Developing better enzymes for organic synthesis.
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Carbohydrates↗
Biomedical subjects
Y Ichikawa
Publications and source records attributed to Y Ichikawa.
Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages.
Analysis of mice carrying mutant T-cell antigen receptor (TCR) genes indicates that TCR-beta gene rearrangement or expression is critical for the differentiation of CD4-CD8- thymocytes to CD4+CD8+ thymocytes, as well as for the expansion of the pool of CD4+CD8+ cells. TCR-alpha is irrelevant in these developmental processes. The development of gamma delta T cells does not depend on either TCR-alpha or TCR-beta.
Animals↗
Neurotoxins: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3,4-tetrahydroisoquinoline and 1-methyl-6,7-dihydroxy-tetrahydroisoquinoline as substrates for FAD-containing monooxygenase of porcine liver microsomes.
The activity of FAD-containing monooxygenase (FMO) (EC 1.14.13.8) of porcine liver microsomes was examined with the neurotoxins, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-6,7-dihydroxy-tetrahydroisoquinoline (MDTIQ), as substrates. FMO catalyses these neurotoxins. The kinetic parameters of FMO for the neurotoxins and electron donors were determined. Km values for MPTP, TIQ and MDTIQ were determined to be 47 microM, 6.9 mM and 5.6 mM, respectively. The Km for the electron donor, NADPH, was variable from 31 to 200 microM depending on the substrate used. The activities of FMO for these neurotoxins were comparable with that for dimethylaniline.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
Multivalent ligand binding by serum mannose-binding protein.
The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP.
Acute-Phase Proteins↗
Effects of interleukin 1 alpha on the activities and gene expressions of the cytochrome P450IID subfamily.
The mechanism by which recombinant human interleukin 1 alpha (rhIL-1 alpha) inhibits the activities of drug-metabolizing enzymes of rat liver microsomes, especially debrisoquine monooxygenase and bufuralol monooxygenase (both cytochrome P450IID supported reactions), as well as other enzymes, was investigated by injecting IL-1 alpha into rats. rhIL-1 alpha suppressed the activities of various P450-linked monooxygenase systems such as aminopyrine N-demethylase, benzphetamine N-demethylase, and 7-ethoxycoumarin O-deethylase. It also suppressed the activities of debrisoquine monooxygenase and bufuralol monooxygenase. On the other hand, IL-1 alpha had little effect on the activity of p-nitroanisole N-demethylase. The suppression of debrisoquine monooxygenase and bufuralol monooxygenase activities was caused by a decrease in the amounts of immunoreactive P450IID protein and its mRNA. The reduction rates in the level of immunoreactive P450IID protein and its mRNA were comparable. These results suggest that at the mRNA level, the enzymatic activities of debrisoquine monooxygenase and bufuralol monooxygenase are down-regulated by IL-1 alpha.
7-Alkoxycoumarin O-Dealkylase↗
Absorbance spectral change of the cytochrome P-450S21-phenylisocyanide complex upon binding of reduced NADPH-cytochrome-P-450 reductase.
Reduction of cytochrome P-450S21 (SF) (SF, substrate-free; purified from bovine adrenocortical microsomes) with sodium dithionite (Na2S2O4) in the presence of phenylisocyanide produced a ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex with Soret absorbance maxima at 429 and 456 nm. On the other hand, when a preformed ferric cytochrome P-450S21 (SF)-NADPH-cytochrome-P-450 reductase (Fp2) complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum of the ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex changed drastically, as characterized by an increase in absorbance intensity at 429 nm and a decrease at 456 nm. Similar spectral changes were observed by addition of reduced Fp2 to the preformed ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex. Experiments to reduce a ferric cytochrome P-450S21 (SF)-phenylisocyanide complex with sodium dithionite in the presence of various amounts of Fp2 showed that; (1), the spectral change reached maxima for both absorption increase at 429 nm and decrease at 456 nm when cytochrome P-450S21 and Fp2 were previously mixed at the cytochrome P-450S21:Fp2 ratio of 1:5; (2), the spectral change was suppressed in 300 mM potassium phosphate buffer (pH 7.4). These results suggest that the absorbance spectral change is due to a conformational change around the heme moiety induced by association with reduced Fp2.
Adrenal Cortex↗
Effects of cholesterol side-chain groups and adrenodoxin binding on the vibrational modes of carbon monoxide bound to cytochrome P-450scc: implications of the productive and nonproductive substrate bindings.
Effects of the bindings of cholesterol and its hydroxylated analogues on the Fe-CO stretching and the C-O stretching vibrations of cytochrome P-450scc-CO complex were examined by resonance Raman and FT-IR spectroscopies to reveal the spatial relationship between the steroid side-chain groups and the heme-bound C-O moiety at the active center. These C-O and Fe-CO vibrations exhibited considerable variations depending on the steroids used; however, analyses on the nu Fe-CO vs nu C-O plot for cytochrome P-450scc indicated the absence of the negative correlation between these two vibrations, which is common among various Fe(2+)-porphyrin-CO complexes having imidazole ligands. Rather, we noticed the existence of two groups depending on substrates, the one exhibiting C-O infrared absorption bands in the region from 1930 to 1940 cm-1 and higher enzymatic turnover numbers in the reconstituted enzymatic systems and the other exhibiting C-O infrared absorption bands in the region above 1945 cm-1 and lower enzymatic turnover numbers. Thus, the former substrate group is likely to be fitted into the substrate binding site in the efficient "productive substrate binding" structure, whereas the latter group may be bound to the enzyme in the structure not suitable for the efficient enzymatic reaction ("nonproductive substrate binding" conformation).(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenal Cortex↗
Characterization of the cytochrome P-450IID subfamily in bovine liver. Nucleotide sequences and microheterogeneity.
To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P-450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P-450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P-450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restriction-enzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P-450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences. The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group. The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P-450IID subfamily in bovine liver. A full-length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N-terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P-450IID subfamily members showed the highly conserved C-terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.
Amino Acid Sequence↗
Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures.
Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.
Adrenal Cortex↗
Localization of metastasis suppressor gene(s) for prostatic cancer to the short arm of human chromosome 11.
Previous studies using somatic cell hybridization of highly metastatic and nonmetastatic rat prostatic cancer cells demonstrated that the resultant hybrids were nonmetastatic if all of the parental chromosomes were retained. Somatic hybrid segregants which underwent nonrandom chromosomal losses reexpressed high metastatic ability. These results demonstrated that there are gene(s) the expression of which can suppress metastatic ability of prostatic cancer cells. To identify the location of homologous gene(s) in the human, specific human chromosomes were introduced into highly metastatic rat prostatic cancer cells using the microcell-mediated chromosome transfer. Introduction of human chromosome 11 into highly metastatic rat prostate cancer cells results in suppression of metastatic ability without suppression of the in vivo growth rate or tumorigenicity of the hybrid cells. Spontaneous deletion of portions of human chromosome 11 in some of the clones delineated the minimal portion of human chromosome 11 capable of suppressing prostatic cancer metastases as the region between 11p11.2-13 but not including the Wilms' tumor-1 locus.
Animals↗
Debrisoquine 4-monooxygenase and bufuralol 1'-monooxygenase activities in bovine and rabbit tissues.
The tissue distributions of debrisoquine 4-monooxygenase and bufuralol 1'-monooxygenase activities in microsomes from bovine and rabbit tissues were analysed. Debrisoquine 4-monooxygenase and bufuralol 1'-monooxygenase activities were found in liver, and at low levels in cerebral cortex, kidney cortex, lung, small intestine and spleen. Other tissues, such as kidney medulla, adrenocortex, adrenomedulla, blood vessels, thyroid gland, heart, ovary, uterus and testis, showed low levels of bufuralol 1'-monooxygenase activity but not detectable debrisoquine 4-monooxygenase activity. The bufuralol/debrisoquine monooxygenase activity ratios were higher in kidney and lung, and lower in cerebral cortex and spleen than in liver. Both monooxygenase activities in several bovine tissues including liver were inhibited strongly by phenylisocyanide (0.1 mM) and quinidine (0.5 mM), moderately by metyrapone (1 mM), and not at all by KCN (1 mM). NaN3 (5 mM) and sodium cholate (0.5% w/v) inhibited debrisoquine 4-monooxygenase activity strongly and moderately, but bufuralol 1'-monooxygenase activity moderately and strongly, respectively. No effect of a hydroxyl radical scavenger or of superoxide dismutase on either monooxygenase activity was observed. It was concluded from these results, as well as the NADPH dependency of the reactions, that the two monooxygenase reactions observed in these tissues were catalysed by cytochrome P450s.
Animals↗
Enzyme-catalyzed oligosaccharide synthesis.
Cell-surface carbohydrates and their conjugates are involved in many types of molecular recognition. This review describes recent developments in enzyme-catalyzed oligosaccharide synthesis, with particular focus on glycosyltransferase and glycosidase reactions. With the increasing availability of glycosyltransferases via recombinant DNA technology, glycosyltransferase-catalyzed glycosylation with in situ regeneration of sugar nucleotides appears to be the most effective method for large-scale stereocontrolled oligosaccharide synthesis.
Carbohydrate Sequence↗
Expression cloning of a sheep adreno-ferredoxin using the polymerase chain reaction.
In addition to the selective amplification of cDNA from total RNA by the PCR method, the distinctive properties of ferredoxin-expressing colonies can be used for cloning a ferredoxin cDNA. This strategy for cloning and expressing cDNA in E. coli was applied to a sheep adreno-ferredoxin. The expressed sheep ferredoxin showed a spectral pattern typical of [2Fe-2S] proteins. The amino acid sequence deduced from the DNA sequence showed that the mature form of sheep ferredoxin consists of 128 amino acid residues. This rapid and simple method for cloning and expressing cDNA can be applied to other ferredoxins.
Adrenal Cortex↗
Genetic factors and suppression of metastatic ability of v-Ha-ras-transfected rat mammary cancer cells.
Following v-Ha-ras transfection of nonmetastatic dimethylbenz(( a ))anthracene-induced rat mammary cancer (RMC1) cells, occasional transfectants were isolated that acquired high metastatic ability. High metastatic ability is not a simple process regulated by v-Ha-ras p21 levels alone in these v-Ha-ras transfectants but involves the development of cytogenetic changes. If such cytogenetic changes involve only gain in gene expression, then all hybrids formed by fusing highly metastatic v-Ha-ras RMC1 transfectants with the parental nonmetastatic RMC1 should be highly metastatic. If loss of a metastatic suppressor gene(s) is also involved, then such hybrids should be nonmetastatic since chromosomes from the nonmetastatic parental cells should supply the suppressor function. To test this possibility, a highly metastatic cloned v-Ha-ras transfectant was fused with the nonmetastatic parental RMC1 cells. Five hybrid clones were isolated that conserved the chromosomes from their parental cells. When these hybrid clones were injected into animals, primary tumors developed with the same tumor-doubling time as that of the highly metastatic parental v-Ha-ras transfectant (i.e., approximately 2 days). High metastatic ability was, however, suppressed in these hybrid clones. All hybrid clones continued to express v-Ha-ras p21. Thus, suppression of metastatic ability in the hybrids can occur even in the presence of an elevated v-Ha-ras p21 level. This suggests that the acquisition of metastatic ability following v-Ha-ras transfection involves loss of metastasis suppressor gene function in rat mammary cancer cells.
Animals↗
Role of steroid 11 beta-hydroxylase and steroid 18-hydroxylase in the biosynthesis of glucocorticoids and mineralocorticoids in humans.
A gene encoding steroid 18-hydroxylase (P-450C18) was isolated from a human genomic DNA library. It was identified as CYP11B2, which was previously postulated to be a pseudogene or a less active gene closely related to CYP11B1, the gene encoding steroid 11 beta-hydroxylase (P-45011 beta) [Mornet, E., Dupont, J., Vitek, A. & White, P. C. (1989) J. Biol. Chem. 264, 20961-20967]. The nucleotide sequence of the promoter region of the P-450C18 gene is strikingly different from that of the P-45011 beta gene, although the sequences of their exons are 93% identical. The transient expression in Y-1 adrenal tumor cells of CAT constructs with a series of deletion mutants of promoter regions of both genes indicated that the two genes are regulated differently. P-450C18 as expressed in COS-7 cells exhibits steroid 18-hydroxylase activity to catalyze the synthesis of aldosterone and 18-oxocortisol and exhibits steroid 11 beta-hydroxylase activity as well. In contrast, P-45011 beta as expressed in the cultured cells exhibits steroid 11 beta-hydroxylase activity exclusively but fails to catalyze the synthesis of aldosterone and 18-oxocortisol. These results indicate that P-45011 beta and P-450C18 are products of two different genes and that the former participates in the synthesis of glucocorticoids whereas the latter participates in the synthesis of mineralocorticoids in humans.
Amino Acid Sequence↗
Differential expressions of CD25 or CD71 on CD4+ and/or CD8+ tumor cells from a patient with adult T-cell leukemia/lymphoma: three color flowcytometric analysis of peripheral blood and lymph node.
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Adult↗
Immunohistological analysis in the distribution of cells in the fetal porcine adenohypophysis.
Adenohypophyses of porcine fetuses from 25 to 110 days of gestation were studied by immunohistochemical staining to ascertain the ontogeny of specific cell types and their spatial distribution in the pars distalis. No hormone-containing cells were found before 30 days of gestation. ACTH cells were observed first at 40 days, while GH and LH cells appeared first at 60 days. PRL cells were initially detected at 105 days. ACTH immunoreactive cells were also observed in the pars intermedia at 40 days. Blood capillaries were interposed between cell cords of the pars distalis after 40 days of gestation. ACTH cells were evenly distribution in all areas of the pars distalis except the rostral area (sex zone). GH cells were densely distributed in lateral wings of the pars distalis and immediately anterior to Rathke's lumen. PRL cells resembled GH cells in their distribution pattern, but PRL cells were fewer in number. LH cells were scattered in the sex zone of the pars distalis from 60 to 80 days of gestation. After 90 days, they became scattered throughout the pars distalis but were more numerous in the sex zone than in other areas. The inductive elements of adenohypophysial cells from Rathke's pouch epithelia are discussed. We hypothesize that cell cords of specific areas facing Rathke's lumen may differentiate into specific cell types of the pars distalis during fetal life.
Adrenocorticotropic Hormone↗
Cross-reactivity of highly purified okadaic acid (OA), synthetic, spiroketal East sphere of OA and ciguatoxin.
This study presents data from the cross-reactivity analysis of purified ciguatoxin (CTX), okadaic acid (OA), and the East sphere or Fragment B-C of OA with their homologous antibodies, monoclonal antibodies to ciguatoxin (MAb-CTX) and okadaic acid (MAb-OA). The test system used was the stick enzyme immunoassay. MAb-CTX gave peak titers of 1.5ng, 10ng and 50ng respectively for CTX, East sphere and OA. Competitive inhibition analysis showed that 4ng purified CTX blocked completely MAb-CTX reaction with crude CTX, OA and East sphere of OA blocked at similar concentrations (approximately 50ng). The activity with MAb-OA in the homologous system with OA and East sphere was insignificant. This may be attributable to the improper concentrations used. The cross-reactivity between MAb-CTX with OA and its Fragment B-C may cause difficulty in the test system in its application to assess toxic fish due to ciguatoxin.
Animals↗