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Biomedical subjects

K Sakaguchi

Publications and source records attributed to K Sakaguchi.

At least 127 records · Page 7Linked to original sources
PubMed1998

Thyroid hormones are involved in insulin-like growth factor-I (IGF-I) production by stimulating hepatic growth hormone receptor (GHR) gene expression in the chicken.

Effect of thyroid status on IGF-I production in growing chickens was studied. Serum concentrations of GH were not affected by propylthiouracil (PTU) or thyroxine (T4) treatments, whereas serum IGF-I levels were significantly decreased in PTU-treated chickens. The lowered serum IGF-I levels in the PTU-treated group were completely restored to the control levels by T4 injections. In the liver, the messenger RNA (mRNA) expressions both for GH receptor (GHR) and IGF-I were significantly repressed by PTU treatment, and were restored again by T4 replacement. In addition, the results of analysis on radiolabelled GH binding to the liver membrane were consistent with the levels of hepatic GHR mRNA expression. Serum concentrations of IGF-I were positively correlated with hepatic IGF-I mRNA and GHR mRNA expressions. The correlation coefficient between serum T3 levels and hepatic IGF-I mRNA expressions was also significant. These results indicate that thyroid hormones regulate IGF-I production in the chicken by affecting hepatic GHR expression.

Animals↗
PubMed1998

An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli.

A cost-effective protocol for uniform 15N and/or 13C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.

Carbon Isotopes↗
PubMed1998

Inverse effects of 20K and 22K human growth hormones on the growth of T-47D human breast cancer cells in culture and in nude mice.

The major form of human growth hormone (22K hGH) stimulates the growth of T-47D human breast cancer cells in culture and in nude mice by binding to their receptors for growth hormone and prolactin. Another isoform of hGH having a smaller molecular mass (20K hGH) is known to show different binding affinities to these receptors. In this study, we have analyzed the effects of 20K hGH on the growth of T-47D cells in vitro and in vivo. 20K hGH (50 ng/ml) inhibited the proliferation and DNA synthesis of T-47D cells cultured in the presence and absence of 17 beta-estradiol (100 ng/ml), while 22K hGH (50 ng/ml) promoted the cellular growth. In estradiol-treated nude mice, 22K hGH (100 micrograms) remarkably promoted the growth of T-47D tumor, but 20K hGH again suppressed the tumor growth significantly. The results suggest the presence of different signal pathways for these two hGH isoforms and imply a possible clinical application for 20K hGH.

Animals↗
PubMed1998

Gene and cDNA structures of flounder insulin-like growth factor-I (IGF-I): multiple mRNA species encode a single short mature IGF-I.

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.

Amino Acid Sequence↗
PubMed1998

Permeability of gadolinium-DTPA through two types of hemodialysis membrane.

RATIONALE AND OBJECTIVES: Gadolinium diethylenetriamine penta-acetic acid (Gd-DTPA) was injected intravenously for magnetic resonance imaging in patients with impaired renal function, and the permeabilities of two types of general hemodialysis (HD) membrane to this agent were compared. METHODS: One of the HD membranes, Nipro FB-210-U membrane (U membrane), has a larger pore size than the other, Nipro FB-210-M membrane (M membrane). On the same day as magnetic resonance imaging was performed, the patient underwent a 4-hour HD session. Blood was sampled before and at five points after the start of HD. RESULTS: Mean dialysance of Gd-DTPA was 175.8 to 185.8 mL/min with the U membrane and 120.5 to 130.1 mL/min with the M membrane. The elimination rate was 91.1% with the U membrane and 81.1% with the M membrane 4 hours after the start of HD. The permeability was significantly higher with the U membrane than with the M membrane. CONCLUSION: Although it is more expensive, the U membrane should be chosen rather than the M membrane for HD to remove Gd-DTPA from the body as efficiently as possible.

Adult↗
PubMed1998

Phosphorylation of p53: a novel pathway for p53 inactivation in human T-cell lymphotropic virus type 1-transformed cells.

Inhibition of p53 function, through either mutation or interaction with viral or cellular transforming proteins, correlates strongly with the oncogenic potential. Only a small percentage of human T-cell lymphotropic virus type 1 (HTLV-1)-transformed cells carry p53 mutations, and mutated p53 genes have been found in only one-fourth of adult T-cell leukemia cases. In previous studies, we demonstrated that wild-type p53 is stabilized and transcriptionally inactive in HTLV-1-transformed cells. Further, the viral transcriptional activator Tax plays a role in both the stabilization and inactivation of p53 through a mechanism involving the first 52 amino acids of p53. Here we show for the first time that phosphorylation of p53 inactivates p53 by blocking its interaction with basal transcription factors. Using two-dimensional peptide mapping, we demonstrate that peptides corresponding to amino acids 1 to 19 and 387 to 393 are hyperphosphorylated in HTLV-1-transformed cells. Moreover, using antibodies specific for phosphorylated Ser15 and Ser392, we demonstrate increased phosphorylation of these amino acids. Since HTLV-1 p53 binds DNA in a sequence-specific manner but fails to interact with TFIID, we tested whether phosphorylation of the N terminus of p53 affected p53-TFIID interaction. Using biotinylated peptides, we show that phosphorylation of Ser15 alone inhibits p53-TFIID interaction. In contrast, phosphorylation at Ser15 and -37 restores TFIID binding and blocks MDM2 binding. Our studies provide evidence that HTLV-1 utilizes the posttranslational modification of p53 in vivo to inactivate function of the tumor suppressor protein.

Binding Sites↗
PubMed1998

Shc phosphotyrosine-binding domain dominantly interacts with epidermal growth factor receptors and mediates Ras activation in intact cells.

The adaptor protein Shc contains a phosphotyrosine binding (PTB) domain and a Src homology 2 (SH2) domain, both of which are known to interact with phosphorylated tyrosines. We have shown previously that tyrosine 1148 of the activated epidermal growth factor (EGF) receptor is a major binding site for Shc while tyrosine 1173 is a secondary binding site in intact cells. In the present study, we investigated the interaction between the PTB and SH2 domains of Shc and the activated human EGF receptor. Mutant 52-kDa Shc with an arginine-to-lysine substitution at residue 175 in the PTB domain (Shc R175K) or 397 in the SH2 domain (Shc R397K) was coexpressed in Chinese hamster ovary cells overexpressing the wild-type or mutant EGF receptors that retained only one of the autophosphorylation sites at tyrosine 1148 (QM1148) or 1173 (QM1173). Shc R397K was coprecipitated with the QM1148 and QM1173 receptors, was tyrosine-phosphorylated, and associated with Grb2 and Sos. In contrast, coprecipitation of Shc R175K with the mutant receptors was barely detectable. In cells expressing the QM1173 receptor, Shc R175K was tyrosine-phosphorylated and associated with Grb2, while association of Sos was barely detectable. In cells expressing the QM1148 receptor, tyrosine phosphorylation of Shc R175K was markedly reduced. When both Shc R175K and 46-kDa Shc R397K were coexpressed with the mutant receptors, p46 Shc R397K was dominantly tyrosine-phosphorylated. In cells expressing the wild-type receptor, Shc R397K, but not Shc R175K, translocated to the membrane in an EGF-dependent manner. In addition, Ras activity stimulated by the immunoprecipitates of Shc R397K was significantly higher than that by the immunoprecipitates of Shc R175K. The present results indicate that tyrosine 1148 of the activated EGF receptor mainly interacts with the Shc PTB domain in intact cells. Tyrosine 1173 interacts with both the PTB and SH2 domains, although the interaction with the PTB domain is dominant. In addition, Shc bound to the activated EGF receptor via the PTB domain dominantly interacts with Grb2-Sos complex and plays a major role in the Ras-signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1998

An ergosterol peroxide, a natural product that selectively enhances the inhibitory effect of linoleic acid on DNA polymerase beta.

As described previously (Mizushina Y., Tanaka N., Yagi H., Kurosawa T., Onoue M., Seto H., Horie T., Aoyagi N., Yamaoka M., Matsukage A., Yoshida S., and Sakaguchi K., Biochim. Biophys. Acta, 1308, 256-262, 1996), linoleic acid (LA) inhibits the activities of mammalian DNA polymerases. We found a natural product from a basidiomycete, Ganoderma lucidum, that enhances this effect of LA in a special manner. The structure was identified to be an ergosterol peroxide, 5,8-epidioxy-5alpha,8alpha-ergosta-6,22E-dien -3beta-ol by spectroscopic analyses. The ergosterol peroxide (EPO) itself scarcely inhibited the activities of calf thymus DNA polymerase alpha (pol. alpha) or rat DNA polymerase beta (pol. beta). However, when EPO at 0.25 mM was present, 10 microM or less of LA almost completely inhibited the pol. beta activity, while almost complete inhibition by LA itself was achieved at 80 microM or higher. Interestingly, under the same conditions, EPO did not affect the LA-effect on pol. alpha. The action mode of the EPO was discussed.

Animals↗
PubMed1998

Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1 from a marine red alga, Gigartina tenella.

A new sulfolipid, KM043, which belongs to the 6-sulfo-alpha-D-quinovopyranosyl-(1-->3')-1',2'-diacylglycerol (SQDG) class of compounds, has been isolated from a marine red alga, Gigartina tenella, as a potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1. Its structure was determined on the basis of spectroscopic and gas chromatographic analyses. The inhibition was dose-dependent, and complete (more than 90%) inhibition of DNA polymerase alpha (pol. alpha), DNA polymerase beta (pol. beta) and HIV-reverse transcriptase type 1 (HIV-RT) was observed at concentrations of 5, 10, and 30 microM, respectively.

Chromatography, Gas↗
PubMed1998

[Enterohemorrhagic Escherichia coli O157 infection in an elderly patient with secondary hemolytic uremic syndrome who developed recurrent acute exacerbation of chronic cholecystitis].

We encountered a patient with enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection and secondary hemolytic uremic syndrome (HUS). The patient was a 79-year-old woman with hypertension, constipation, and asymptomatic cholelithiasis. She complained of nausea and abdominal pain, and had bloody stool EHEC O157 was detected by fecal culture. The bloody stool resolved after treatment with antibiotics, but the patient was hospitalized on July 23, 1996 because of abdominal distention. HUS was diagnosed because of proteinuria, hematuria, thrombocytopenia, hemolytic anemia, fragmentation of red blood cells, and increased serum LDH. Treatment was focused on plasma exchange, administration of antibiotics, large doses of gamma-globulin, haptoglobin replacement, and anticoagulation. Within about 2 weeks, the level of hemoglobin, the number of platelets, and the serum LDH had normalized, and the patient recovered from HUS. The decreased intestinal movement continued. On August 23, acute cholecystitis was diagnosed, and percutaneous transhepatic gall bladder drainage was done. Another exacerbation was noted on October 13, and cholecystectomy was done on November 12, when the patient's status had improved after instillation of antibiotics. Macroscopically, the gallbladder wall was thickened. Histopathological examination showed diffuse infiltration of lymphocytes into the mucosa, chronic cholecystitis was diagnosed. Because the postoperative course was satisfactory, the patient was discharged from the hospital on December 15. Acute exacerbation of chronic cholecystitis might have been caused by decreased cholic excretion after the marked decrease in intestinal movement due to O157 infection and secondary HUS. Because elderly people frequently have anamnesis of the digestive system, considerably attention should be paid to the management of anamnesis, as well as O157 infection and secondary HUS.

Aged↗
PubMed1998

A 5'-monophosphate form of bredinin selectively inhibits the activities of mammalian DNA polymerases in vitro.

Bredinin is an immunosuppressive drug which is used clinically in Japan. In this study, we investigated bredinin's molecular mode of action to clarify its immunosuppressive effects. We focused on the DNA polymerases in the somatic DNA synthesis which may be required in the process of lymphocyte differentiation. We found that bredinin-5'-monophosphate (breMP) could be a potent inhibitor of mammalian DNA polymerase alpha(pol.alpha) and (pol.beta) in vitro, although bredinin itself has no such effects. BreMP inhibited the pol. alpha activity at less than 7 micrograms/ml and the pol. activity at 7 micrograms/ml. Neither breMP nor bredinin influenced the activities of a plant DNA polymerase, prokaryotic DNA polymerases such as E. coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as DNase I, indicating that breMP selectively suppressed the activities of the mammalian DNA polymerases. For pol., beta breMP acted by competing with both the substrate and template-primer. For pol. alpha, it acted by competing only with the substrate, and non-competitively with the template-primer. The ribose of bredinin is quickly and quantitatively converted to its ribose-5'-phosphate form in vivo as soon as it is incorporated into cells. The action mode of bredinin and its use as an immunosuppressive drug are discussed based on these results.

Animals↗
PubMed1998

Carboxyflavins, novel inhibitors of Taq DNA polymerase.

Carboxyflavins were found to be potent selective inhibitors of Taq DNA polymerase in a polymerase chain reaction. The inhibitions were dose-dependent, and complete inhibitions were observed at the concentration of 3.0 microM. Carboxyflavins were much less, or not sensitive to the DNA polymerases tested such as calf thymus DNA polymerase alpha, rat DNA polymerase beta, human immunodeficiency virus type 1 reverse transcriptase, the Klenow Fragment of E. coli DNA polymerase I and T4 DNA polymerase. To our knowledge, there is no other report of an agent that selectively inhibits only a thermophilic polymerase. Interestingly, the carboxyflavins were able to prevent DNA synthesis in the murine lymphoid leukemia cell line L1210 in vitro; almost complete inhibitory levels were achieved in the range of less than 10 microM.

Animals↗
PubMed1998

Effect of combined fenthion and cimetidine use in rats on lethality, blood cholinesterase activities, and serum cholinesterase isoenzymes.

H2-receptor antagonists inhibit cholinesterase (ChE) activity. We examined perturbations in ChE isoenzyme patterns and ChE activities of rats from the combined effects of fenthion (FEN) and cimetidine (CIM). Sixty-four female Sprague-Dawley rats were divided into 8 groups. Four rat groups were given FEN or gum arabic solution and each group divided into 2 small groups according to the CIM or gum arabic administration. FEN was administered po at 12.3 mg/kg (1/20 LD50) or 24.5 mg/kg (1/10 LD50) for 14 days or 49 mg/kg (1/5 LD50) every 4 days. CIM was given po at 1,500 mg/kg from days 7 to 13. Samples were collected 3 h after CIM administration on days 8 and 13. CIM did not influence ChE isoenzyme patterns or ChE activity. FEN inhibited both the ChE isoenzyme patterns and ChE activities without producing clinical signs. Although 1 rat in the 12.5 mg FEN/kg + CIM group died on day 10, all rats in other FEN (24.5 mg/kg or 49 mg/kg) + CIM groups died on days 8-10. Differences in suppression of ChE isoenzyme patterns were detectable between the FEN-dosed and FEN + CIM-dosed groups. There were no differences in ChE activities between the FEN-dosed and FEN + CIM-dosed groups. The i.p. administration of 500 mg CIM/kg (LD50) did not suppress ChE activities.

Animals↗
PubMed1997

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence.

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.

Brassica↗