Search PubMed⌕ Search

Biomedical subjects

K Ohtsuki

Publications and source records attributed to K Ohtsuki.

At least 55 records · Page 3Linked to original sources
PubMed1998

The use of technetium Tc 99m annexin V for in vivo imaging of apoptosis during cardiac allograft rejection.

OBJECTIVE: Apoptosis, or programmed cell death, has been suggested as a mechanism of immunologic injury during cardiac allograft rejection. We tested the hypothesis that technetium Tc 99m annexin V, a novel radiopharmaceutical used to detect apoptosis, can be used to detect cardiac allograft rejection by nuclear imaging. METHODS: Untreated ACI rats served as recipients of allogeneic PVG rat (n = 66) or syngeneic ACI rat (n = 30) cardiac grafts. Untreated recipient animals underwent 99mTc-annexin V imaging daily for 7 days. Region of interest analysis was used to quantify the uptake of 99mTc-annexin V. Immediately after imaging grafts were procured for histopathologic analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling of apoptotic nuclei. One group was treated with 10 mg/kg/d cyclosporine (INN: ciclosporin) commencing on day 4 after transplantation (n = 6). RESULTS: Untreated allografts showed histologic signs of rejection 4 days after transplantation. Apoptotic nuclei could be demonstrated in myocytes, endothelial cells, and graft-infiltrating cells of all rejecting allografts. Nuclear imaging revealed a significantly greater uptake of 99mTc-annexin V in rejecting allogeneic grafts than in syngeneic grafts on day 4 (P = .05), day 5 (P < .001), day 6 (P < .001), and day 7 (P = .013) after transplantation. A correlation between the histologic grade of acute rejection and uptake of 99mTc-annexin V was observed (r2 = 0.87). After treatment of rejection with cyclosporine, no apoptotic nuclei could be identified in allografts and uptake of 99mTc-annexin V decreased to baseline. CONCLUSIONS: Apoptosis occurs during acute cardiac allograft rejection and disappears after treatment of rejection. 99mTc-annexin V can be used to detect and monitor cardiac allograft rejection.

Animals↗
PubMed1998

Biochemical characterization of recombinant HIV-1 reverse transcriptase (rRT) as a glycyrrhizin-binding protein and the CK-II-mediated stimulation of rRT activity potently inhibited by glycyrrhetinic acid derivative.

By means of successive Mono Q and glycyrrhizin (GL)-affinity column chromatography (HPLC), recombinant HIV-1 RT (rRT) was purified to apparent homogeneity from the Superdex 200 pg fraction of the crude protein extract of E. coli BL21 transfected with pET 21a(+)/HIV-1 PR-RT. It was found that (i) rRT functioned as an effective phosphate acceptor for recombinant human casein kinase II (rhCK-II) in vitro; (ii) this phosphorylation was inhibited by anti-HIV-1 substances [a glycyrrhetinic acid derivative (oGA) and quercetin] and a high dose (100 microM) of GL; (iii) RNA-dependent DNA polymerase (RDDP) activity was stimulated about 2.5-fold after full phosphorylation of rRT by rhCK-II; and (iv) oGA as well as NCS-chromophore effectively prevented the CK-II-mediated stimulation of RDDP activity. These results suggest that the anti-HIV-1 effect of oGA may be involved in the selective inhibition of the CK-II-mediated stimulation of HIV-1 RT at the cellular level.

Anti-HIV Agents↗
PubMed1998

Separation of phospholipase A2 in Habu snake venom by glycyrrhizin (GL)-affinity column chromatography and identification of a GL-sensitive enzyme.

By means of glycyrrhizin (GL)-affinity and Mono S column chromatographies (HPLC), at least four GL-binding proteins (p25, p17, p15-1 and p15-2) in the two Superdex fractions (P-II and P-III fractions) from Habu snake venom were selectively purified. By determination of their N-terminal partial amino acid sequences, a metalloprotease (p25) and three GL-binding phospholipases A2 (gbPLA2s) [PA2Y (p17), PA21 (p15-1) and PA2B (p15-2)] were identified. PA2B (lysine-49 PLA2) was found to be the most sensitive to GL because (i) it strongly bound to a GL-affinity column; and (ii) its enzyme activity was selectively inhibited by low dose (ID50 = approx. 1.5 microM) of GL, but not by GA. Furthermore, these three gbPLA2s were phosphorylated by casein kinase II (CK-II) in vitro and GL inhibited the CK-II-mediated stimulation of their enzyme activities in vitro.

Amino Acid Sequence↗
PubMed1998

Characterization of an apoptosis-inducing factor in Habu snake venom as a glycyrrhizin (GL)-binding protein potently inhibited by GL in vitro.

By means of successive heparin-affinity and glycyrrhizin (GL)-affinity column chromatographies (HPLC), a 55 kDa GL-binding protein (gp55) was purified to apparent homogeneity from the Superdex P-I fraction of Habu snake venom. This gp55 was identified as an apoxin I-like protein, because (i) its 20 N-terminal amino acid residues (AHDRNPLEEYFRETDYEEFL) are 95% identical with the corresponding sequence of apoxin I (apoptosis-inducing factor, approx. 55 kDa) in the venom of the western diamondback rattlesnake; and (ii) L-amino acid oxidase (LAO) activity of gp55 is detected when incubated with L-leucine, but not with D-leucine. GL inhibited the LAO activity of gp55 in a dose-dependent manner, but had no effect on the activity of a 65 kDa LAO also purified from Habu snake venom. In addition, GL reduced the ability of gp55 to induce the hemolysis of sheep red blood cells. These results suggest that GL is a potent inhibitor of apoxin I-like proteins in harmful snake venoms.

Amino Acid Oxidoreductases↗
PubMed1998

Comparative bio-antimutagenicity of common vegetables and traditional vegetables in Kyoto.

Traditional vegetables in Kyoto are a unique group of vegetables that have been cultivated in limited areas near Kyoto city. We compared the traditional vegetables in Kyoto with common vegetables for the bio-antimutagenicity of their extracts against UV-induced mutation of E. coli B/r WP2. Among the traditional vegetables in Kyoto, Kamo eggplant (Solanaceae) and Katsura oriental pickling melon (Cucurbitaceae) showed higher bio-antimutagenicity and yield in the n-hexane, chloroform and ethyl acetate fractions than their common vegetable counterparts. Shishigatani pumpkin (Cucurbitaceae) possessed bio-antimutagenicity in the chloroform and ethyl acetate fractions, but common pumpkin did not. Polyphenolic compounds in the ethyl acetate fraction of plants are known to be related to antimutagenicity. However, the intensity of bio-antimutagenicity was not correlated with the polyphenol content in the ethyl acetate fractions of the present vegetables. In particular, Kamo eggplant possessed both polyphenolic and non-polyphenolic bio-antimutagenic sub-fractions in the ethyl acetate fraction. In the aqueous fraction, taro (Dioscoreaceae) was the most capable among our samples, whether being of common or traditional origin. Consequently, it is considered, that some traditional vegetables in Kyoto are superior to common vegetables in their bio-antimutagenicity and that these could be used as starting materials to identify new bio-antimutagens.

Antimutagenic Agents↗
PubMed1997

Immunohistochemical study on the nm23 gene produce (NDP kinase) in oral squamous cell carcinoma.

The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. Immunohistochemical investigation on the role of nm23 gene product (nucleoside diphosphate kinase: NDPK) in squamous cell carcinomas of the oral cavity was performed and compared to clinical and histopathological factors. The stainability of NDPK did not correlate to tumour extent, differentiation and mode of invasion. However, the NDPK positive group tended to have a lower frequency of lymph node metastasis, and a better prognosis than the NDPK negative group. Consequently, nm23/NDPK is suggested to be a metastasis suppressor factor that may be useful for predicting tumour metastasis and prognosis in oral squamous cell carcinoma.

Biomarkers, Tumor↗
PubMed1997

Purification and characterization of glutaredoxin (thioltransferase) from rice (Oryza sativa L.).

We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants. The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose. Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da. Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins. Inactivation assay also indicated that cysteine residues are responsible for enzyme activity. Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics. The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate. In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.

Amino Acid Sequence↗
PubMed1997

Biochemical characterization of glycyrrhizin as an effective inhibitor for hyaluronidases from bovine testis.

The inhibitory effects of several anti-inflammatory agents, including glycyrrhizin (GL), on the activities of hyaluronidases (HAses) purified from bovine testes and Streptomyces were investigated in vitro. It was found that (i) GL inhibits the activity of HAse (p55) from bovine testes in a dose-dependent manner, but does not affect HAse from Streptomyces; (ii) GL was the most effective of the compounds tested on bovine testis HAse activity (50% inhibition with approx. 3 microM GL); and (iii) glycyrrhetinic acid (GA), a derivative (oGA) of GA and diglucuronic acid had no detectable effects on HAse activity at 9.0 microM. The GL-induced inhibition of HAse activity is uncompetitive for its substrates. Data are provided to support the contentions that (i) bovine testis HAse (p55) is a GL-binding protein; and (ii) GL acts as a potent inhibitor of HAse in vitro.

Animals↗
PubMed1997

Effects of growth hormone-releasing hormone (GRF) analogs, bovine and rat GRF on growth hormone secretion in cattle in vivo.

Effects of bovine and human growth hormone-releasing hormone (GRF) analogs (bGRF(1-29)-NH2: bGRF-29, [D-Ala2, Ala15]-bGRF-29, [D-Ala2]-hGRF(1-29)-NH2: [D-Ala2]-hGRF-29), bovine GRF (bGRF(1-44)-NH2: bGRF-44), as well as rat GRF (rGRF) on GH release were studied in female calves. Intravenous (i.v.) bolus injections of 0.25 microg/kg BW of bGRF-29, [D-Ala2, Ala15]-bGRF-29, and [D-Ala2]-hGRF-29 stimulated GH release. Plasma GH levels began to rise 10 min after the injection of each peptide, and significant increases in GH concentrations were obtained at 60, 180 and 150 min after the injection of bGRF-29, [D-Ala2, Ala15]-bGRF-29 and [D-Ala2]-hGRF-29, respectively. The concentrations of GH 80 min after the injection of [D-Ala2, Ala15]-bGRF-29 were significantly higher than those after the injection of [D-Ala2]-hGRF-29 (except at 80 and 90 min) or bGRF-29. The i.v. bolus injections of 0.25 microg/kg BW of bGRF-44 and rGRF stimulated GH release, and the GH-releasing potency of rGRF was approximately equal to that of bGRF-44. The plasma GH responses to the repeated i.v. injection of bGRF-29 or [D-Ala2, Ala15]-bGRF-29 at 2-h intervals were examined. bGRF-29 acutely increased plasma GH levels after each injection, and the high GH levels decreased to the basal values within 2 h. In contrast, high GH levels induced by [D-Ala2, Ala15]-bGRF-29 were gradually decreased but not lowered to basal values throughout the experiment. These results show that [D-Ala2, Ala15]-bGRF-29 has longer-lasting and greater GH-releasing activity than the other GRF analogs in female calves, and the GH-releasing potency of rat GRF is approximately equal to that of bovine GRF in cattle in vivo.

Animals↗
PubMed1997

Enzymatic properties, substrate specificities and pH-activity profiles of two kiwifruit proteases.

Kiwifruit (Actinidia chinensis) contains abundant protease, actinidin, and two possible components which were named A1 and A2. However, a comparison of the two components has not been thoroughly conducted. We have previously shown the presence of six proteases named KP1, KP2, KP3, KP4, KP5 and KP6 in kiwifruit, and that each purified kiwifruit protease was chromatographically pure. It was also indicated that the two representative components, KP4 and KP6, must be A1 and A2. To establish whether or not the two proteases, KP4 and KP6, have the same specificity in proteolytic activity, their enzymatic properties were compared. Between the two proteases, differences in substrate specificity against several protein-substrates (casein, gelatin, collagen, ovalbumin and bovine serum albumin) were not observed by digestion-product analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinetic parameters of KP4 against N-alpha-carbobenzoxyl-lysine p-nitrophenyl esters were different from those of KP6. The pH-activity profiles of KP4 and KP6 against S-3-trimethylaminopropyl-lysozyme, a wide-pH range soluble substrate, and N-alpha-carbobenzoxyl-lysine p-nitrophenyl esters were different.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

[Biochemical mechanisms of NCS-chromophore-induced DNA cleavage and inhibition of protein kinase activity].

A NCS-chromophore (molecular weight: approximately 695) can be extracted from an antitumor antibiotic neocarzinostatin (NCS) (approximately 11,000, pI 3.3) with methanol as a DNA cleavaging molecule. Recently, the Goldberg research group (Harvard University, USA) proposed two distinct mechanisms of DNA cleavages (thiol-dependent DNA cleavage and thiol-independent base-catalyzed (bc) cleavage of ssDNA) by NCS-chromophore in vitro. Therefore, it is concluded that the ability of NCS-chromophore to cleave DNA is its primary action, which selectively inhibits DNA synthesis in cultured cells. Furthermore, we found that NCS-chromophore inhibits protein phosphorylation by CK-II (casein kinase II) involved in transcriptional regulation in a dose-dependent manner. Disruption of the repair systems of the NCS-chromophore-induced biological damages results in the positive induction of apoptosis, because the drug is greatly activated by thiols at the intracellular level, and it inhibits the activities of several transcriptional factors through their specific phosphorylation by nuclear kinases, such as CK-II. Taken together, all these biological and biochemical data suggest that the NCS-chromophore could be an effective chemotherapeutic drug for human cancer if its toxicity can be appropriately controlled.

Antibiotics, Antineoplastic↗
PubMed1996

An inhibitory effect of actin on casein kinase II activity in vitro.

The inhibitory effect of actin on protein phosphorylation by three distinct protein kinases (CK-II, A-kinase and MAP-kinase) was examined in vitro. It was found that: (i) actin inhibits the activities of alpha-monomeric CK-II (CK-IIalpha) as well as oligomeric CK-II (alpha2beta2) in a dose-dependent manner, but has no effect on the activities of the two other kinases; and (ii) actin-induced inhibition of CK-II activity is due to the binding of actin to the alpha-subunit of CK-II and is non-competitive with its phosphate acceptors. In addition, it is demonstrated that actin binds directly to CK-II: both actin and CK-II are coprecipitated by anti-serum against Drosophila CK-IIbeta or by specific IgG against Ascaris suum muscle actin. The results presented here suggest that actin can suppress CK-II-mediated signal transduction.

Actins↗
PubMed1996

Identification of glycyrrhizin-binding protein kinase as casein kinase II and characterization of its associated phosphate acceptors in mouse liver.

Two forms (G-I and G-II kinases) of casein kinase II(CK-II) in a partially purified CK-II fraction (Mono Q fraction) of mouse liver were separated by means of glycyrrhizin (GL)-affinity column chromatography. Biochemical characterization revealed that these two GL-binding kinases were identical to CK-II. Two phosphate acceptors [p99 (pI 7.0) and p56] copurified with CK-II were identified as ERp99 (Hsp-90-family protein) and calreticulin, respectively. Another protein [p100 (pI 9.0)], which crossreacted with anti-serum against human glucocorticoid receptor (GR), was associated with ERp99. Phosphorylation of p99 [a hetero-complex of p99 (pI 7.0) and p100 (pI 9.0)] and p56 by CK-II in vitro was stimulated significantly by low levels (1-3 microM) of GL, but inhibited significantly at doses above 20 microM. However, no effect of GL on autophosphorylation of ERp99 was detected. The data provided here suggest that GL can regulate CK-II-mediated phosphorylation involved in the GL-induced biological effects in mammalian cells.

Animals↗
PubMed1996

Physiological correlation between glycyrrhizin, glycyrrhizin-binding lipoxygenase and casein kinase II.

By means of glycyrrhizin (GL)-affinity column chromatography, a GL-binding lipoxygenase (gbLOX) was selectively purified from the partially purified soybean LOX-1 fraction. Polypeptide analysis of the purified gbLOX by SDS-PAGE detected two distinct polypeptides (p96 and p94), which were identical to LOX-3 as determined by their partial N-terminal amino acid sequences. Moreover, it was found that (i) phosphorylation of gpLOX by casein kinase II (CK-II) is significantly stimulated by 3 microM GL, but inhibited by 30 microM GL or 10 microM oGA; and (ii) gbLOX activity is enhanced when the enzyme is phosphorylated by CK-II in the presence of 3 microM GL. These results suggest that (i) CK-II is a kinase responsible for the activation of gbLOX through its specific phosphorylation; and (ii) GL is one of the regulatory substances for specific phosphorylation of gbLOX (LOX-3) by CK-II in plant cells.

Amino Acid Sequence↗
PubMed1996

DNA-binding sperm proteins with oligo-arginine clusters function as potent activators for egg CK-II.

The stimulatory effect of DNA-binding sperm proteins (histone and protamine) on the phosphorylation of p98 (ERp99/GRp94, one of the Hsp-90 family of proteins) by egg casein kinase II (CK-II) was investigated in vitro. It was found that (i) phosphorylation of p98 by egg CK-II in vitro is greatly stimulated by poly-Arg, but not by poly-Lys; and (ii) similar stimulation is observed with sperm histones H2B2 and H2B3 (sea urchin) and fish protamines, such as salmine A1 (salmon) and protamine 3a (rainbow trout). These findings suggest that these DNA-binding sperm proteins function as potent activators for CK-II in fertilized eggs. All of these DNA-binding sperm proteins contain at least an oligo-Arg cluster as a common feature, which can interact with an acidic amino acid cluster of the regulatory beta-subunit CK-II.

Amino Acid Sequence↗
PubMed1996

[Gynecological management of climacteric syndrome with psychiatric disorders].

PURPOSE: To discriminate patients with severe mental disorders from those complaining of menopausal symptoms, psychological tests were performed on patients who visited the menopausal clinic. The effectiveness of hormone replacement therapy (HRT) in patients with slight mental disorders was also evaluated. SUBJECTS AND METHODS: Patients with menopausal symptoms (n = 150, 41-59 yr. old) were interviewed by a psychiatrist (according to DSM-III-R) and classified as the climacteric syndrome group (C) and the mental disorder group (P). The patients were also evaluated by the Menopausal index (MI), Maudsley Personality Inventory (MPI) and Self-rating depression Scale (SDS). The patients with severe mental disorders were eliminated and the remaining patients with menopausal symptoms were treated with conjugated equine estrogen (0.625 mg/day) and medroxyprogesterone acetate (2.5mg/day) for 24 weeks. RESULTS: 1) Thirty six patients were diagnosed as P (24%) and seven of them were diagnosed as having major depression (4.7% of the all patients). 2) MPI-N (neurosis scale) in C was 16.2 +/- 10, and in P was 30.5 +/- 9.0, and SDS in C and P were 39.0 +/- 8.0, and 51.4 +/- 8.9, respectively. In both psychological test, P shows a significantly higher value than C (p < 0.01). 3) Ninety point six % of the patients responded. HRT was not effective in 63.2% of P, but when psychotropic drugs were combined, significant improvement (p < 0.05) was observed. CONCLUSION: One fourth of the patients were suspected of having mental disorders in the menopausal clinic, and 5% of the patients needed the care of the psychiatrist. And the application of MPI and SDS was useful in helping the gynecologist to differentiate and classify the mental disorders to some extent. The applicability and effectiveness of HRT for patients with mild mental disorders were suggested.

Adult↗