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H Wada

Publications and source records attributed to H Wada.

At least 361 records · Page 20Linked to original sources
PubMed1997

Regulation of myosin phosphatase through phosphorylation of the myosin-binding subunit in platelet activation.

Human platelets were found to contain myosin phosphatase consisting of a 38-kD catalytic subunit of protein phosphatase type 1delta, a 130-kD myosin-binding subunit (MBS) and a 20-kD subunit, all of which cross-reacted with antibodies against these subunits of smooth muscle myosin phosphatase. Anti-MBS antibody coimmunoprecipitated RhoA and Rho-kinase of human platelets. Platelets MBS is a substrate for Rho-kinase and phosphorylation of MBS decreases the activity of myosin phosphatase. Treatment of intact platelets with 9, 11-epithio-11,12-methano-thromboxane A2 led to a dramatic increase in phosphorylation of MBS and a significant decrease in the activity of myosin phosphatase. These findings suggest a putative mechanism for agonist-induced regulation of myosin phosphatase activity in platelets.

Binding Sites↗
PubMed1997

T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice.

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF -/- mice. To investigate the responses of CD8(+) and CD4+ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8+ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF -/- mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF -/- mice. Purified CD4+ T cells from GM-CSF -/- mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-gamma (IFN-gamma) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4+ T cells from GM-CSF -/- mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-gamma and IL-4. To analyze the rescue effect of DC on CD4(+) T cells, supernatants from (i) CD4(+) T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4(+) T cells and KLH-pulsed spleen cells from GM-CSF -/- mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-gamma production of CD4(+) T cells from GM-CSF -/- mice.

Animals↗
PubMed1997

Clonal analysis of high-grade squamous intra-epithelial lesions of the uterine cervix.

We previously reported that invasive squamous cell carcinomas of the uterine cervix are of monoclonal composition. In the current study, we extended our previous work to determine the clonal composition of cases of high-grade squamous intra-epithelial lesion (HSIL). Clonal analysis targeting the HUMARA locus was performed on cervical tissue from 9 cases, 8 showing heterozygosity at the HUMARA locus and being, therefore, informative for clonality analysis. Uterine cervices were cut into 12 blocks, fixed with formalin and embedded in paraffin, and DNA was extracted from targeted lesions of each block. A total of 30 samples of cervical intra-epithelial neoplasia 3 (CIN3) (14 samples of carcinoma in situ and 16 samples of severe dysplasia) and 1 sample of CIN2 (moderate dysplasia) were analyzed. Monoclonal composition of the lesions was demonstrated in 30/30 cases of CIN3. Polyclonal composition was seen in the single case of CIN2. In 6 uterine cervices, in which dysplastic lesions were present in more than 3 blocks, the pattern of X-chromosome inactivation was the same in all lesions, suggesting that these individual lesions were derived from a single cell, with intraepithelial extension within the cervical mucosa. By contrast, one uterus contained 2 discontinuous dysplastic foci with different patterns of X-chromosome inactivation, indicating that the 2 lesions developed independently from each other. Our results demonstrate that (i) lesions of CIN3 (severe dysplasia and carcinoma in situ) are composed of a clonal neoplastic population of cells and (ii) most cases of HSIL are unifocal in origin.

Carcinoma in Situ↗
PubMed1997

Dilazep, an antiplatelet agent, inhibits tissue factor expression in endothelial cells and monocytes.

Dilazep, an antiplatelet agent, is generally used as an antithrombotic drug in clinical practice. Dilazep is also known to exert cytoprotective and antioxidant effects on endothelial cells. However, its effect on the endothelial or monocyte procoagulant activity is unknown. In the current study, the effect of dilazep on the expression of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) after the stimulation with tumor necrosis factor-alpha (TNF), thrombin, or phorbol 12-myristate 13-acetate (PMA) was evaluated. We also evaluated the effect of dilazep on TNF (1,000 U/mL)-induced TF expression on monocytes. Dilazep inhibited TF activity induced on HUVECs by each stimulant, TNF (1000 U/mL), thrombin (25 nmol/L), or PMA (5 nmol/L) in a dose-dependent fashion (1 to 100 microg/mL). TF activity decreased to approximately 10% after treating with 100 microg/mL of dilazep. Dilazep also blocked the expression of TF antigen induced by each stimulant on the surface of HUVECs as determined by flow cytometric analysis. In addition, in HUVECs, it significantly decreased the expression of TF mRNA and the total TF antigen induced by thrombin or PMA, but not those induced by TNF, suggesting that dilazep blocks the TF expression induced by PMA or thrombin at a transcriptional level and that induced by TNF at a posttranscriptional level. Western blot analysis showed that dilazep reduces the accumulation of native TF but increases that in lower molecular weight TF derivatives. The adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline, partially counteracted the anticoagulant activity of dilazep on HUVECs, thereby suggesting that the inhibitory effect of dilazep on TF expression in HUVECs depends, at least in part, on its adenosine potentiating activity. Dilazep also inhibited TNF-induced TF expression on monocytes in a dose-dependent fashion (0.1 to 100 microg/mL). In brief, the current study showed for the first time that dilazep, a commonly used antiplatelet drug, strongly inhibits the TF expression in HUVECs and monocytes. Dilazep may have a potent therapeutic value in patients with hypercoagulable state for its inhibitory property on the procoagulant activity of endothelial cells and monocytes.

Adenosine↗
PubMed1997

Characterization of tumor necrosis factor-deficient mice.

Although tumor necrosis factor (TNF) initially came to prominence because of its anti-tumor activity, most attention is now focused on its proinflammatory actions. TNF appears to play a critical role in both early and late events involved in inflammation, from localizing the noxious agent and amplifying the cellular and mediator responses at the local site and systemically, to editing (e.g., apoptosis) injured cells or effete immune cells and repairing inflammatory damage. We have generated mice deficient in TNF (TNF-/- mice) and have begun to examine the multiple functions attributed to TNF. TNF-/- mice develop normally and have no gross structural or morphological abnormalities. As predicted, they are highly susceptible to challenge with an infectious agent (Candida albicans), are resistant to the lethality of minute doses of lipopolysaccharide (LPS) following D-galactosamine treatment, have a deficiency in granuloma development, and do not form germinal centers after immunization. Phagocytic activity of macrophages appears relatively normal, as do T cell functions, as measured by proliferation, cytokine release, and cytotoxicity. B cell response to thymus-independent antigens is normal, but the Ig response to thymus-dependent antigen is reduced. Surprisingly, cytokine production induced by LPS appears essentially intact, with the exception of reduced colony-stimulating factor activity. Other unexpected findings coming from our initial analysis are as follows. (i) TNF has low toxicity in TNF-/- mice. (ii) TNF-/- mice show an anomalous late response to heat-killed Corynebacterium parvum. In contrast to the prompt response (granuloma formation, hepatosplenomegaly) and subsequent resolution phase in C. parvum-injected TNF+/+ mice, similarly treated TNF-/- mice show little or no initial response, but then develop a vigorous, disorganized inflammatory response leading to death. These results suggest that TNF has an essential homeostatic role in limiting the extent and duration of an inflammatory process-i.e., an anti-inflammatory function. (iii) In contrast to the expectation that TNF+/+ mice and TNF+/- mice would have identical phenotypes, TNF+/- mice showed increased susceptibility to high-dose LPS lethality, increased susceptibility to Candida challenge, and delayed resolution of the C. parvum-induced inflammatory process, indicating a strong gene dose requirement for different actions of TNF.

Animals↗
PubMed1997

The effects of lactate and beta-hydroxybutyrate on the energy metabolism and neural activity of hippocampal slices from adult and immature rat.

We investigated the correlation between energy metabolism and neural activity during glucose deprivation and during replacement of glucose with lactate and beta-hydroxybutyrate (OHBA) in neural tissue from rats of different ages. Hippocampal slices were prepared from 4-, 7-, 10-, 13- and 16-day-old and adult rats. The population spikes (PS) were recorded in the pyramidal cell layer of the CA3 area as the index of neural activity. ATP and creatine-phosphate (CrP) levels in each slice were determined during glucose deprivation and during replacement of glucose with lactate or OHBA. After deprivation of glucose, the PS of the slices from 4-, 7- and 10-day-old and adult rats decayed and extinguished in 30 min and the decay time was shortened according to the age of the rat. The levels of ATP and CrP in the slices also decreased, but to a lesser extent than the amplitudes of PS. After substitution of lactate or beta-hydroxybutyrate (OHBA) for glucose, PS of the adult rat disappeared as was the case with glucose deprivation, although the levels of high energy phosphates were well maintained. In the case of the immature rat, however, PS decayed more slowly. Especially in the case of 4-day-old rat, ATP and CrP in the slices were maintained as high as those under the initial concentrations and PS amplitude showed no decay even after 60 min. These results indicate that the presence of glucose is essential for neural activity in the adult rat, and lactate or OHBA cannot replace it for the maintenance of neural activity. In the immature rat, glucose metabolites such as lactate and OHBA are available for both neural activity as well as maintaining the levels of high-energy phosphates in the tissue slice.

3-Hydroxybutyric Acid↗
PubMed1997

Neural tube is partially dorsalized by overexpression of HrPax-37: the ascidian homologue of Pax-3 and Pax-7.

The origin and elaboration of the central nervous system played an important role in chordate and vertebrate history. All chordates possess a dorsal tubular central nervous system, but elaboration of dorsoventral and segmental pattern is far more pronounced in cephalochordates and vertebrates than in the more basal urochordates. Analysis of the urochordates, therefore, should allow deduction of the neural organization and neuronal patterning mechanisms that predated overt dorsoventral and segmental complexity. Here we report functional studies of the ascidian Pax gene (HrPax-37). The spatiotemporal expression pattern of HrPax-37 has suggested involvement in two distinct developmental processes: specification of dorsal cell fates of ectoderm during neurulation, and regional differentiation of the neural tube in later stages. Here we show that HrPax-37 is descendent from the precursor of the Pax-3 and Pax-7 genes implicated in specification of dorsal fate in the vertebrate neural tube. We also demonstrate that injection of HrPax-37 RNA into fertilized eggs causes ectopic expression of the dorsal neural marker tyrosinase gene in neurulae, confirming a regulatory role in dorsal patterning of the neural tube comparable to its vertebrate homologues. These results suggest that dorsal specification in the neural tube by Pax-3/7 subfamily genes was established in the ancestors of extant chordates during emergence of the dorsal tubular nervous system.

Amino Acid Sequence↗
PubMed1997

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002.

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C18 fatty acids increased in cells grown at lower temperature for all lipid classes, and omega 3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the omega 3 position of C18 fatty acids increased gradually during a 12-h period after a temperature shift-down to 22 degrees C. However, the mRNA levels of the desA (delta 12 desaturase), desB (omega 3 desaturase) and desC (delta 9 desaturase) genes increased within 15 min after a temperature shift-down to 22 degrees C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38 degrees C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22 degrees C and 38 degrees C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.

Cold Temperature↗
PubMed1997

A novel monoclonal antibody which reacts with a high molecular weight neuronal cytoplasmic protein and myelin basic protein (MBP) in a patient with macroglobulinemia.

We report on the case of a 70-year-old man with primary macroglobulinemia who showed cranial polyneuropathy and extensive radiculoneuropathy. His serum contained an IgM lambda monoclonal antibody which reacted with both a high molecular weight protein in grey matter and purified myelin basic protein (MBP) on immunoblotting. In an immunohistochemical study, strong immunoreactivity was detected only in the cytoplasm of neurons and weak immunoreactivity was detected in myelin. These findings suggest that this antibody may be related to the pathogenesis of neuronal damage in patients with macroglobulinemia.

Aged↗
PubMed1997

A patient with paroxysmal nocturnal hemoglobinuria bearing four independent PIG-A mutant clones.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by clonal blood cells that are deficient in the surface expression of glycosylphosphatidylinositol-anchored proteins due to somatic mutation in the X-linked gene PIG-A. In some patients, more than one abnormal clone may be present. Analysis of bulk DNA/RNA from granulocytes has been useful in identifying the predominant PIG-A mutation in each patient. However, it is often not useful in determining the presence of minor clones. Many patients have cells with partial deficiency. Here, we analyzed the PIG-A gene in two B-cell lines bearing complete or partial deficiencies, cells of hematopoietic progenitor colonies and peripheral blood granulocytes from the same patient. We found that two B-cell lines had different mutations, the granulocytes contained at least two mutants, and the hematopoietic progenitors contained four mutants. Three of the four were shared by B cells and/or granulocytes whereas the other one was found only in the hematopoietic progenitors. The partial deficiency was caused by a point mutation near an alternative splice site within exon 2 that resulted in partial decreases of activity and quantity of the full-length transcript. These results further show the oligoclonal nature of PNH and differences in extent of expansion among mutant clones.

Alternative Splicing↗
PubMed1997

Alteration of p16 and p15 genes in common epithelial ovarian tumors.

We have examined the roles of 2 putative tumor-suppressor genes, the p16 and p15 inhibitor-of-cyclin-dependent-kinase genes, in the most commonly occurring epithelial tumors of the human ovary. Expression of p16 mRNA, examined by RT-PCR, was significantly reduced in 15 of the 48 tumors. Aberrant expression of p16 protein, detected by immunohistochemistry, occurred in 22 of 60 tumors, more frequently in low-grade tumors, and had significant correlation with low p16 mRNA expression. Hypermethylation of a site within the 5'-CpG island of the p16 gene was significantly associated with loss of p16 mRNA and protein expression. Homozygous gene deletion, evaluated by differential PCR analysis, was found in 2 tumors for the p16 gene and in 1 tumor for the p15 gene among 70 ovarian tumors examined. PCR-SSCP analysis detected point mutations in p16 in 4 tumors and in p15 in 1 tumor. One was a 38-bp deletion, from codons 48 to 60, in a mucinous tumor of low malignant potential; another was a non-sense mutation in codon 60 in a mucinous adenocarcinoma. The remaining 2 mutations were mis-sense mutations, one in codon 58 and the other in codon 60, in 2 endometrioid adenocarcinomas. We conclude that inactivation of p16, by loss of p16 mRNA and protein expression as a consequence of hypermethylation of the 5'-CpG island, rather than by gene deletion or point mutation, may play an important role in the genesis of human ovarian epithelial tumors.

Actins↗
PubMed1997

Why do mitochondria synthesize fatty acids? Evidence for involvement in lipoic acid production.

The function of acyl carrier protein (ACP) in mitochondria isolated from pea leaves has been investigated. When pea leaf mitochondria were labeled with [2-14C] malonic acid in vitro, radioactivity was incorporated into fatty acids, and, simultaneously, ACP was acylated. [1-14C]Acetate was much less effective as a precursor for fatty acid synthesis, suggesting that mitochondria do not possess acetyl-CoA carboxylase. The incorporation of radioactivity from [2-14C]malonate into fatty acids and the labeling of ACP were inhibited by cerulenin and required ATP and Mg2+. These findings indicate that plant mitochondria contain not only ACP, but all enzymes required for de novo fatty acid synthesis. Over 30% of the radioactive products from pea mitochondria labeled with [2-14C]malonate were recovered in H protein, which is a subunit of glycine decarboxylase and contains lipoic acid as an essential constituent. In similar experiments, the H protein of Neurospora mitochondria was also labeled by [2-14C]malonate. The labeling of pea H protein was inhibited by addition of cerulenin into the assay medium. Together, these findings indicate that ACP is involved in the de novo synthesis of fatty acids in plant mitochondria and that a major function of this pathway is production of lipoic acid precursors.

Acyl Carrier Protein↗
PubMed1997

Plasma-activated factor VII level in patients positive for lupus anticoagulant.

We examined plasma levels of activated factor VII (F VIla) in 50 patients positive for lupus anticoagulant (LA), in 83 patients negative for LA, and in 10 healthy volunteers as controls. Plasma F VIIa was present in healthy volunteers; its level was significantly increased, compared to the level in the controls, in patients with thrombosis, collagen diseases, and disseminated intravascular coagulation (DIC), suggesting that it reflected a thrombotic state. Plasma F VIIa was correlated with thrombin-antithrombin complex (TAT) in patients negative for LA but showed no such correlation in those positive for LA. Plasma F VIIa was negatively correlated with activated partial thromboplastin time (APTT) in patients positive for LA, but not in those negative for LA, suggesting that LA could inhibit the F VIIa assay system. Plasma F VIIa level was significantly increased in patients with thrombotic diseases; however, in patients positive for LA, it is possible that increased plasma F VIIa level may not be correlated with thrombogenicity.

Collagen Diseases↗
PubMed1997

Plasma tissue factor and tissue factor pathway inhibitor levels in patients with disseminated intravascular coagulation.

We measured the plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in patients with disseminated intravascular coagulation (DIC) to examine the relationship between TFPI and vascular endothelial cell injury. Plasma TF (273 +/- 90 pg/ml) and TFPI (252 +/- 125 ng/ml) levels were significantly increased in patients with DIC compared with non-DIC patients. Plasma TF antigen level was significantly increased in pre-DIC patients (285 +/- 85 pg/ml), while the plasma TFPI level (152 +/- 54 ng/ml) was not markedly increased in such a state. The plasma TF/TFPI ratio was high in the pre-DIC patients (2.10 +/- 0.90), and low in the DIC patients (1.40 +/- 0.87) and healthy volunteers (0.84 +/- 0.26). There was no significant difference between the DIC patients with a good outcome and those with a poor outcome in terms of plasma TF levels, although the plasma TFPI level in the DIC patients with a good outcome (289 +/- 133 ng/ml) was significantly higher than those with a poor outcome (187 +/- 75 ng/ml). During the clinical course of DIC, plasma TF antigen was increased first, and an increase of the plasma TFPI level followed the increase in plasma TF level. These findings suggest that plasma TFPI is released from vascular endothelial cells and it may reflect vascular endothelial cell injury. It is conceivable that TF and TFPI may play an important role in the onset of DIC.

Anticoagulants↗
PubMed1997

Increased tissue factor pathway inhibitor in patients with acute myocardial infarction.

We examined hemostatic abnormalities in 23 patients with acute myocardial infarction (AMI), 10 with pulmonary embolism (PE), and 10 with deep vein thrombosis (DVT). At the onset of AMI, plasma levels of tissue-type plasminogen activator (t-PA), PA inhibitor-I (PAI-I), fibrin-D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PPIC) were significantly increased. Both the plasma total TFPI and free-TFPI levels in the AMI patients were significantly higher than those in the healthy volunteers, PE patients, and DVT patients. There was no significant difference in total TFPI or free-TFPI among patients with PE, those with DVT, and healthy volunteers. One hour after percutaneous transluminal coronary angioplasty (PTCA) in the AMI group, the total TFPI level was further increased, and it was significantly reduced 24 hr after PTCA, to a level similar to that in healthy volunteers. Free-TFPI showed a pattern similar to that of total TFPI. The ratio of free-TFPI/total TFPI was highest 1 hr after PTCA. Increased TFPI in AMI patients might be released from ischemic tissues.

Adult↗