Biomedical subjects

T Kusano

Publications and source records attributed to T Kusano.

At least 19 recordsLinked to original sources

PubMed2000

Specific binding of a 14-3-3 protein to autophosphorylated WPK4, an SNF1-related wheat protein kinase, and to WPK4-phosphorylated nitrate reductase.

WPK4 is a wheat protein kinase related to the yeast protein kinase SNF1, which plays a role in catabolite repression. To identify proteins involved in signal transduction through WPK4, we performed yeast two-hybrid screens and isolated two cDNA clones designated as TaWIN1 and TaWIN2. Both encode 14-3-3 proteins that, upon autophosphorylation, bind the C-terminal regulatory domain of WPK4. Mutational analysis through amino acid substitution revealed that TaWIN1 and TaWIN2 primarily bind WPK4 through phosphoserines at the positions 388 and 418, both located in the C-terminal region. Mutations in the conserved residues of the TaWIN1 amphipathic groove impaired the ability of TaWIN1 to bind to WPK4. A screen for in vitro phosphorylation of proteins involved in nutrient metabolism revealed a putative WPK4 substrate, nitrate reductase; its hinge 1 region was efficiently phosphorylated by WPK4. Subsequent far Western blots showed that it specifically bound TaWIN1. Since nitrate reductase has been shown to be inactivated by phosphorylation upon 14-3-3 binding, the present findings strongly suggest that WPK4 is the protein kinase responsible for controlling the nitrogen metabolic pathway, assembling the nitrate reductase and 14-3-3 complex through its phosphorylation specificity.

14-3-3 Proteins↗

PubMed2000

Expression of ZmMET1, a gene encoding a DNA methyltransferase from maize, is associated not only with DNA replication in actively proliferating cells, but also with altered DNA methylation status in cold-stressed quiescent cells.

A cDNA fragment encoding part of a DNA methyltransferase was isolated from maize. The putative amino acid sequence identically matched that deduced from a genomic sequence in the database (accession no. AF063403), and the corresponding gene was designated as ZmMET1. Bacterially expressed ZmMET1 actively methylated DNA in vitro. Transcripts of ZmMET1 could be shown to exclusively accumulate in actively proliferating cells of the meristems of mesocotyls and root apices, suggesting ZmMET1 expression to be associated with DNA replication. This was confirmed by simultaneous decrease of transcripts of ZmMET1 and histone H3, a marker for DNA replication, in seedlings exposed to wounding, desiccation and salinity, all of which suppress cell division. Cold stress also depressed both transcripts in root tissues. In contrast, however, accumulation of ZmMET1 transcripts in shoot mesocotyls was not affected by cold stress, whereas those for H3 sharply decreased. Such a differential accumulation of ZmMET1 transcripts was consistent with ZmMET1 protein levels as revealed by western blotting. Expression of ZmMET1 is thus coexistent, but not completely dependent on DNA replication. Southern hybridization analysis with a methylation-sensitive restriction enzyme revealed that cold treatment induced demethylation of DNA in the Ac/Ds transposon region, but not in other genes, and that such demethylation primarily occurred in roots. These results suggested that the methylation level was decreased selectively by cold treatment, and that ZmMET1 may, at least partly, prevent such demethylation.

Blotting, Southern↗

PubMed2000

Rice gibberellin-insensitive gene homolog, OsGAI, encodes a nuclear-localized protein capable of gene activation at transcriptional level.

This paper reports isolation and properties of a rice gene, OsGAI, a putative homolog of the GAI of Arabidopsis thaliana. OsGAI encodes a polypeptide of 625 amino acids, which shows 53-55% identity to GAI and RGA from A. thaliana, and 85% identity to wheat rht-D1a and maize d8. Genomic DNA blot analysis indicated the OsGAI to be a single-copy gene in the rice genome. RNA blot hybridization showed that OsGAI transcripts increased within 6h upon GA(3) but not ABA application. This GA-induced increment in OsGAI transcripts did not require de novo protein synthesis. High levels of OsGAI transcripts were detected in nodes, internodes, leaf sheaths and ears of adult plants and leaf sheaths of young seedlings, where GA enhances cell elongation and division. Transiently expressed OsGAI-GFP fusion protein located to the nucleus in onion epidermal cells. Transactivation assays clearly indicated that OsGAI protein is a transcriptional activator or a coactivator.

Amino Acid Sequence↗

PubMed2000

Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding.

An immediate-early, transiently activated wound-responsive gene was identified in tobacco by fluorescent differential display screening. The full-length cDNA encodes a polypeptide of 356 amino acids with a relative molecular mass of 39,082 Da. The deduced amino acid sequence shows two characteristic features; a leucine-zipper motif found in the more N-terminal region and a WRKY domain containing a zinc-finger motif located in the central region. The gene was designated as wizz (wound-induced leucine zipper zinc finger). Northern analysis showed that upon wounding wizz transcripts were locally and systemically accumulated within 10 min, reached a maximum level by 30 min, and decreased thereafter to the basal level. Analyses of a WIZZ-GFP fusion protein clearly indicated that WIZZ is a nuclear factor. WIZZ specifically binds to sequences containing two TTGAC core motifs that are separated by a spacer of appropriate length. The binding activity was dependent on bivalent cations, most probably zinc. In transient reporter assays, however, WIZZ did not show transactivation activity in tobacco suspension cells, suggesting that it functions together with other components. The results indicate that WIZZ is a new transcription factor which participates in early stages of the wound response.

Amino Acid Sequence↗

PubMed2000

Diverse response of rice and maize genes encoding homologs of WPK4, an SNF1-related protein kinase from wheat, to light, nutrients, low temperature and cytokinins.

The wheat gene WPK4 encodes a 56-kDa protein kinase that belongs to group 3 of the SNF1-related protein kinase family (SnRK3), and is up-regulated by light and cytokinins and down-regulated by sucrose. In order to determine whether or not this particular regulation pattern is general among plant species, we isolated and characterized homologous genes from rice and maize. Two rice genes, OsPK4 and OsPK7, encode proteins comprising 508 and 520 amino acids, and show, respectively, 75% and 76% sequence similarity to WPK4. OsPK4 and OsPK7 proteins produced in Escherichia coli were able to phosphorylate themselves and myelin basic proteins, the reaction requiring magnesium and/or manganese ions. Transcripts of OsPK4 were detected in all tissues tested, and amounts were increased upon illumination, nutrient deprivation and treatment with cytokinins. In contrast, transcripts of OsPK7 were not found in any tissues except in mature leaves at low levels, and did not accumulate under any of the stress conditions examined. A maize gene, ZmPK4, encodes a protein with 518 amino acids that shows 74% similarity to WPK4. Its transcripts were constitutively expressed in all tissues, regardless of light, nutrient and cytokinin status, but were increased upon exposure to low temperature. These results indicate that, despite the sequence similarity between their products, genes for SnRK3 proteins are differentially regulated in response to environmental stimuli.

Amino Acid Sequence↗

PubMed2000

Secreted proteins of tobacco cultured BY2 cells: identification of a new member of pathogenesis-related proteins.

Cultured cells of tobacco BY2 secrete more than 100 proteins into culture medium. Six major proteins were purified, and partial protein sequences were determined. Five of them were found to be similar to an ascorbic acid oxidase, three peroxidase isozymes and a beta-1,3-exoglucanase, respectively. A cDNA clone encoding the remaining polypeptide, whose amino acid sequence showed no similarity with earlier reported proteins, was isolated. It encoded a putative 27 kDa protein of 242 amino acids with resemblance to WCI-5, a wheat protein induced by benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) which activates genes involved in systemic acquired resistance. Transcripts of this clone accumulated upon tobacco mosaic virus infection, mechanical wounding and drought treatment, an induction profile that satisfies the definition of pathogenesis-related (PR) proteins by van Loon et al. (Plant Mol. Biol. Rep. 12 (1994) 245). No similar PR proteins have so far been reported, and therefore our newly designated NtPRp27 points to the existence of a novel PR protein family in tobacco plants.

Abscisic Acid↗

PubMed2000

A tobacco NtMET1 cDNA encoding a DNA methyltransferase: molecular characterization and abnormal phenotypes of transgenic tobacco plants.

A cDNA encoding a DNA methyltransferase, with a predicted polypeptide of 1556 amino acid residues containing all motifs conserved in this enzyme family, was isolated from tobacco plants, and the corresponding gene was designated as NtMET1. RNA blot analysis indicated NtMET1 transcripts to accumulate in dividing tissues of tobacco plants, and they could be detected during the S phase in synchronized dividing BY2 cells. In situ hybridization revealed the transcripts to be localized exclusively in actively proliferating tissues around axillary apical meristem. In order to ascertain physiological roles, transgenic tobacco plants that had the antisense construct were made and examined for phenotypes. Methylation levels of genomic DNA from transgenic plants significantly decreased in comparison with wild-type levels, and distinct phenotypic changes including small leaves, short internodes and abnormal flower morphology were noted. Microscopic observation revealed that leaf structure differed between transgenic and wild-type plants. These results suggest that NtMET1 functions during DNA replication, and that DNA methylation plays an important role in plant morphogenesis.

Cell Cycle↗

PubMed2000

Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase.

Three cDNA clones encoding putative cysteine synthases (O-acetylserine (thiol) lyase, EC 4.2.99.8) were isolated from Arabidopsis thaliana and designated AtcysC1, AtcysD1 and AtcysD2, respectively. Southern blot analyses suggested that the corresponding genes were present as a single copy, or at most two copies, in the A. thaliana genome. Escherichia coli complementation analyses confirmed that the cDNAs encode cysteine synthase and the corresponding proteins produced in E. coli clearly showed cysteine synthase activity. In addition, AtcysC1 protein showed beta-cyanoalanine synthase (EC 4.4.1.9) activity, but the other two did not. Kinetic analysis suggests that AtcysC1 actually functions as beta-cyanoalanine synthase rather than cysteine synthase in vivo. The mRNA accumulation of AtcysC1, AtcysD1 and AtcysD2 differed in various organs, but did not change markedly when A. thaliana seedlings were subjected to various stresses, including nutrient deprivation. In vivo targeting experiments indicated that AtcysD1 and AtcysD2 are cytoplasmic isozymes, and AtcysC1 is a mitochondrial isozyme.

Amino Acid Sequence↗

PubMed2000

Screening of wound-responsive genes identifies an immediate-early expressed gene encoding a highly charged protein in mechanically wounded tobacco plants.

In order to identify genes that are temporally and spatially regulated during wound response, a cDNA population in mechanically wounded tobacco leaves was screened by the fluorescence differential display method. Of 28 clones initially identified to have altered levels of transcripts within 3 h of wounding, eight were characterized. Although each clone showed a unique pattern of transcript accumulation, one distinct clone was further characterized because of its immediate-early response. Its transcripts began to accumulate 10 min after wounding, reached a maximum level within 1 h and disappeared after 2 h. The response, which occurred repeatably and systemically, was observed by the treatment with propionic acid or erythrosin B, indicating that cytosolic acidification could be one of the signals for immediate-early response of this gene. The cDNA encodes a polypeptide of 513 amino acids with a relative molecular mass of 60,952. The putative polypeptide is rich in lysine (K), glutamic acid (E) and aspartic acid (D), which constitute up to 70% of total amino acids, and was therefore designated as KED. The KED polypeptide is composed of a highly hydrophilic N-terminal region and a relatively hydrophobic C-terminal region, suggesting that KED may function through electrostatic interactions with cellular components.

Amino Acid Sequence↗

PubMed2000

Prostaglandin E(1) continuous hepatic arterial infusion in the treatment of postoperative acute liver failure: basic study on hepatic hemodynamics and clinical application.

AIM: In the treatment of severe liver damage, it is of greater advantage to administer prostaglandin E(1) (PGE(1)) directly to the liver compared with systemic intravenous infusion, because of its high inactivation rate in the lungs. In comparison with intraportal infusion, hepatic arterial infusion is more advantageous because of its easier and safer accessibility. This study was designed to prove the superiority of hepatic arterial infusion to intravenous infusion. METHODS: Changes in hepatic hemodynamics and oxygen delivery accompanying PGE(1) infusion using both methods were investigated in pigs. In addition, continuous hepatic arterial infusion was applied in 3 cases of postoperative acute liver failure, for patients in whom other conventional treatments like plasma exchange failed to improve the functioning of the liver. RESULTS: Hepatic arterial flow increased significantly accompanying hepatic arterial infusion of PGE(1) at a rate of 0.1 microg/kg/min compared with intravenous infusion at the same rate in pigs. Such an increase resulted in elevation of total hepatic blood flow and oxygen delivery to the liver. Correspondingly, bile flow significantly increased accompanying hepatic arterial infusion of PGE(1). Continuous hepatic arterial infusion was applied in 3 cases of postoperative acute liver failure. The infusion was continued for 7-9 days at a rate of 0.01 microg/kg/min without any complications through heparin-coated catheters inserted via the femoral artery. Significant increase in bile flow was observed in 2 cases in whom bile was collected, serum total bilirubin began to decrease in all these 3 cases, and the patients recovered from acute liver failure. CONCLUSION: Hepatic arterial infusion of PGE(1) is very useful and effective in the treatment of acute liver failure.

Aged↗

PubMed1999

Characteristics of hepatocellular carcinoma in patients with negative virus markers: clinicopathologic study of resected tumors.

Fifty-one cases of resected hepatocellular carcinoma (HCC) were retrospectively analyzed to evaluate the clinicopathologic features of HCC in patients with negative virus markers. The data were compared between three groups: hepatitis B surface antigen positive (HB, n = 11), hepatitis C virus antibody positive (HC, n = 21), and non-BC (both HbsAg and HCVAb negative, n = 12). Seven patients were excluded from the study because of operative death (n = 3), a history of alcohol abuse (n = 3), or the presence of dual positive HB and HC virus markers (n = 1). The data were analyzed by either an analysis of variance (ANOVA) or a contingency table. The age of the non-BC patients was higher (63.0 +/- 4.1, +/- SE) than that of HB patients (54.0 +/- 3.2, p < 0.05) but was identical to that of the HC group (62.0 +/- 1.8). Among the preoperative laboratory data, the serum glutamic oxaloacetate and glutamate pyruvate transaminoses (GOT, GPT) levels were statistically lower in the non-BC patients (32.8 +/- 4.8 and 28.0 +/- 4.4 IU/L, respectively) than in the HB and HC patients. The pathologic features of the resected specimens in the non-BC patients showed more invasive growth than in specimens from the HB or HC patients. The clinical stages (defined based on the criteria of the Japanese Association of Hepatocellular Carcinoma) were also more advanced in the non-BC patients than in the other groups. Postoperative survival time showed no significant difference among the groups. In conclusion, the non-BC patients had comparatively greater invasive growth and more advanced clinical stages than the HB and HC patients, despite the absence of liver cirrhosis, and so demonstrated the same poor survival data as observed in the HB and HC patients.

Adult↗

PubMed1999

Involvement of a MAP kinase, ZmMPK5, in senescence and recovery from low-temperature stress in maize.

Four species of protein kinase were identified in senescent maize leaves using a gel assay for kinase activity with myelin basic protein (MBP) as the substrate. Most of these kinases were also found in healthy green leaves that had been exposed to low-temperature stress (5 degrees C) and then returned to 25 degrees C. A 41-kDa protein was activated in senescent leaves, whereas a 45-kDa protein was activated 3 h after up-shift from 5 degrees C to 25 degrees C as well as in senescent leaves. A 39-kDa protein was activated by cold stress. The other two proteins, of 35 kDa and 52 kDa, constitutively phosphorylated MBP during senescence and temperature up-shift. Judging from their molecular masses, cation requirements and substrate specificities, it seemed likely that the 39-kDa, 41-kDa and 45-kDa proteins represented mitogen-activated protein kinases (MAPKs). Subsequently two MAPK cDNAs were isolated from a cDNA library constructed using mRNAs from senescent leaves. Northern analysis showed that the transcript corresponding to one of the cDNAs, designated ZmMPK5, accumulated in healthy leaves 3 h after the up-shift to 25 degrees C as well as in senescent leaves, suggesting that the 45-kDa protein kinase is encoded by ZmMPK5. Western analysis using an antiserum against the C-terminal region of ZmMPK5 showed that the level of the ZmMPK5 protein increased in senescent leaves. These results indicate that a 45-kDa MAPK is involved in the process of senescence and in recovery from low-temperature stress in maize plants.

Amino Acid Sequence↗

PubMed1999

Common bile duct blood clot: an unusual cause of ductal filling defects for calculi.

We report a case of obstructive jaundice caused by a blood clot in the common bile duct in a 75-year-old man with cirrhosis. Five years prior to his admission, he had undergone a left hepatectomy for hepatocellular carcinoma. At the present admission, he appeared icteric, and endoscopic retrograde cholangiography revealed filling defects in the common bile duct. Choledochotomy was therefore performed for possible common duct stones, and exploration of the duct showed blood clot casts filling the duct. The casts were easily removed, and the patient's postoperative course was uneventful. However, he developed ascites and jaundice 1 month later and died of liver failure approximately 3 months after undergoing the choledochotomy. Autopsy revealed hemorrhagic necrosis in the proximal intrahepatic duct of the posterior segment, which was considered to be the cause of the observed hemobilia, as well as the blood clot in the common bile duct at surgery. We report this rare case and discuss the cause of hemobilia.

Aged↗

PubMed1999

Gibberellin-responsive genes: high level of transcript accumulation in leaf sheath meristematic tissue from Zea mays L.

In order to identify genes that are related to the gibberellin (GA) response in maize (Zea mays L.), mRNA species from wild-type and single-gene dwarf mutants, d5 and D8, were compared by fluorescent differential display. The d5 mutant is unable to produce biologically active GA, but recovers its tall stature on exogenous application of GA. D8 is insensitive to GA, despite the accumulation of a high level of endogenous GA, suggesting it to be a receptor mutant or a mutant in signal transduction pathway(s). After screening 7000 cDNA populations, one clone was isolated, for which transcripts were rare in d5 shoots but accumulated within 1 h after GA3 application. This clone, designated as ZmGR1a, encodes a polypeptide with a relative molecular mass of ca. 13 kDa, which shows significant homology to proline-rich proteins from several plant species. A similar experiment with D8 identified a clone, ZmGR2a, with low transcript levels, but accumulation within 6 h after GA3 treatment of d5 shoots. ZmGR2a encodes a polypeptide with a relative molecular mass of ca. 19 kDa, which shows no significant homology with any known protein. Southern blot analysis indicated that ZmGR1a and ZmGR2a form a small multigene family within the maize genome. In situ hybridization with wild-type seedlings showed transcripts on both to be abundant in leaf sheath meristematic tissue, in which GA enhances cell elongation and cell division.

Amino Acid Sequence↗

PubMed1999

Microsatellite instability in double cancers of the esophagus and head and neck.

It is generally accepted that patients with squamous cancers of the esophagus are known to have a high risk of concomitant head and neck cancer. However, there have been only a few reports describing microsatellite instability (MSI) in patients with both esophageal squamous cell carcinoma and head and neck cancers. To evaluate the role of genetic instability in carcinogenesis in such patients, we analyzed six microsatellite loci in 21 tumors from 10 patients who had developed primary cancers of both the esophagus and the head and neck. MSI was detected in 6 out of 10 patients. In five patients with double cancer, MSI was observed at the same microsatellite loci in both the esophageal and the head and neck tumors obtained from the same individuals. These data suggest that such patients may have the same underlying defect in the mismatch repair system, providing insight into possible mechanisms for field carcinogenesis.

Carcinoma, Squamous Cell↗

PubMed1999

Use of Tc-99m DTPA galactosyl human serum albumin to predict postoperative residual liver function.

PURPOSE: Residual liver uptake at 15 minutes (RLU15), a new index for predicting residual liver function after excision of hepatocellular carcinoma, was evaluated using dynamic liver images and SPECT by Tc-99m DTPA galactosyl human serum albumin. MATERIALS AND METHODS: In 13 cases of hepatectomy, RLU15 was compared with postoperative serum prothrombin levels, serum bilirubin levels, and ascites. RESULTS: RLU15 showed good correlation with both the prothrombin activity and the serum bilirubin, with correlation coefficients of r = 0.829 and r = -0.757. CONCLUSION: This new index appears useful for predicting residual liver function after hepatectomy.

Ascites↗

PubMed1999

Sucrose and Cytokinin Modulation of WPK4, a Gene Encoding a SNF1-Related Protein Kinase from Wheat.

WPK4, a gene encoding a putative protein kinase, was initially identified in wheat (Triticum aestivum) and shown to be up-regulated by light, nutrient deprivation, and cytokinins. To confirm that WPK4 has protein kinase activity, the protein was produced in Escherichia coli as a fusion protein with glutathione S-transferase. The purified protein exhibited autophosphorylation activity and phosphorylated both myelin basic protein and a peptide fragment of rice 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Levels of WPK4 transcripts in wheat seedlings were increased and decreased by the removal and addition of sucrose (Suc), respectively, to the culture medium. The introduction of the N-terminal kinase region of WPK4 into the yeast snf1 mutant cells, which cannot utilize Suc as a carbon source, rescued growth in Suc-containing medium. Cytokinins up-regulated the accumulation of WPK4 transcripts, but their effects were cancelled by the addition of Suc. Our results suggest that Suc negatively regulates the signaling pathway in which transcriptional activation of WPK4 is mediated by cytokinins.

Journal Article↗

PubMed1999

Radiation-associated rectal cancer: report of four cases.

BACKGROUND/AIMS: Radiation-associated rectal cancer is a remarkable clinical entity. We demonstrate 4 patients (mean age 68 years, range 63-74) who had undergone pelvic radiotherapy for cervical cancer. We indicate some characteristics of radiation-associated rectal cancer. RESULTS: Two patients had received intracavitary and external pelvic radiotherapy, while the remaining 2 had external pelvic radiotherapy following hysterectomy. The mean total radiation dose was 63 Gy, though radiation dose information was not available for 1 patient. Colorectal cancer developed at a mean time of 20.7 years (range 11-30) after radiation therapy. All patients presented with chronic radiation colitis, and 3 demonstrated abnormal tumor markers. Colonoscopy revealed an ulcerative, localized well-differentiated adenocarcinoma of the rectosigmoid colon in 1 patient, and diffusely infiltrating cancers of the lower rectum, one signet-ring cell carcinoma and two mucinous carcinomas in the remaining 3. One case was stage I, 2 were stage IIIa, and the remaining case was stage IV. Three patients underwent abdominoperineal resection. The remaining patient was felt to be inoperable. The colorectal wall demonstrated the changes of chronic radiation injury. Two patients died within a short time because of their advanced cancers. CONCLUSION: Radiation-associated rectal cancer has a tendency to be diagnosed in the advanced stage and to have a poor prognosis. A literature review and our case report suggest that since there are no reliable clinical or laboratory indicators of the presence of a curable colorectal cancer in the setting of chronic radiation proctocolitis, surveillance with a colonoscope should be done 10 years after irradiation in patients with previous pelvic radiotherapy.

Aged↗