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H Ishida

Publications and source records attributed to H Ishida.

At least 433 records · Page 24Linked to original sources
PubMed1996

Molecular cloning and expression of rabbit sterol 12alpha-hydroxylase.

Sterol 12alpha-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid. The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) J. Biol. Chem. 267, 21319-21323), and we here describe the cloning and sequencing of a cDNA coding for this enzyme. After tryptic digestion of purified protein in a polyacrylamide gel, eight different peptides were isolated and sequenced. Using oligonucleotides deduced from the amino acid sequences, clones were isolated from a rabbit liver cDNA library. In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 amino acids, corresponding to a molecular mass of 57 kDa. All of the eight peptides and the reported NH2-terminal amino acid sequence were matched against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the rate-limiting enzyme in over-all synthesis of bile acids, the cholesterol 7alpha-hydroxylase (CYP7) of the rabbit. The similarity with most other sterol cytochrome P-450 hydroxylases was less. Thus, this species of cytochrome P-450 should belong to a group of its own, here denoted CYP12. Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of sterol 12alpha-hydroxylase activity toward 7alpha-hydroxy-4-cholesten-3-one. Northern blotting showed that the enzyme was exclusively expressed in the liver. The major mRNA fraction in rabbit liver had a size of approximately 2.9 kilobases, and those found in rat and human liver were about 2.5 and 4.5 kilobases, respectively. Fasting of rats and mice led to a severalfold increase in both enzyme activity and mRNA levels. In contrast, starvation of rabbits had little or no stimulatory effect on enzyme activity and mRNA levels.

Amino Acid Sequence↗
PubMed1996

MAGE-1 and MAGE-3 or -6 expression in neuroblastoma-related pediatric solid tumors.

MAGE-1 and MAGE-3 or -6 are genes encoding melanoma-rejection antigens recognized by cytotoxic T lymphocytes in an HLA-A1 restriction manner. MAGE-1 and MAGE-3 or -6 were expressed in 5/14 (36%) and 6/14 (43%) neuroblastoma (NB) cell lines, and in 20/41 (49%) and 24/41 (59%) clinical NB-related tumors, respectively. Additionally, they were also expressed in pediatric tumors of other types such as rhabdomyosarcoma and Wilms' tumor. MAGE-1 expression at a functional level in tumor cells was confirmed by the cytotoxicity assay using MAGE-1-specific tumor-infiltrating lymphocytes (TIL). In clinical NB-related tumors, MAGE-3 or -6 expression demonstrated an inverse correlation to clinical stage. Furthermore, although the sample number was small, the incidence of MAGE-1 and/or MAGE-3 or -6 expression was significantly correlated to the absence of metastasis and a more favorable clinical outcome (p < 0.05). These results may suggest that NB cells silent for the expression of MAGE genes escape from the host anti-tumor immune response and consequently retain a growth advantage. Finally, NB-related tumors could be reliable candidates for immunotherapy targeted towards MAGE gene products.

Antigens, Neoplasm↗
PubMed1996

Secondary structure and thermostability of the photosystem II manganese-stabilizing protein of the thermophilic cyanobacterium Synechococcus elongatus.

The secondary structure of the manganese-stabilizing protein of the thermophilic cyanobacterium Synechococcus elongatus in solution was investigated by Fourier-transform infrared (FT-IR) and circular dichroism (CD) spectroscopies. Both methods showed a high proportion of disordered structure (40-43%) and a relatively small amount of beta-sheet (23-24%) and alpha-helix (17-19%). The conformation of the protein remained essentially unchanged at temperatures up to 70 degrees C. Unfolding of the protein occurred at higher temperatures and FT-IR spectroscopy revealed that beta-sheet was more strongly unfolded than alpha-helix at 76 degrees C. The protein largely lost the ordered secondary structures at 90 degrees C, but, when cooled down to 30 degrees C, regained its original conformation. Thus, the cyanobacterial protein is very thermostable and its denaturation at an extremely high temperature is reversible.

Bacterial Proteins↗
PubMed1996

Novel 5-aminoflavone derivatives as specific antitumor agents in breast cancer.

In the course of our search for new antitumor agents in breast cancer, novel amino-substituted flavone derivatives were synthesized and examined for antitumor activities. Among them, 5,4'-diaminoflavone and some of its congeners showed remarkable antiproliferative activity against the estrogen receptor (ER)-positive and estrogen-responsive human breast cancer cell line MCF-7. The activity was observed irrespective of the presence or absence of estrogen. The 5-aminoflavone derivatives (5-AFs) are not classical anti-estrogens because they did not compete with [3H]estradiol to bind the estrogen receptor. Moreover, 5-AFs showed antitumor activity highly selective to the ER-positive breast cancer cell line, and they showed no effects against the ER-negative human cancer cell lines HeLa S3, WiDr, and MDA-MB-453. Although the mechanism of their selective antitumor activity to ER-positive breast cancer cells is unclear, 5-AFs are expected to be a new type of antitumor agents in breast cancer.

Antineoplastic Agents↗
PubMed1996

Regulation of phosphorylation of Gi2 alpha protein controls the secretory response to isoproterenol in rat parotid tissues.

Treatment of rat parotid tissues with 1 microM isoproterenol (IPR) for 10 min caused a 60% decrease in pertussis toxin (IAP)-catalyzed ADP-ribosylation of Gi alpha and resulted in supersensitivity of amylase secretion from the tissues. However, conversely, IPR treatment for 30 min caused a 40% increase in IAP-catalyzed ADP-ribosylation of Gi alpha, coupled with desensitization of amylase secretion. No changes in Gs function were observed in IPR-induced phenomena. Pretreatment with okadaic acid induced enhancement of the supersensitivity of amylase secretion and disappearance of the desensitization. These phenomena were accompanied with decreases in IAP-catalyzed ADP-ribosylation of Gi alpha. IPR treatment for 30 min caused a 50% decrease in phosphorylation of Gi2 alpha immunoprecipitated with anti-G protein antiserum (AS/7) from [32P]Pi-labeled cells, but such treatment for 10 min caused a 40% increase in phosphorylation in the cells pretreated with okadaic acid. Phosphorylation and dephosphorylation of immunoprecipitates with AS/7 by protein kinase A (PKA) and alkaline phosphatase caused decreases and increases in IAP-catalyzed ADP-ribosylation, respectively, indicating the presence of PKA-mediated phosphorylation sites on Gi2 alpha. Thus, the control of the phosphorylation of Gi2 alpha is of importance and relevance in the regulation of biological processes and cellular responses.

Adenosine Diphosphate Ribose↗
PubMed1996

Peroxynitrite formation from activated human leukocytes.

We showed direct evidence of peroxynitrite formation from polymorphonuclear cells (PMN) with the nitration of 4-hydroxyphenylacetic acid (HPA) to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA). Human PMN from healthy volunteers was stimulated with phorbol-12-myristate-13-acetate (PMA, 10 ng/ml) at 37 degrees C in 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid-buffered Hank's balanced salt solution (pH 7.4) with HPA (1 mM). NO2HPA was detected under PMA stimulation only in the presence of myeloperoxidase inhibitor. NO2HPA was eliminated by N-monomethyl-L-arginine (100 microM). The inhibition of myeloperoxidase appears to be essential to demonstrate the production of NO2HPA since myeloperoxidase itself or its product, hypochlorite, reacted with peroxynitrite and hampered the formation of NO2HPA.

Animals↗
PubMed1996

Status of microchimerism in recipients 15 years after living related kidney transplantation.

To study the relevance of microchimerism to the long-term outcome of renal allografting, we analyzed the frequency of microchimerism in kidney transplant recipients who had stable graft function for 15 years or longer. Among the 104 recipients who underwent kidney transplantation between 1971 and 1980, 27 renal allografts (26%) are still functioning. Among these 27 patients, 13 recipients whose donor was still alive and cooperative were investigated for the presence of microchimerism in the peripheral blood and for their immunological status. Microchimerism was tested using the polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) method. To test the sensitivity of PCP-SSCP, the peripheral blood obtained within 5 weeks after transplantation (four kidney transplants, three liver transplants) was also examined. Microchimerism was detectable in five patients within 5 weeks of transplantation (kidney transplantation, 3/4; liver transplantation 2/3. However, in the patients studied 15 years after transplantation, microchimerism was detected in only one recipient (1/13). In this chimeric patient, mixed lymphocyte response revealed high responsiveness against donor antigen. In contrast, some patients who did not have chimerism showed donor-specific hyporesponsiveness in mixed lymphocyte response assay and did not develop antidonor antibody, according to flow cytometric analysis. Microchimerism is an infrequent state in the long-term survivors of kidney allografting, and this state is irrelevant to donor-specific unresponsiveness.

Adult↗
PubMed1996

High affinity antibodies against Lex and sialyl Lex from a phage display library.

Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.

Amino Acid Sequence↗
PubMed1996

Alterations in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta cells of non-insulin-dependent diabetes mellitus GK rats.

In genetically occurring non-insulin-dependent diabetes mellitus (NIDDM) model rats (GK rats), the activities of L- and T-type Ca2+ channels in pancreatic beta cells are found to be augmented, by measuring the Ba2+ currents via these channels using whole-cell patch-clamp technique, while the patterns of the current-voltage curves are indistinguishable. The hyper-responsiveness of insulin secretion to nonglucose depolarizing stimuli observed in NIDDM beta cells could be the result, therefore, of increased voltage-dependent Ca2+ channel activity. Perforated patch-clamp recordings reveal that the augmentation of L-type Ca2+ channel activity by glucose is markedly less pronounced in GK beta cells than in control beta cells, while glucose-induced augmentation of T-type Ca2+ channel activity is observed neither in the control nor in the GK beta cells. This lack of glucose-induced augmentation of L-type Ca2+ channel activity in GK beta cells might be causatively related to the selective impairment of glucose-induced insulin secretion in NIDDM beta cells, in conjunction with an insufficient plasma membrane depolarization due to impaired closure of the ATP-sensitive K+ channels caused by the disturbed intracellular glucose metabolism in NIDDM beta cells.

Animals↗
PubMed1996

Studies on selectin blockers. 2. Novel selectin blocker as potential therapeutics for inflammatory disorders.

As a part of our studies of selectin blockers, we prepared 1-(2-tetradecylhexadecyl)-3'-O-sulfo Le(X) 1 and 1-(2-tetradecylhexadecyl) sLe(X) 2 and examined their inhibitory activities against natural ligand (sLe(X)) binding to E-, P-, and L-selectins. Compounds 1 and 2 were 2 times more potent than the sLe(X) tetrasaccharide toward E-selectin binding and up to 4 times more potent than sLe(X) toward P- and L-selectin binding. Interestingly, compound 1 provided dose-dependent protective effects against an immunoglobulin E-mediated skin reaction in mouse ears. This protective effect was associated with diminished tissue accumulation of neutrophils in the ear (as assessed by myeloperoxidase). These findings indicate that the modification of sLe(X) or 3'-O-sulfo Le(X) with a "branched anchor", a 2-tetradecylhexadecyl group, is useful in the design of a more potent selectin blocker, which has broad inhibitory activities toward all selectins.

Animals↗
PubMed1996

Systematic synthesis of alpha-sialyl-(2 --> 3)- and -(2 --> 6)-isoglobopentaosylceramides (V3Neu5AciGb5Cer and V6Neu5AciGb5Cer).

Systematic syntheses of the isoglobo-series gangliosides, alpha-sialyl-(2 --> 3)- and -(2 --> 6)-isoglobopentaosyl ceramides (21 and 24, V3Neu5AciGb5Cer and V6Neu5AciGb5Cer) are described. 2-(Trimethylsilyl)ethyl 2-O-benzyl-4,6-O-benzylidene-alpha-D-galactopyranosyl-(1 --> 3)- 2,4,6,tri-O-benzyl-beta-D-galactopyranosyl-(1--> 4) -2,3,6-tri-O-benzyl-bet a-D- glucopyranoside (4), the core structure of the isoglobo-series gangliosides was prepared by the glycosylation of 2-(trimethylsilyl)ethyl 2,4,6-tri-O-benzyl-(1 --> 4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside (2) with methyl 3-O-acetyl-2-O-benzyl-4,6-O-benzylidene-1-thio-beta-D-galactopyranoside (1), and subsequent O-deacetylation. Coupling of 4 and methyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-beta-D- galactopyranoside gave an isoglobotetraoside derivative 6, from which the phthaloyl and O-acetyl groups were removed. N-Acetylation then gave a tetrasaccharide acceptor 7. Dimethyl(methylthio)sulfonium triflate-promoted coupling of 7 with methyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D- galacto)-(2 --> 3)-2,4,6-tri-O-benzoyl-1-thio-beta-D-galactopyranoside or -(2 --> 6)-2,4-di-O-benzoyl-3-O-benzyl-1-thio-beta-D-galactopyranoside gave the pentasaccharide derivative 9 and 14 in good yields. Compounds 9 and 14 were converted into the corresponding alpha-trichloroimidates 12 and 17 which, on coupling with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol (18), gave the beta-glycoside 19 and 22 respectively, Finally, 19 and 22 were transformed, via selective reduction of azide group, condensation with octadecanoic acid, O-deacylation, and hydrolysis of the methyl ester group into 21 and 24, respectively.

Acetylation↗
PubMed1996

Two distinct quinoprotein amine oxidases are induced by n-butylamine in the mycelia of Aspergillus niger AKU 3302. Purification, characterization, cDNA cloning and sequencing.

Two distinct quinoprotein amine oxidases were found in Aspergillus niger mycelia grown on n-butylamine medium and purified using chromatographic techniques. The respective enzymes were termed AO-I, which had already been isolated, and AO-II, a new enzyme found in this study. HPLC indicated that their molecular masses are 150 kDa and 80 kDa, respectively. On SDS/PAGE, the enzymes gave a similar but distinct mobility, which corresponds to 75 kDa for the subunit dimeric AO-I and 80 kDa for monomeric AO-II. The absorption spectra of both enzymes were different from each other; the absorption maxima in the visible region were at 490 nm for AO-I and 420 nm for AO-II. The enzymes showed positive quinone staining, comparable substrate specificity, and sensitivity to inhibitors typical for copper/topa quinone-containing amine oxidases, but they had different copper contents and also differed in their N-terminal sequences. Their peptide maps showed almost identical patterns, with the exception of two additional bands for AO-II. Among the peptides obtained from digestion of AO-II, peptides with sequences corresponding to the N-terminal part of AO-I were detected. Polyclonal antibodies raised against AO-I and AO-II recognized both enzymes, but with different specificities. Using precipitation with AO-I, the antibody prepared against AO-II was purified and was shown to be specific only for AO-II. The cDNA of AO-I was cloned and sequenced. A highly conserved tetrapeptide sequence, Asn-Tyr-Glu-Tyr, was identified in which the first tyrosine residue (Tyr404) that could be converted to topa quinone was present in the 670-residue deduced amino acid sequence. Northern blot analysis indicated that AO-I was highly expressed in A. niger grown on n-butylamine as a single nitrogen source. Genomic Southern blot analysis confirmed that both enzymes are likely to be encoded by the same gene.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Studies on selectin blocker. 1. Structure-activity relationships of sialyl Lewis X analogs.

As part of our studies of selectin blockers, we prepared 1-deoxy-3'-O-sulfo LeX analogs (1-3), 1-deoxy-3'-O-phosphono LeX analogs (4), and 1-deoxy sLeX analogs (5-7), and examined their inhibitory activities against natural ligand (sLeX) binding to E-selectin, P-selectin, and L-selectin. The 1-deoxy sLeX 5 was up to 20 times more potent an inhibitor than the sLeX tetrasaccharide toward P- and L-selectin binding. This indicates that the modification of the 1 or 2 position of sLeX is useful in the design of a more potent selectin blocker.

E-Selectin↗
PubMed1996

Total synthesis of VIM-2 ganglioside isolated from human chronic myelogenous leukemia cells.

A total synthesis of the tumor-associated glycolipid antigen, VIM-2, is described [2]. Phenyl 2,3,4-tri-O-benzoyl-6-O-benzyl-beta-D-galactopyranosyl-(1-->4)-6-O-benzy l-2- deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (7), a key intermediate prepared by condensation of phenyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (6) and 2,3,4-tri-O-benzoyl-6-O-benzyl-alpha-D-galactopyranosyl bromide (5), was glycosylated with methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside (8) to give the trisaccharide donor 9, which, on coupling with 2-(trimethylsilyl)ethyl 2,4,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside (10), afforded the pentasaccharide 11. The regioselective glycosylation of 12 (derived by O-debenzoylation of 11) with 7 gave the heptasaccharide 13, which was converted by treatment with hydrazine monohydrate and subsequent N-acetylation into the hexasaccharide acceptor 14. The stereo- and regio-selective glycosylation of 14 with methyl (phenyl 5-acetamido-4,7,8,9-O-benzoyl-3,5-dideoxy-2-thio-D-glycero-beta-D-galact o-2- nonulopyranosid)onate (16) gave the desired octasaccharide 18. Hydrogenolytic removal of the benzyl groups in 18 and successive O-acetylation, removal of the 2-(trimethylsilyl)ethyl group, and treatment with trichloroacetonitrile gave the alpha-trichloro-acetimidate 21, which was then coupled with (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol (22) to give 23. Compound 23 was transformed, via selective reduction of the azido group, N-introduction of octadecanoic acid, O-desilylation, O-deacylation, and saponification of the methyl ester group, into the title VIM-2 ganglioside 26.

Carbohydrate Sequence↗