the dominant-negative effect on NEDD8 conjugation to substrates, including the C-terminal fragment of cullin-2 (Cul-2-DeltaN), full-length cullin-1, and also other uncharacterized target proteins. Interestingly, UBC12(C111S) formed a heterodimeric conjugate with NEDD8. This conjugate was stable under stringent conditions, including 6 m guanidine HCl, 8 m urea, 2% SDS, or 5% beta-mercaptoethanol. Our results are consistent with the hypothesis that UBC12(C111S) sequesters the NEDD8 monomer by forming a UBC12(C111S)-NEDD8 conjugate and, in turn, inhibits the subsequent transfer of NEDD8 to its targets. To examine the biological role of NEDD8 conjugation, this dominant-negative form of UBC12 was applied to a cell growth assay. Overexpression of UBC12(C111S) led to inhibition of growth in U2OS and HEK293 cells. Thus, this dominant-negative form of UBC12 could be useful in defining the role of NEDD8 modification in other biological systems.

Amino Acid Sequence↗
PubMed2000

Expression of polysialic acid and STX, a human polysialyltransferase, is correlated with tumor progression in non-small cell lung cancer.

Polysialic acid (PSA) is a carbohydrate composed of a linear homopolymer of alpha-2-8-linked sialic acid residues and is mainly attached to the neural cell adhesion molecule (NCAM). Because of the large negative charge of PSA, presence of PSA attenuates the adhesive property of NCAM and increases the cellular motility. PSA expression on NCAM is developmentally regulated, and PSA plays important roles in formation and remodeling of the neural system through regulation of the adhesive property of NCAM. Expression of the polysialated form of NCAM has been also demonstrated in some malignant tumors, such as Wilms' tumor and small cell lung cancer. Despite the possible importance as an onco-developmental antigen, however, significance of PSA expression in most malignant tumors has not been revealed. Therefore, PSA expression in non-small cell lung cancer was assessed in the present study. PSA was expressed only in 5 (20.8%) of 24 pathological stage I cases, whereas it was expressed in most stage IV cases (76.8%, 11 of 14 cases). PSA expression was correlated with nodal metastasis and distant metastasis, but not with local extent of the primary tumor. Next, expression of polysialyltransferase genes (PST and STX genes) which controlled formation of PSA, was examined. The PST gene was constantly expressed in both normal lung tissue and tumor tissue of all cases. In contrast, the STX gene was not expressed in normal lung tissue of any case, and STX gene expression in tumor tissue was closely correlated with tumor progression. The STX gene was expressed only in 1 (4.2%) of 24 stage I cases, whereas it was expressed in most stage IV cases (85.7%, 12 of 14 cases). These results suggested that the PSA and STX genes could be new targets of cancer therapy as well as important clinical markers.

Adult↗
PubMed2000

Reperfusion lung injury after cold preservation correlates with decreased levels of intrapulmonary high-energy phosphates.

BACKGROUND: To evaluate the role of energy state in primary graft dysfunction, which is crucial in lung transplantation, we investigated the relationship between intrapulmonary high-energy phosphate compounds and reperfusion lung injury after cold preservation. METHODS: Using an isolated rat lung perfusion model with fresh rat blood as perfusate, rat lungs were exposed to various cold preservation periods (0, 6, 9, and 12 hr) and reperfused. RESULTS: We found that extending the preservation period exacerbated the pulmonary hemodynamics after reperfusion. The levels of intrapulmonary high-energy phosphate compounds did not change during cold preservation, but these levels after reperfusion decreased as the preservation period was prolonged. The pulmonary hemodynamics after reperfusion were inversely correlated with the intrapulmonary high-energy phosphate compound levels after reperfusion. Total adenine nucleotide and ATP were sensitive indicators of reperfusion lung injury after cold preservation. Energy charge was not a sensitive indicator. The decreased levels of intrapulmonary high-energy phosphate compounds after reperfusion following cold preservation period were partially caused by their decreased production. CONCLUSIONS: These results demonstrated that reperfusion lung injury after cold preservation was closely correlated with decreased levels of intrapulmonary high-energy phosphate compounds after reperfusion, although the levels of the intrapulmonary high-energy phosphate compounds did not change during cold preservation of up to 12 hr.

Adenine Nucleotides↗
PubMed2000

Phosphorylation of GATA-4 is involved in alpha 1-adrenergic agonist-responsive transcription of the endothelin-1 gene in cardiac myocytes.

The expression of endothelin-1 (ET-1) in cardiac myocytes is markedly induced during the development of heart failure in vivo and by stimulation with the alpha(1)-adrenergic agonist phenylephrine in culture. Although recent studies have suggested a role for cardiac-specific zinc finger GATA factors in the transcriptional pathways that modulate cardiac hypertrophy, it is unknown whether these factors are also involved in cardiac ET-1 transcription and if so, how these factors are modulated during this process. Using transient transfection assays in primary cardiac myocytes from neonatal rats, we show here that the GATA element in the rat ET-1 promoter was required for phenylephrine-stimulated ET-1 transcription. Cardiac GATA-4 bound the ET-1 GATA element and activated the ET-1 promoter in a sequence-specific manner. Stimulation by phenylephrine caused serine phosphorylation of GATA-4 and increased its ability to bind the ET-1 GATA element. Inhibition of the extracellularly responsive kinase cascade with PD098059 blocked the phenylephrine-induced increase in the DNA binding ability and the phosphorylation of GATA-4. These findings demonstrate that serine phosphorylation of GATA-4 is involved in alpha(1)-adrenergic agonist-responsive transcription of the ET-1 gene in cardiac myocytes and that extracellularly responsive kinase 1/2 activation plays a role upstream of GATA-4.

Adrenergic alpha-1 Receptor Agonists↗
PubMed2000

Monitoring CD8 T cell responses to NY-ESO-1: correlation of humoral and cellular immune responses.

NY-ESO-1, a member of the cancer-testis family of antigens, is expressed in a subset of a broad range of different human tumor types. Patients with advanced NY-ESO-1-expressing tumors frequently develop humoral immunity to NY-ESO-1, and three HLA A2-restricted peptides were defined previously as targets for cytotoxic CD8(+) T cells in a melanoma patient with NY-ESO-1 antibody. The objectives of the present study were (i) to develop enzyme-linked immunospot (ELISPOT) and tetramer assays to measure CD8(+) T cell responses to NY-ESO-1, (ii) to determine the frequency of CD8(+) T cell responses to NY-ESO-1 in a series of HLA-A2 patients with NY-ESO-1 expressing tumors, (iii) to determine the relation between CD8(+) T cell and humoral immune responses to NY-ESO-1, and (iv) to compare results of NY-ESO-1 ELISPOT assays performed independently in two laboratories with T cells from the same patients. NY-ESO-1 ELISPOT and tetramer assays with excellent sensitivity, specificity, and reproducibility have been developed and found to correlate with cytotoxicity assays. CD8(+) T cell responses to HLA-A2-restricted NY-ESO-1 peptides were detected in 10 of 11 patients with NY-ESO-1 antibody, but not in patients lacking antibody or in patients with NY-ESO-1-negative tumors. The results of ELISPOT assays were concordant in the two laboratories, providing the basis for standardized monitoring of T cell responses in patients receiving NY-ESO-1 vaccines.

Antibody Formation↗
PubMed2000

Purification and characterization of bovine cone arrestin (cArr).

To elucidate the quenching mechanism of phototransduction in vertebrate cone photoreceptors, a cDNA clone encoding cone specific arrestin (cArr) was isolated from a bovine retinal cDNA library using a human cArr cDNA probe. Affinity-purified anti-peptide antibody specific to cArr was prepared. Immunohistochemical staining displayed specific labeling of cArr in cone photoreceptors and immunoblotting identified a 46 kDa protein band. We purified cArr from bovine retinas by sequential column chromatography using DEAE-cellulose, gel filtration and mono Q columns. Binding studies revealed no binding of cArr to rhodopsin regardless of whether it was bleached and/or phosphorylated. cArr also failed to bind to heparin-Sepharose under conditions which rod arrestin (rArr) bound to the column. The present data suggest that cArr may play a role in the quenching of phototransduction in cone photoreceptors and that its activity therein is different to that of rArr.

Amino Acid Sequence↗
PubMed2000

Prognostic significance of cyclin E overexpression in resected non-small cell lung cancer.

Cyclin E plays a pivotal role in the regulation of G1-S transition and relates to malignant transformation of the cells. However, the clinical significance of cyclin E expression in patients with non-small cell lung cancer remains unknown. We examined the expression of cyclin E in 242 resected non-small cell lung cancer in pathological stages I-IIIa and analyzed its relation to clinicopathological factors. Cylin E overexpressions were observed frequently in deeply invasive tumors. Multivariate analysis revealed that complete resection, pathological stage, and cyclin E expression were independent prognostic indicators. When cyclin E and proliferating cell nuclear antigen are combined, the cases negative for both had a significantly better prognosis than the other cases. We concluded that cyclin E overexpression relates to deeply invasive tumors and is correlated with poor prognosis. New therapeutic options may be provided by combination of cyclin E expression and proliferating cell nuclear antigen overexpression.

Adenocarcinoma↗
PubMed2000

FHIT alterations in cancerous and non-cancerous cervical epithelium.

We have reported a significant frequency of an alteration of the fragile histidine triad (fhit) gene in squamous-cell carcinoma of the uterine cervix (series 1). To further define the role of fhit alteration in the development of cervical carcinoma, we surveyed 36 normal cervical epithelium, 22 cervical intra-epithelial neoplasias (CINs) and 20 additional cases of invasive cervical carcinomas (series 2). fhit transcripts were analyzed using reverse-transcription-polymerase-chain-reaction amplification and sequencing. Loss of expression of fhit was observed in 14 of 48 (29%) invasive carcinomas (8/28, series 1; 6/20, series 2) but not in any normal squamous epithelia or CINs analyzed. Abnormal fhit transcripts, including deletions and/or insertions, were observed in 12 of 48 (25%) invasive carcinomas (9/28, series 1; 3/20, series 2), 6 of 22 (27%) CINs, and 10 of 40 (25%) normal squamous epithelia (0/4, series 1; 10/36, series 2). Point mutation was detected in 9 of 48 (19%) cervical carcinomas (8/28, series 1; 1/20, series 2). Inactivation in both alleles was observed in 18 of 48 cervical carcinomas (38%), but not in any of 22 CINs or 40 normal squamous epithelia. Loss or impaired expression of the fhit-gene product was detected in 13 of 30 (43%) cervical carcinomas by immunohistochemistry, whereas all 6 normal cervical epithelia, or 22 CINs, expressed fhit protein. There was a strong association of impaired fhit protein expression with the disruption of normal fhit transcript in cervical carcinoma. No apparent correlation was observed between fhit inactivation and HPV infection. Our results suggest that fhit-gene inactivation occurs, not as an initiating event, but rather as a later event in cervical carcinogenesis, when the cervical tumor has acquired an invasive character.

Acid Anhydride Hydrolases↗
PubMed2000

Human thioredoxin attenuates hypoxia-reoxygenation injury of murine endothelial cells in a thiol-free condition.

The adult T cell leukemia-derived factor (ADF), or human thioredoxin (hTRX), has a radical scavenging effect similar to that of N-acetyl cysteine (NAC). We have recently shown that ADF/hTRX protects the lung and the heart from ischemia-reperfusion induced injury. To elucidate mechanisms of the protective effect, a hypoxia-reoxygenation (H-R) injury model was developed using a murine endothelial cell line, cultured in a thiol-free medium. In this condition, cells became much more vulnerable to H-R injury. The viability of cells decreased significantly after 1 h of hypoxic incubation followed by 1 h of reoxygenation. The injury was reduced by ADF/hTRX (100 microM) or NAC (10 mM). These two agents also demonstrated an additive protective effect. When cells were cultured in thiol-free medium for 2 h in a normoxic condition, intracellular hydrogen peroxide production was increased, which was associated with a decrease in glutathione level. NAC (10 mM) attenuated these changes whereas ADF/hTRX (100 microM) did not. These results suggest that although both ADF/hTRX and NAC protected cells from H-R injury, the underlying mechanisms are different. Because the cytoprotective effect of ADF/hTRX occurs in the thiol-free condition, it must be mediated via a novel mechanism other than enhancing thiol uptake. The additive cytoprotective effect between ADF/hTRX and NAC suggests that we should combine these two agents clinically.

Acetylcysteine↗
PubMed2000

Decreased tissue factor and tissue-plasminogen activator antigen in relapsed acute promyelocytic leukemia.

This study evaluated hemostatic data in 28 patients with newly diagnosed acute promyelocytic leukemia (APL) and 15 patients with relapsed APL. Activated partial thromboplastin time and prothrombin time were prolonged at initial onset of APL. Plasma level of fibrinogen was significantly decreased in patients with initial disease of APL, but it was not decreased significantly during the relapse of APL. Plasma fibrin and fibrinogen degradation products levels were significantly increased and platelet counts significantly decreased in both groups. Plasma levels of antiplasmin significantly decreased at initial onset but not during relapse. Plasma levels of antithrombin were within normal range in patients with initial disease but significantly decreased in those with relapse. Plasma levels of D-dimer, soluble fibrin monomer (sFM), plasmin-plasmin inhibitor complex (PPIC), and thrombin antithrombin complex (TAT) levels were significantly high in both groups. Plasma levels of PPIC, sFM, and D-dimer were significantly higher at initial onset of APL than during relapse. However, there was no significant difference in DIC score between patients with initial onset and those with relapse; plasma levels of tissue factor (TF) significantly increased in both groups, but they were significantly higher at initial onset of APL than during relapse. TF and tissue type plasminogen activator (t-PA) antigen levels in leukemic cell lysate were significantly increased in both groups, and they were significantly lower during relapse than at initial onset. Hemostatic abnormalities occurring in patients with relapsed APL might be the result of the decrease of TF and t-PA in leukemic cells. These findings suggest that DIC in APL patients with relapse might not be caused only by TF and t-PA and thus should be treated with different therapy from patients with initial onset of APL.

Adolescent↗
PubMed2000

Plasma levels of activated protein C-protein C inhibitor complex in patients with hypercoagulable states.

Plasma levels of activated protein C (APC)-protein C inhibitor (PCI) were significantly increased in patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), acute myocardial infarction (AMI), pulmonary embolism (PE), or deep vein thrombosis (DVT) and in patients undergoing hemodialysis (HD). Plasma levels of APC-alpha(1)-antitrypsin (AT) complex were significantly increased in patients with DIC and in those with TTP. Plasma levels of PCI were significantly decreased in patients with DIC, non-DIC, or TTP and in those undergoing HD. In the pre-DIC stage, the plasma levels of APC-PCI complex were significantly increased but not those of APC-alpha(1)-AT complex. These data suggest that measurements of APC-PCI complex and APC-alpha(1)-AT complex may be useful for the diagnosis of DIC. After treatment of DIC, the plasma levels of APC-PCI complex and APC-alpha(1)-AT complex were significantly decreased, but not those of PCI. Plasma levels of thrombin-antithrombin complex (TAT), plasmin-alpha(2)-plasmin complex (PPIC), D-dimer, and soluble fibrin monomer (SFM) were markedly increased in patients with DIC or pre-DIC and were moderately increased in patients with non-DIC, TTP, AMI, PE, or DVT and in those undergoing HD. The receiving operating characteristic (ROC) analysis showed that SFM and the APC-PCT complex are useful markers for diagnosis of DIC. The specificity of plasma TAT and PPIC levels was low. The positive rate of APC-PCI complex was higher than 90% with DIC, TTP, AMI, PE, and it was higher than 60% with DVT and HD. Since the APC-PCI complex was elevated not only in patients with venous thrombosis but also in those with arterial thrombosis, components of the protein C pathway might be useful markers for the diagnosis of arterial thrombosis.

Antithrombin III↗
PubMed2000

Increased plasma levels of tissue factor pathway inhibitor-activated factor X complex in patients with disseminated intravascular coagulation.

Plasma levels of tissue factor pathway inhibitor (TFPI)-activated factor Xa (FXa) complex were measured in patients with disseminated intravascular coagulation (DIC), pre-DIC, and DIC. Plasma levels of plasmin-plasmin inhibitor complex (PPIC), D-dimer, and soluble fibrin monomer (SFM) were significantly higher in patients with DIC than in those with pre-DIC or non-DIC; the levels of these hemostatic markers were significantly higher in patients with pre-DIC than in those with non-DIC. Plasma levels of thrombin-antithrombin complex (TAT) were significantly higher in patients with DIC or pre-DIC than in those with non-DIC. Plasma levels of tissue factor (TF), total TFPI, free TFPI, and TFPI-Xa complex were significantly higher in patients with DIC than in those with non-DIC. Plasma levels of TFPI-Xa complex were significantly increased in patients with pre-DIC as compared to those with non-DIC; however, plasma free TFPI levels were significantly decreased in patients with pre-DIC as compared to those with non-DIC. These findings suggest that free TFPI might be consumed in the pre-DIC state, thereby confirming the activation of the extrinsic pathway. Plasma levels of TFPI-Xa complex were significantly correlated with TF, free TFPI, and total TFPI. Increased plasma TFPI-Xa complex levels might be useful for the diagnosis of DIC or pre-DIC, particularly that occurring by activation of the extrinsic pathway of blood coagulation.

Anticoagulants↗