Biomedical subjects

T Sugimura

Publications and source records attributed to T Sugimura.

At least 91 records · Page 5Linked to original sources

PubMed1999

Regression and prevention of autochthonous tumors induced by 3-methylcholanthrene after injection of a T-cell receptor alpha /beta positive and CD4/CD8 double negative T-cells.

Both the therapeutic and preventative effects of a murine T-cell line, tMK-2, with T-cell receptor (TCR) alpha/beta positive and CD4-/8- double negative (DN) phenotype against autochthonously tumors induced by subcutaneous (s.c.) injection of 3-methylcholanthrene (MC) were examined. Complete regression of the tumor was observed when administration of tMK-2 cells was begun on tumors 5 mm in diameter. The tumor mass in five out of five mice was reduced in size after the administration of tMK-2 cells regardless of the routes of administration: s.c. injection of tMK-2 cells (5 x 10(7) cells) once a week around tumors, intraperitoneal (i.p.) injection (5 x 10(7) cells), or intravenous (i.v.) injection (1 x 10(7) cells). The tumors regressed to the status of a scar within 1 month of initial injection, and this status was maintained throughout the remainder of the 3 months period of tMK-2 cell injection. One month after discontinuation of tMK-2 cell administration, the diameter of the tumors had not increased regardless of the route of injection. The control groups consisted of either untreated mice, mice with i.v. injection of 1 microg of recombinant murine interleukin (IL)-12 once a week, or mice with s.c. injection of autologous splenocytes (5 x 10(7)) from BALB/c mice once a week. Continuous growth of tumors was observed in each group and all control mice died due to bleeding ulcerations of the tumors. Tumor development was effectively prevented when tMK-2 cells were administrated 1 week after the s.c. injection of MC. In the groups receiving s.c., i.p., and i.v. injection of tMK-2 cells, no MC-induced tumors developed, whereas four out of five of the control mice developed autochthonous tumors. The tMK-2 cells also exerted in vitro NK-like cytotoxic activity, and their killing activity was strongly increased in the presence of both IL-2 and IL-12. These results suggest that the injected T-cells with TCR alpha/beta positive and CD4- /8- DN phenotype and NK-like activity are important in the therapy as well as the prevention of tumor development.

Animals↗

PubMed1999

Genetic analysis of the susceptibility in rats to aberrant crypt foci formation by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine, PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine produced while cooking fish and meat, induces aberrant crypt foci (ACF) and colon cancers in rats. We previously reported that F344 rats were sensitive and ACI rats resistant to ACF formation by PhIP, and that the genetic susceptibility in F344 rats to ACF formation by PhIP was autosomally dominant over ACI rats. To identify candidate susceptibility genes in F344 rats, a preliminary genome-wide linkage analysis was employed using a subset of 170 progeny of (F344 x ACI)F1 x ACI backcross rats with either high or low sensitivity to ACF formation by PhIP. Three chromosomes, 1, 6 and 16, demonstrated the presence of loci with a logarithm of the odds (lod) scores of more than 1.0, and a susceptible gene for ACF formation by PhIP was suggested to reside on chromosomes 16.

Animals↗

PubMed1999

Carcinogenicity of heterocyclic amines for the pancreatic duct epithelium in hamsters.

Effects of eight heterocyclic amines (HCAs) on pancreatic duct carcinogenesis were investigated in a rapid production model in hamsters. N-Nitrosobis(2-oxopropyl)amine (BOP) was given to effect initiation, followed by augmentation pressure consisting of four daily i.p. injections of 500 mg/kg DL-ethionine, a choline-deficient (CD) diet, a single i.p. injection of 800 mg/kg L-methionine and a s.c. injection of 20 mg/kg BOP. After two cycles of augmentation pressure, the HCAs 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) or 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [4,8-DiMeIQx) were administered in the diet for 50 or 70 days. The numbers of pancreatic ductal hyperplasias (H) and a total lesions including, atypical hyperplasia (AH), carcinomas in situ (CIS) and invasive carcinomas, were increased in hamsters given the diet containing 0.02% Trp-P-1 for 50 days. This result was confirmed and extended by the finding of increased numbers of invasive carcinomas in hamsters given 0.02% Trp-P-1 for 70 days. The number and incidence of invasive carcinomas were also elevated in hamsters given the diet containing 0.06% 4,8-DiMeIQx for 50 days. These results suggest a possible involvement of Trp-P-1 and 4,8-DiMeIQx in pancreatic duct carcinogenesis.

Animals↗

PubMed1999

Quantification of the co-mutagenic beta-carbolines, norharman and harman, in cigarette smoke condensates and cooked foods.

Co-mutagenic beta-carbolines, such as norharman and harman, were quantified in mainstream and sidestream smoke condensates of six Japanese brands of cigarettes, and also in 13 kinds of cooked foods, using a combination of blue cotton treatment and HPLC. Norharman and harman were detected in all the cigarette smoke condensate samples. Their levels in the mainstream smoke case were 900-4240 ng per cigarette for norharman, and 360-2240 ng for harman, and in sidestream smoke, 4130-8990 ng for norharman and 2100-3000 ng for harman. These beta-carbolines were also found to be present in all the cooked food samples, at levels of 2.39-795 ng for norharman and 0.62-377 ng for harman per gram of cooked food. The observed concentrations are much higher than those found for mutagenic and carcinogenic heterocyclic amines (HCAs), suggesting that humans are exposed to norharman and harman in daily life to a larger extent than to HCAs.

Carbolines↗

PubMed1999

Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro.

It has been suggested that DNA-dependent protein kinase (DNA-PK) is a central component of DNA double-strand-break repair. The mechanism of DNA-PK action, however, has not been fully understood. Poly(ADP-ribose) polymerase (PARP) is another nuclear enzyme which has high affinity to DNA ends. In this study, we analysed the interaction between these two enzymes. First, DNA-PK was found to suppress the PARP activity and alters the pattern of poly(ADP-ribosyl)ation. Although DNA-PK phosphorylates PARP in a DNA-dependent manner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP. Conversely, PARP was found to ADP-ribosylate DNA-PK in vitro. However, the auto-phosphorylation activity of DNA-PK was not influenced by this modification. In a competitive electrophoretic mobility shift assay, Ku 70/80 complex, the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP. Furthermore, co-immunoprecipitation analysis suggested direct or close association between Ku and PARP. Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or sequestration of DNA-ends which serve as an important stimulator for both enzymes.

Antigens, Nuclear↗

PubMed1999

Identification of receptor genes in renal cell carcinoma associated with angiogenesis by differential hybridization technique.

To screen the receptor genes in renal cell carcinoma (RCC) associated with angiogenesis, we performed differential hybridization of the cDNA library of membrane-type protein tyrosine kinases (mPTKs). Three thousand plaques of a mPTKs-enriched cDNA library were screened with mPTKs mixture probes produced from hypervascular RCC tissues and RCC cell lines. Six different cDNA fragments of the PTK genes were isolated, and the sequence analysis showed that these represented cDNAs for TIE1, KDR, FMS, FGFR-4, JAK1 and HCK. Of these genes, the expression of TIE1, KDR, and FGFR-4 was studied in RCC tissue and cell lines by Northern blot analysis. We also investigated the expression of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptor FLT-1. In all the hypervascular RCC tissues, the amounts of mRNAs for KDR and FLT-1 were increased compared to adjacent normal tissues. The TIE1 and FGFR-4 genes were also overexpressed in most of the hypervascular RCC tissues, while no mRNA of KDR, FLT-1, or TIE1 could be detected in any of the four human RCC cell lines. The amounts of the VEGF and PlGF mRNAs were increased in hypervascular RCC tissues, while VEGF mRNA was detected in the four cell lines but PlGF mRNA was not. FGFR-4 mRNA was expressed in three of the four cell lines. These results suggest that KDR, FLT-1, PlGF and TIE1 mRNAs are present in the mesenchymal cells of RCC, while VEGF and FGFR-4 genes are expressed in RCC cells themselves in vivo.

Blotting, Northern↗

PubMed1999

Cytotoxic activity of pierisin, from the cabbage butterfly, Pieris rapae, in various human cancer cell lines.

Pierisin, a protein purified from pupae of the cabbage butterfly, Pieris rapae, exhibits cytotoxic effects against the human gastric cancer TMK-1 cell line, inducing apoptosis. The present study was performed to determine whether pierisin might exert a similar influence on nine other human cancer cell lines and human umbilical vein endothelial cells (HUVECs). Pierisin showed cytotoxic effects in all the human cells tested, with IC50 values ranging from 0.043 ng/ml to 150 ng/ml. Among the target cells, the cervical carcinoma cell line, HeLa, was the most sensitive to pierisin, showing a 1000-fold less IC50 value than that of HUVECs. While pierisin clearly induced apoptotic cell death in most cancer cell lines and HUVECs, the pathway appeared to be probably different from that involving anti-Fas, TNF-alpha and p53. Pierisin may thus be a promising new candidate for cancer therapy.

ADP Ribose Transferases↗

PubMed1999

Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes.

Streptozotocin (STZ), a glucose analogue known to induce diabetes in experimental animals, causes DNA strand breaks and subsequent activation of poly(ADPribose) polymerase (Parp). Because Parp uses NAD as a substrate, extensive DNA damage will result in reduction of cellular NAD level. In fact, STZ induces NAD depletion and cell death in isolated pancreatic islets in vitro. Activation of Parp therefore is thought to play an important role in STZ-induced diabetes. In the present study, we established Parp-deficient (Parp-/-) mice by disrupting Parp exon 1 by using the homologous recombination technique. These mice were used to examine the possible involvement of Parp in STZ-induced beta-cell damage in vivo. The wild-type (Parp+/+) mice showed significant increases in blood glucose concentration from 129 mg/dl to 218, 370, 477, and 452 mg/dl on experimental days 1, 7, 21, and 60, respectively, after a single injection of 180 mg STZ/kg body weight. In contrast, the concentration of blood glucose in Parp-/- mice remained normal up to day 7, slightly increased on day 21, but returned to normal levels on day 60. STZ injection caused extensive necrosis in the islets of Parp+/+ mice on day 1, with subsequent progressive islet atrophy and loss of functional beta cells from day 7. In contrast, the extent of islet beta-cell death and dysfunction was markedly less in Parp-/- mice. Our findings clearly implicate Parp activation in islet beta-cell damage and glucose intolerance induced by STZ in vivo.

Animals↗

PubMed1999

Development of the arbitrarily primed-representational difference analysis method and chromosomal mapping of isolated high throughput rat genetic markers.

Linkage mapping of quantitative trait loci requires analysis of a large number of animals. Although genetic markers isolated by representational difference analysis (RDA) and its modifications meet the needs, the number of these markers has been limited. In the present study, we established the arbitrarily primed (AP)-RDA method to isolate virtually an unlimited number of the high throughput genetic markers. A representation of the genome, an AP-amplicon, was prepared by AP-PCR with a single primer or with a combination of primers using genomic DNA of the ACI/N (ACI) or BUF/Nac (BUF) rat as a template. By subtracting the AP-amplicon of ACI from that of BUF, a total of 40 polymorphic and independent markers were isolated in seven series of AP-RDA using a single primer. Two series of AP-RDA with primer combination yielded seven additional independent markers. All of the markers gave clear positive/negative signals by hybridization of a filter where AP-amplicons from F2 rats of ACI and BUF were dot-blotted at a high density without any concentration or purification. All of the 47 independent markers were mapped to unique chromosomal positions by linkage analysis, even though some arbitrary primers had very similar sequences. The markers were also informative between other strains of rats. Simultaneous hybridization of multiple filters made it possible to genotype a large number of rats simultaneously for multiple genetic loci. The AP-RDA method promises isolation of a large number of high throughput genetic markers in any species and is expected to facilitate linkage mapping of subtle quantitative trait loci.

Animals↗

PubMed1999

Frequent mutations of the rat beta-catenin gene in colon cancers induced by methylazoxymethanol acetate plus 1-hydroxyanthraquinone.

Recent evidence suggests that the beta-catenin gene (CTNNB1) acts as an oncogene, and some human colon tumors with an intact APC gene have activating mutations in CTNNB1. In this study, mutations in the region corresponding to N-terminal phosphorylation sites (codons 1-51) of the rat Ctnnb1 gene were investigated in 20 colon tumors associated with ulcerative colitis and induced with methylazoxymethanol acetate and 1-hydroxyanthraquinone. Ninety percent (18 of 20) of the tumors induced in male F344 rats harbored mutations, which were detected in three of four adenomas (75%) and 15 of 16 adenocarcinomas (94%). Of 18 total missense mutations, 13 (72%) were G-->A transitions at position 101, three were G-->A transitions at position 94, and two were C-->T transitions at position 122, resulting in the amino acid substitutions Gly34-->Glu, Asp32-->Asn, and Thr41-->Ile, respectively. Although there were no mutations in the Apc gene, as we previously reported in the same tumor samples, the results obtained in this study strongly implicate the Apc-beta-catenin-T-cell factor (Tcf) signaling pathway in methylazoxymethanol acetate, 1-hydroxyanthraquinone-induced colon carcinogenesis.

Adenocarcinoma↗

PubMed1999

Rare mutations of p53, Ki-ras, and beta-catenin genes and absence of K-sam and c-erbB-2 amplification in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach cancers.

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) have been widely used as a model for human stomach cancers of the differentiated type. However, there has been little information regarding their molecular basis. In this study, we examined the genetic alterations reported in human stomach cancers in 10 rat stomach cancers that had been induced in male ACI/N rats by administering MNNG in the drinking water. One of the 10 cancers had a mutation of the p53 gene at the second position of codon 171 (Val --> Glu). However, none of the 10 cancers had mutations in codons 12, 13, or 61 of Ki-ras or in the N-terminal phosphorylation sites of the beta-catenin gene. Southern blot analysis showed no amplification of K-sam or c-erbB-2 in the seven cancers examined. Finally, we searched for microsatellite alterations in 12 loci in nine cancers, but no alterations were observed. As these genetic alterations are observed in only a minor fraction of human stomach cancers, further analysis of genetic and epigenetic alterations in MNNG-induced rat stomach cancers is needed to disclose the major mechanisms of stomach carcinogenesis.

Animals↗

PubMed1999

Functional and phenotypical characteristics of hepatic NK-like T cells in NK1.1-positive and -negative mouse strains.

We previously reported and partially characterized a unique monoclonal antibody (mAb), U5A2-13, which recognizes a T cell subset similar to NK1.1+ T cells, not only in NK1.1-positive mouse strains but also in NK1.1-negative strains. In NK1.1-positive C57BL/6 mice, U5A2-13+ TCRalphabeta+ cells produced abundant IL-4 as well as extremely high levels of IFN-gamma upon CD3 cross-linking, but this did not occur with U5A2-13- TCRalphabeta+ cells. In NK1.1-negative C3H/He mice, U5A2-13+ TCRalphabeta+ cells produced high levels of IL-4 and IFN-gamma upon CD3 cross-linking, but this was not observed with U5A2-13- TCRalphabeta+ cells. To the best of our knowledge, this is the first direct evidence of the presence of NK-like T cells defined phenotypically by U5A2-13 mAb and functionally by IL-4/IFN-gamma production in NK1.1-negative mouse strains. We also demonstrated that U5A2-13- NK1.1+ T cells and U5A2-13+ NK1.1- T cells in C57BL/6 mice could produce both IL-4 and IFN-gamma. In addition, Vbeta8 or Vbeta7 usage by U5A2-13+ NK1.1- T cells was lower than that by U5A2-13+ NK1.1+ T cells, but remained higher than that by U5A2-13- NK1.1- T cells. Based on the present results, U5A2-13 mAb appears to be a valuable tool in the study of NK-like T cells.

Animals↗

PubMed1999

Motor coordination in a multi-muscle system as revealed by principal components analysis of electromyographic variation.

The variation in electromyographic output of twelve trigeminal muscles of the rabbit was studied to test the hypothesis that they are under the control of a small number of independent neural factors. Jaw muscle electromyograms (EMGs) of eight animals were recorded in 95 chewing sequences, each consisting of 40-75 chewing cycles. The within-sequence correlations of the EMG burst amplitudes (integrated per cycle) and burst onsets were calculated between the muscles. The correlation matrix was subjected to a principal components analysis. This method aims at describing the variation in EMG amplitude and timing by means of the smallest possible set of newly defined variables, or principal components. Of the variation in EMG amplitude values of the twelve muscles, 75-90% could be accounted for by only three principal components. Each principal component was characterized by a group of muscles with high mutual positive correlations; they had zero correlation with other principal components. The first component represents the jaw closers: most of the bilateral masseter and the medial pterygoid muscles. The second represents the openers: the bilateral digastric and lateral pterygoid muscles. This demonstrates the tight control of both the jaw openers and closers, each by a single neural factor; these two factors are independent of one another. They most likely originate from the specific inputs from primary afferents to the opener and closer motoneurons. Unexpectedly, a third independent principal component appeared to control the closing activity of the non-chewing side, posterior deep masseter muscle. It was hypothesized that this muscle acts independently of the other closers to disengage the teeth and resets the jaw for a new chewing cycle. Principal components analysis of variation in timing of EMG onset revealed a grouping of all masticatory muscles in a single cluster, independent of EMG amplitude. This supports the hypothesis that timing and amplitude of masticatory EMG patterns are controlled independently.

Animals↗

PubMed1999

Selective high dose gamma-globulin treatment in Kawasaki disease: assessment of clinical aspects and cost effectiveness.

BACKGROUND: High-dose intravenous gamma-globulin (IVGG) plus aspirin (ASA) treatment is effective in preventing coronary artery complications in acute Kawasaki disease (KD). However, gamma-globulin is very expensive, especially in Japan. Furthermore the indication for IVGG treatment and the optimal dose of gamma-globulin remain controversial. OBJECTIVES: To examine these two issues, we used Harada's scoring system to investigate whether a single 2 g/kg dose therapy has any advantage over the 5 day 400 mg/kg per day therapy. METHODS: We studied 203 patients with KD who had no coronary artery complications on admission. Of these, 145 patients scored 4 or more on Harada score within the first 9 days of illness and were treated with IVGG treatment. Using a random number table, 72 patients were selected to receive a single 2 g/kg dose (2 g group), while the remaining 73 patients were treated with 400 mg/kg per day for 5 consecutive days (400 mg group). Those who had a Harada score of three or less received no IVGG (non-IVGG group) treatment (58 patients). RESULTS: The incidence rate of coronary artery complications in the 2 g group was significantly lower than in the 400 mg group. The duration of high fever, positive duration of C-reactive protein and the number of hospital days in the 2 g group were each significantly shorter than in the 400 mg group. The total medical expense in the 2 g group was significantly lower than in the 400 mg group. There were no coronary artery complications in the non-IVGG group. CONCLUSIONS: It was found to be clinically more effective and more cost effective to select a patient by Harada's scoring system and, where a score of four or more was obtained, to administer a single 2 g/kg intravenous dose of gamma-globulin for acute KD.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Preferential induction of guanine deletion at 5'-GGGA-3' in rat mammary glands by 2-amino- 1-methyl-6-phenylimidazo[4,5-b]pyridine.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is known to induce a characteristic mutation, G deletion at the 5'-GGGA-3' site, preferentially in the lacI transgene of the colonic mucosa of Big Blue((R)) rats (BBR) and mice and specifically in the Apc gene of rat colon tumors. In this study, lacI mutations of the mammary glands in PhIP-treated rats were investigated. Six-week-old female (BBRxSprague-Dawley)F(1) rats were administered 10 gavages of 65 mg/kg/day PhIP. Mammary ducts were collected from the macroscopically normal mammary tissue of PhIP-treated and untreated rats at 56-69 weeks of age by collagenase treatment. The mutant frequencies were 25 +/- 2.1x10(-6) in control rats and 323 +/- 44x10(-6) in the PhIP-treated rats. By sequencing 40 and 177 mutants in the control and PhIP-treated groups, respectively, 34 and 149 mutations were considered independent mutations. In the control group, G:C-->A:T transitions at CpG sites dominated and no G:C deletions were detected. In the PhIP-treated group, G:C-->T:A transversions were most frequent (43%), followed by single base pair deletions of G:C (21%). A total of nine deletions were at 5'-GGGA-3' sites, accounting for 29% of the G:C deletions and 6% of the 149 total mutations. Clusters of more than three mutations at one nucleotide position were observed at 12 positions and two were G deletions at 5'-GGGA-3' sites. Comparison of the PhIP-induced mutations in the mammary glands with those previously reported in the colon revealed that G:C-->T:A transversions occurred at a significantly higher frequency in the mammary glands and that G:C deletions occurred at a significantly lower frequency. However, the signature mutation, G deletion at the 5'-GGGA-3' site, was commonly observed in both tissues.

Animals↗

PubMed1999

Strain differences of rats in the susceptibility to aberrant crypt foci formation by 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine: no implication of Apc and Pla2g2a genetic polymorphisms in differential susceptibility.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine contained in cooked food, induces colon tumors in F344 male rats when administered orally. In the present study, PhIP was introduced to various rat strains, and susceptibility to the induction of aberrant crypt foci (ACFs) was analyzed as a biomarker for colon carcinogenesis. BUF/Nac rats were highly susceptible, giving rise to 12.2 +/- 1.7 ACFs per rat. F344 rats were intermediate and ACI/N rats were resistant, giving 3.5 +/- 1.8 and 0.9 +/- 0.7 ACFs per rat, respectively. In spite of this, the extent of DNA damage by PhIP in F344, in terms of the level of PhIP-DNA adducts, was significantly lower than that in ACI/N. The differences in formation of ACFs could be, in some part, implicated in the differential susceptibility to colon carcinogenesis induced by PhIP, especially in a step later than adduct formation. In an attempt to determine the genetic factors implicated in the susceptibility to formation of ACFs, a possible involvement of the adenomatous polyposis gene (Apc) and its modifier secretory phospholipase A2 (Pla2g2a) was analyzed. No genetic polymorphisms in either Apc or Pla2g2a showed a significant correlation to susceptibility to formation of ACFs among rat strains.

Animals↗

PubMed1999

A new modification of the 32P-post-labeling method to recover IQ-DNA adducts as mononucleotides.

To obtain accurate estimates of DNA adduct levels yielded by genotoxic compounds, it is essential to completely digest adducted nucleotides to mononucleotides. We previously developed a suitable method, called modified method I, to obtain DNA adducts of heterocyclic amines as 32P-labeled-mononucleoside adduct 5'-phosphate forms, by use of nuclease P1 (NP1) and phosphodiesterase I (PDEI) to digest adducted oligonucleotides. In this study, we applied method I to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA adduct analysis and found that one of the IQ-DNA adducts, 5-(deoxyguanosin-N2-yI)-2-amino-3-methylimidazo[4,5-f]quinoline 3',5'-diphosphate (pdGp-N2-IQ), was resistant to the 3'-phosphatase activity of NP1, but sensitive to that of T4 polynucleotide kinase (PNK). DNA obtained from the liver of rats fed IQ was 32P-labeled by the standard method and the 32P-labeled nucleotides obtained were incubated with PNK and NP1 to remove 3'-phosphate groups and then digested with PDEI. Three spots were obtained. One major spot was identified as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 5'-phosphate (pdG-C8-IQ) and a second abundant adduct as pdG-N2-IQ. The third spot, of which the structure is unknown, was minor. The new method is called modified method II. Modified method II could be applicable to a wide variety of chemicals.

Animals↗

PubMed1999

Isolation and cloning of rat poly(ADP-ribose) glycohydrolase: presence of a potential nuclear export signal conserved in mammalian orthologs.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme of poly(ADP-ribose) degradation. To understand its structure-and-function relationship, we purified Parg from rat testis 9,740-fold using an improved affinity column; the purified product was a 60 kDa protein. Based on the determined sequences of three peptide fragments, degenerated primers were synthesized and a Parg cDNA comprising 3,974 nucleotides, encoding a 109 kDa protein, was isolated. The 60 kDa Parg purified from rat testes corresponded to the C-terminal half of the 109 kDa deduced peptide. When recombinant rat Parg was expressed as a glutathione S-transferase fusion protein in Escherichia coli, Parg activity was observed for the full-length and C-terminal half proteins but not in for the N-terminal half protein. Taken together, these data indicate that the catalytic domain of Parg is located in the C-terminal half. Further, we newly identified the presence of a potential nuclear export signal in the N-terminal half in addition to the previously reported nuclear localization signals in rat and other mammalian Pargs. Northern blot analysis showed the ubiquitous expression of a single 4.0 kb Parg mRNA in various rat tissues. The findings suggest that the 60 kDa Parg is produced by post-transcriptional processing.

Amino Acid Sequence↗