Sample records for s-mephenytoin 4-hydroxylase studies
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Stereoselectivity of the arene epoxide pathway of mephenytoin hydroxylation in man.
Küpfer, A; Lawson, J; Branch, R A
1984-02-01
Stereoselective metabolism of mephenytoin has been investigated in four normal subjects by comparing urinary recoveries of hydroxylated metabolites after administration of racemic RS-mephenytoin (1.4 mmol/day) and R-mephenytoin (0.7 mmol/day) on separate occasions. Gas chromatography-mass spectrometry was employed to measure the urinary recovery of 3-methyl-5-(4-hydroxyphenyl)-5-ethylhydantoin (4-OH-M) and mephenytoin catechol, methylcatechol, and dihydrodiol metabolites. Following a single oral dose of racemic mephenytoin, 4-OH-M, mephenytoin catechol, and methylcatechol metabolites were identified in urine mainly as conjugates, whereas the dihydrodiol metabolite was recovered mainly in its unconjugated form. Urinary elimination of each metabolite was similar on days 1 and 10 of chronic racemic mephenytoin administration. Following R-mephenytoin administration, urinary recoveries of hydroxylated metabolites were five to 10 times smaller than after administration of the racemic drug. This implies substrate-stereoselective hydroxylation of the S-enantiomer of mephenytoin. In one subject with a genetic deficiency of aromatic mephenytoin hydroxylation deficiency, the excretion of each hydroxylated mephenytoin metabolite after RS-mephenytoin administration was decreased to 5-15% of the values found in the four extensively hydroxylating study volunteers. The impaired formation of hydroxylated mephenytoin metabolites in genetic hydroxylation deficiency, in conjunction with stereoselective hydroxylation of S-mephenytoin via an extensive NIH shift in normal man, is consistent with the hypothesis that the formation of the S-mephenytoin arene oxide is under genetic control and represents the initial enzymatic reaction of stereoselective aromatic mephenytoin hydroxylation. The formation of this potentially reactive metabolite of S-mephenytoin may have implications in mephenytoin-induced toxicity.
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Brøsen, K; de Morais, S M; Meyer, U A; Goldstein, J A
1995-10-01
It has recently been shown that the most common mutation (named m1) in both Caucasian and Japanese poor metabolizers (PM) of S-mephenytoin is a single base pair mutation (G-->A) in exon 5 of the CYP2C19 gene. In Japanese, a second defective allele of CYP2C19 named m2 consists of a G-->A mutation in exon 4. In the present study, we have investigated the inheritance of the CYP2C19 wild type allele (wt) and the two defective alleles (m1 and m2) in families of 11 Danish PM probands. The study was carried out for two principal reasons. First, we wanted to confirm the autosomal recessive inheritance of the defective alleles, and second, we wanted to examine the specificity and sensitivity of the CYP2C19 genotyping test. Individuals were phenotyped by measuring the ratio of S/R mephenytoin excreted in the urine after administration of mephenytoin, and genotyping was carried out by a PCR-based DNA amplification procedure. The genotypes of nine of the 11 probands were consistent with their phenotypes. Eight were homozygous m1/m1, and one was heterozygous m1/m2. The genotypes of two putative PM probands (wt/m1) were not consistent with their phenotypes. On the basis of extended phenotyping (additional late urine collections (24-36 h) and acidification of urine), one of these could probably be reclassified as an extensive metabolizer (EM) while the other was considered to be a true PM. This suggests the presence of an additional unknown mutant allele in the latter. Seven of the 41 phenotyped relatives in the 11 families were phenotyped as PMs, and with the exception of the father of family 10, their genotypes (m1/m1) were consistent with their phenotypes. Extended phenotyping (acidification of urine) suggested that the father of family 10 in fact is an EM and hence that his genotype (wt/m1) is concordant with his phenotype. Thus, the specificity of genotyping tests for PM was 100%, while the sensitivity was 15/16 or 94%. Our study provides unequivocal evidence for autosomal
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Kusaka, M; Setiabudy, R; Chiba, K; Ishizaki, T
1996-02-01
A simple high-performance liquid chromatographic (HPLC) assay method was developed for the measurement of proguanil (PG) and its major metabolites, cycloguanil (CG) and 4-chlorophenyl-biguanide (CPB), in human plasma and urine. The assay allowed the simultaneous determination of all analytes in 1 ml of plasma or 0.1 ml of urine. The detection limits of PG, CG, and CPB, defined as the signal-to-noise ratio of 3, were 1 and 5 ng/ml for plasma and urine samples, respectively. Recoveries of the analytes and the internal standard (pyrimethamine) were > 62% from plasma and > 77% from urine. Intra-assay and interassay coefficients of variation for all analytes in plasma and urine were < 10% except for the values of CG and CPB, which ranged from 10% to 15% at one or two concentrations among 4-5 concentrations studied. The clinical applicability of the method was assessed by the preliminary pharmacokinetic study of PG, CG, and CPB in six healthy volunteers with the individually known phenotypes (extensive and poor metabolizers) of S-mephenytoin 4'-hydroxylation, suggesting that individuals with a poor metabolizer phenotype of S-mephenytoin have a much lower capacity to bioactivate PG to CG compared with the extensive metabolizers.
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Genetic polymorphism of (S)-mephenytoin 4′-hydroxylation in populations of African descent
Xie, H G; Kim, R B; Stein, C M; Wilkinson, G R; Wood, A J J
1999-01-01
Aims The frequency of CYP2C19 poor metabolizers (PMs) in populations of African descent has been reported to range from 1.0% to 35.4%. In order to determine with greater certainty the frequency of CYP2C19 PMs in such black populations we have performed a meta-analysis of the studies. Methods Relevant data on the frequency of both the PM phenotype of probe drugs (mephenytoin, omeprazole, and proguanil), and the distribution frequencies of CYP2C19 alleles and genotypes in black populations were summarized and reanalysed using a meta-analytical approach. Results Of nine reported studies two were excluded because of significant heterogeneity (χ2 = 115, P < 0.0001). The combined data from the remaining seven studies showed that the frequency of the PM phenotype in 922 healthy unrelated black Africans and black Americans ranged from 1.0% to 7.5% (n = 7 for combined data) with an overall frequency being 3.9% (36 of 922; 95%CI: 2.7%–5.2%). The frequency of the PM genotypes in blacks was 3.7% (36 of 966; 95%CI: 2.5%–4.9%), in agreement with the frequency of the PM phenotype. In the extensive metabolizers (EMs) 29% (271 of 930) were heterozygotes (wt/m). The observed frequencies of the three Mendelian genotypes were 0.68 for wt/wt, 0.28 for wt/m, and 0.04 for m/m. The allelic distribution was appropriate at 82.3% (95%CI: 80.5%-83.9%) for CYP2C19*1, 17.3% (95%CI:15.7%–19.0%) for CYP2C19*2 (m1), and 0.4% (95%CI: 0.1%–0.7%) for CYP2C19*3 (m2) in these populations. Conclusions We conclude that subjects of African ancestry have a low frequency of the CYP2C19 PM phenotype and genotype; that the defective CYP2C19 alleles are uncommon, and that a small proportion of heterozygotes exists in the EM subpopulation. PMID:10510153
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Dawoodji, Amina; Chen, Ji-Li; Shepherd, Dawn; Dalin, Frida; Tarlton, Andrea; Alimohammadi, Mohammad; Penna-Martinez, Marissa; Meyer, Gesine; Mitchell, Anna L; Gan, Earn H; Bratland, Eirik; Bensing, Sophie; Husebye, Eystein; Pearce, Simon H.; Badenhoop, Klaus; Kämpe, Olle; Cerundolo, Vincenzo
2016-01-01
The mechanisms behind the destruction of the adrenal glands in autoimmune Addison’s disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in over 90% of patients, but these autoantibodies are not thought to mediate the disease. Here we demonstrate highly frequent 21-hydroxylase specific T cells detectable in 20 patients with Addison’s disease. Using overlapping 18aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8+ and CD4+ T cell responses in a large proportion of Addison’s patients both ex-vivo and after in-vitro culture of peripheral blood lymphocytes up to 20 years after diagnosis. In a large proportion of patients, CD8+ 21-hydroxylase specific T cells and CD4+ 21-hydroxylase specific T cells were very abundant and detectable in ex-vivo assays. HLA class-I tetramer-guided isolation of 21-hydroxylase specific CD8+ T cells showed their ability to lyse 21-hydroxylase positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate strong cytotoxic T lymphocyte responses to 21-hydroxylase often occur in-vivo, and that reactive cytotoxic T lymphocytes have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer. PMID:25063864
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Lewandowski, M; Chui, Y C; Levi, P; Hodgson, E
1991-02-01
A simple and sensitive method for the separation of 14C-labelled acetanilide, 4-hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBA2/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [14C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.
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USDA-ARS?s Scientific Manuscript database
Cinnamic acid 4-hydroxylase (C4H) is the first hydroxylase enzyme of the phenylpropanoid pathway, and its content and activity affects the lignin synthesis. In this study, we isolated a C4H gene SbC4H1 from the suppression subtractive hybridization library of brown midrib (bmr) mutants of Sorghum b...
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Shak, S; Reich, N O; Goldstein, I M; Ortiz de Montellano, P R
1985-10-25
Human polymorphonuclear leukocytes (PMN) not only generate and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation (probably due to the action of a cytochrome P-450 enzyme). To develop pharmacologically useful inhibitors of the LTB4 omega-hydroxylase in human PMN, we devised a general scheme for synthesizing terminal acetylenic fatty acids based on the "acetylenic zipper" reaction. We found that the LTB4 omega-hydroxylase in intact PMN and in PMN sonicates is inactivated in a concentration-dependent fashion by terminal acetylenic analogues of lauric, palmitic, and stearic acids (i.e. 11-dodecynoic, 15-hexadecynoic, and 17-octadecynoic acids). Consistent with a suicidal process, inactivation of the LTB4 omega-hydroxylase requires molecular oxygen and NADPH, is time-dependent, and follows pseudo-first-order kinetics. Inactivation of the omega-hydroxylase by acetylenic fatty acids also is dependent on the terminal acetylenic moiety and the carbon chain length. Saturated fatty acids lacking a terminal acetylenic moiety do not inactivate the omega-hydroxylase. In addition, the two long-chain (C16, C18) acetylenic fatty acids inactivate the omega-hydroxylase at much lower concentrations (less than 5.0 microM) than those required for inactivation by the short-chain (C12) terminal acetylenic fatty acid (100 microM). Potent suicidal inhibitors of the LTB4 omega-hydroxylase in human PMN will help elucidate the roles played by LTB4 and its omega-oxidation products in regulating PMN function and in mediating inflammation.
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USDA-ARS?s Scientific Manuscript database
This study was conducted to clone and analyze the expression pattern of a C4H gene encoding cinnamate 4-hydroxylase from kenaf (Hibiscus cannabinus L.). A full-length C4H ortholog was cloned using degenerate primers and the RACE (rapid amplification of cDNA ends) method. The full-length C4H ortholog...
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Shak, S; Goldstein, I M
1985-09-01
Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.
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Sykes, Robert W.; Gjersing, Erica L.; Foutz, Kirk; ...
2015-08-27
In this study, lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance.
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Jaiswal, A K; Nebert, D W; Eisen, H W
1985-08-01
The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.
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Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase
Gorres, Kelly L.; Pua, Khian Hong; Raines, Ronald T.
2009-01-01
The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change. PMID:19890397
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Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu
DOE Office of Scientific and Technical Information (OSTI.GOV)
Schnicker, Nicholas J.; Razzaghi, Mortezaali; Guha Thakurta, Sanjukta
Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant,more » and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.« less
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Park, Nam Il; Park, Jee Hee; Park, Sang Un
2012-02-01
Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Miller, M.A.; Scott, E.E.; Limburg, J.
2009-05-26
Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 {angstrom} resolution.
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Long-Term Behavioral Recovery in Parkinsonian Rats by an HSV Vector Expressing Tyrosine Hydroxylase
Naegele, Janice R.; O’Malley, Karen L.; Geller, Alfred I.
2006-01-01
One therapeutic approach to treating Parkinson’s disease is to convert endogenous striatal cells into levo-3,4-dihydroxyphenylalanine (l-dopa)–producing cells. A defective herpes simplex virus type 1 vector expressing human tyrosine hydroxylase was delivered into the partially denervated striatum of 6-hydroxydopamine–lesioned rats, used as a model of Parkinson’s disease. Efficient behavioral and biochemical recovery was maintained for 1 year after gene transfer. Biochemical recovery included increases in both striatal tyrosine hydroxylase enzyme activity and in extracellular dopamine concentrations. Persistence of human tyrosine hydroxylase was revealed by expression of RNA and immunoreactivity. PMID:7669103
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John Ralph; Takuya Akiyama; Hoon Kim; Fachuang Lu; Sally A. Ralph; Clint Chapple; Ramesh B. Nair; Armin Wagner; Fang Chen; M.S. Srinivasa Reddy; Richard A Dixon; Heather D. Coleman; Shawn D. Mansfield
2006-01-01
Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in angiosperms massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to the normally dominant syringyl (S) and guaiacyl (G) units. Alfalfa stem levels of up to ~65% P (from wild-type (WT) levels of ~1%) resulting from down-regulation of C3H were measured by...
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Hlouchova, Klara; Rudolph, Johannes; Pietari, Jaana M.H.; Behlen, Linda S.; Copley, Shelley D.
2014-01-01
Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The kcat for PCP (0.024 s−1) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure/activity studies reveal that substrate binding and activity are enhanced by a low pKa for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H2O2 in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 – 100% with different substrates. Uncoupling is increased by the presence of bulky substituents in the 3-, 4-, or 5-position, and lessened by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with TCHQ, a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ. PMID:22482720
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The effects of Urtica dioica L. leaf extract on aniline 4-hydroxylase in mice.
Ozen, Tevfik; Korkmaz, Halil
2009-01-01
The effects of hydroalcoholic (80% ethanol-20% water) extract of Urtica dioica L. on microsomal aniline 4-hydroxylase (A4H) were investigated in the liver of Swiss albino mice (8- 10-weeks-old) treated with two doses (50 and 100 mg/kg body weight, given orally for 14 days ). The activities of A4H showed a significant increase in the liver at both dose levels of extract treatment. The hydroalcoholic extract of Urtica dioica induced the activities of A4H that had been increased by treatment of metal ions (Mg2+ and Ca2+) and the mixture of cofactors (NADH and NADPH). At saturated concentration of cofactor, microsomal A4H exhibited significantly even higher activities in the presence of the mixture of cofactors than NADPH and NADH. Mg2+ and Ca2+ ions acted as stimulants in vitro. The present results suggest that the hydroalcoholic extract of Urtica dioica may have modalatory effect on aniline hydroxylase at least in part and enhance the activity of A4H adding metals ions and cofactors.
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McCormick, Michael S.; Lippard, Stephen J.
2011-01-01
In all structurally characterized bacterial multicomponent monooxygenase (BMM) hydroxylase proteins, a series of hydrophobic cavities in the α-subunit trace a conserved path from the protein exterior to the carboxylate-bridged diiron active site. The present study examines these cavities as a potential route for dioxygen transport to the active site by crystallographic characterization of a xenon-pressurized sample of the hydroxylase component of phenol hydroxylase from Pseudomonas sp. OX1. Computational analyses of the hydrophobic cavities in the hydroxylase α-subunits of phenol hydroxylase (PHH), toluene/o-xylene monooxygenase (ToMOH), and soluble methane monooxygenase (sMMOH) are also presented. The results, together with previous findings from crystallographic studies of xenon-pressurized sMMO hydroxylase, clearly identify the propensity for these cavities to bind hydrophobic gas molecules in the protein interior. This proposed functional role is supported by recent stopped flow kinetic studies of ToMOH variants (Song, et al., 2011). In addition to information about the Xe sites, the structure determination revealed significantly reduced regulatory protein binding to the hydroxylase in comparison to the previously reported structure of PHH, as well as the presence of a newly identified metal binding site in the α-subunit that adopts a linear coordination environment consistent with Cu(I), and a glycerol molecule bound to Fe1 in a fashion that is unique among hydrocarbon-diiron site adducts reported to date in BMM hydroxylase structures. Finally, a comparative analysis of the α-subunit structures of MMOH, ToMOH, and PHH details proposed routes for the other three BMM substrates, the hydrocarbon, electrons, and protons, comprising cavities, channels, hydrogen-bonding networks, and pores in the structures of their α-subunits. PMID:22136180
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Somerville, Chris; Broun, Pierre; van de Loo, Frank
2001-01-01
This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.
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Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar
2016-05-25
Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H.
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Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar
2016-01-01
Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H. PMID:27220407
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Zebrafish have an ethanol-inducible hepatic 4-nitrophenol hydroxylase that is not CYP2E1-like.
Hartman, Jessica H; Kozal, Jordan S; Di Giulio, Richard T; Meyer, Joel N
2017-09-01
Zebrafish are an attractive model organism for toxicology; however, an important consideration in translating between species is xenobiotic metabolism/bioactivation. CYP2E1 metabolizes small hydrophobic molecules, e.g. ethanol, cigarette smoke, and diesel exhaust components. CYP2E1 is thought to only be conserved in mammals, but recent reports identified homologous zebrafish cytochrome P450s. Herein, ex vivo biochemical measurements show that unlike mammals, zebrafish possess a low-affinity 4-nitrophenol hydroxylase (K m ∼0.6 mM) in hepatic microsomes and mitochondria that is inducible only 1.5- to 2-fold by ethanol and is insensitive to 4-methylpyrazole inhibition. In closing, we suggest creating improved models to study CYP2E1 in zebrafish. Copyright © 2017 Elsevier B.V. All rights reserved.
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Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis.
Luthra, Pratibha Mehta; Singh, Satendra
2010-05-01
Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which L: -dopa is formed from the substrate L-tyrosine. L-Dopa concentration and activity of L-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum L-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH(4), and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K (m) value of 808.63 microM for L-tyrosine at 37 degrees C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 microM L-tyrosine. Higher concentrations of the cofactor 6-MPH(4), however, completely inhibited the synthesis of L-dopa. Tyrosine hydroxylase converted specific monophenols such as L-tyrosine (808.63 microM) and tyramine (K (m) 1.1 mM) to diphenols L-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.
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Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R
1992-01-01
The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568
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Ablation of cytochrome P450 omega-hydroxylase 4A14 gene attenuates hepatic steatosis and fibrosis
Zhang, Xiaoyan; Li, Sha; Zhou, Yunfeng; Su, Wen; Ruan, Xiongzhong; Wang, Bing; Zheng, Feng; Warner, Margaret; Gustafsson, Jan-Åke; Guan, Youfei
2017-01-01
Nonalcoholic fatty liver disease (NAFLD) is characterized by simple hepatic steatosis (SS), nonalcoholic steatohepatitis (NASH), hepatic fibrosis, and cirrhosis. Dysregulated fatty acid metabolism in the liver plays a critical role in the pathogenesis of NAFLD. Cytochrome P450 omega-hydroxylase 4A14 (CYP4A14) is a homolog of human CYP4A hydroxylase that catalyzes omega-hydroxylation of medium-chain fatty acids and arachidonic acid in mice. The goal of this study was to determine the role of CYP4A14 in the development and the progression of NAFLD. Here, we showed that hepatic CYP4A expression was up-regulated in the livers of patients and three murine models of NAFLD. Adenovirus-mediated overexpression of CYP4A14 in the livers of C57BL/6 mice resulted in a fatty liver phenotype with a significant increase in hepatic fatty acid translocase (FAT/CD36) expression. In contrast, CYP4A14 gene-deficient mice fed a high-fat diet or a methionine and choline-deficient (MCD) diet exhibited attenuated liver lipid accumulation and reduced hepatic FAT/CD36 expression. In addition, hepatic inflammation and fibrosis was markedly ameliorated in MCD diet-fed CYP4A14-deficient mice. Collectively, CYP4A14 plays an important role in the pathogenesis of both SS and NASH and may represent a potential therapeutic target for the treatment of NAFLD. PMID:28270609
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Regulation and Functional Expression of Cinnamate 4-Hydroxylase from Parsley
Koopmann, Edda; Logemann, Elke; Hahlbrock, Klaus
1999-01-01
A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit. PMID:9880345
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Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase.
Houwaart, Stefanie; Youssar, Loubna; Hüttel, Wolfgang
2014-11-03
Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhang, Yanjun; Zhao, Li; Zhao, Jiangzhe
The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant 6, At5g24530) has been proven essential in plant immunity of Arabidopsis, but its biochemical properties are not well understood. Here in this paper, we report the discovery and functional characterization of DMR6 as a SA 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBAmore » by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (K cat/K m=4.96×10 4 M -1s -1) than the previously reported SA 3-hydroxylase (S3H, K cat/K m=6.09 × 10 3 M -1s -1) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant over accumulate SA and display phenotypes such as a smaller growth size, early senescence and a loss of susceptibility to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). S5H/DMR6 is sensitively induced by SA/pathogen treatment and is widely expressed from young seedlings to senescing plants, whereas S3H is more specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.« less
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S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis
Zhang, Yanjun; Zhao, Li; Zhao, Jiangzhe; ...
2017-09-12
The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant 6, At5g24530) has been proven essential in plant immunity of Arabidopsis, but its biochemical properties are not well understood. Here in this paper, we report the discovery and functional characterization of DMR6 as a SA 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBAmore » by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (K cat/K m=4.96×10 4 M -1s -1) than the previously reported SA 3-hydroxylase (S3H, K cat/K m=6.09 × 10 3 M -1s -1) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant over accumulate SA and display phenotypes such as a smaller growth size, early senescence and a loss of susceptibility to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). S5H/DMR6 is sensitively induced by SA/pathogen treatment and is widely expressed from young seedlings to senescing plants, whereas S3H is more specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.« less
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NASA Astrophysics Data System (ADS)
Timmins, Amy; de Visser, Sam P.
2017-11-01
Prolyl-4-hydroxylase is a vital enzyme for human physiology involved in the biosynthesis of 4-hydroxyproline, an essential component for collagen formation. The enzyme performs a unique stereo- and regioselective hydroxylation at the C4 position of proline despite the fact that the C5 hydrogen atom should be thermodynamically easier to abstract. To gain insight into the mechanism and find the origin of this regioselectivity, we have done a quantum mechanics/molecular mechanics (QM/MM) study on wildtype and mutant structures. In a previous study [Timmins, Saint-André and de Visser, J. Am. Chem. Soc. 2017] we identified several active site residues critical for substrate binding and positioning. In particular, the Glu127 and Arg161 were shown to form multiple hydrogen bonding and ion-dipole interactions with substrate and could thereby affect the regio- and stereoselectivity of the reaction. In this work, we decided to test that hypothesis and report a QM/MM and molecular dynamics (MD) study on prolyl-4-hydroxylase and several active site mutants where Glu127 or Arg161 are mutated for Asp, Gln or Lys. Thus, the R161D and R161Q mutants give very high barriers for hydrogen atom abstraction from any proline C–H bond and therefore will be inactive. The R161K mutant, by contrast, sees the regio- and stereoselectivity of the reaction change but still is expected to hydroxylate proline at room temperature. By contrast, the Glu127 mutants E127D and E127Q show possible changes in regioselectivity with the former being more probable to react compared to the latter.
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Khattab, Ahmed; Haider, Shozeb; Kumar, Ameet; Dhawan, Samarth; Alam, Dauood; Romero, Raquel; Burns, James; Li, Di; Estatico, Jessica; Rahi, Simran; Fatima, Saleel; Alzahrani, Ali; Hafez, Mona; Musa, Noha; Razzghy Azar, Maryam; Khaloul, Najoua; Gribaa, Moez; Saad, Ali; Charfeddine, Ilhem Ben; Bilharinho de Mendonça, Berenice; Belgorosky, Alicia; Dumic, Katja; Dumic, Miroslav; Aisenberg, Javier; Kandemir, Nurgun; Alikasifoglu, Ayfer; Ozon, Alev; Gonc, Nazli; Cheng, Tina; Kuhnle-Krahl, Ursula; Cappa, Marco; Holterhus, Paul-Martin; Nour, Munier A; Pacaud, Daniele; Holtzman, Assaf; Li, Sun; Zaidi, Mone; Yuen, Tony; New, Maria I
2017-03-07
Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1 , a gene encoding 11β-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11β-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11β-hydroxylase deficiency CAH.
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Effects of coumarate 3-hydroxylase down-regulation on lignin structure
John Ralph; Takuya Akiyama; Hoon Kim; Fachuang Lu; Paul F. Schatz; Jane M. Marita; Sally A. Ralph; M.S. Srinivasa Reddy; Fang Chen; Richard A. Dixon
2006-01-01
Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa massively but predictably increased the proportion of p-hydroxyphenyl (P) units relative to thenormally dominant guaiacyl (G) and syringyl (S) units Stem levels of up to ~65% P (from wild-type levels of ~1%) resulting from down-regulation of C3H were measured by traditional degradative...
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Daniels, J.; Williams, J.; Asherson, P.
1995-02-27
It has been suggested that the cytochrome P450 mono-oxygenase, debrisoquine 4-hydroxylase, is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells. It is also thought to be related to the dopamine transporter that acts to take released dopamine back up into presynaptic terminals. The present study used the association approach to test the hypothesis that mutations in the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT) confer susceptibility to schizophrenia. There were no differences in allele or genotype frequencies between patients and controls in the mutations causing the poor metaboliser phenotype inmore » CYP2D6. In addition there was no association found between schizophrenia and a 48 bp repeat within the 3{prime} untranslated region of DAT. 18 refs., 2 tabs.« less
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Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.
Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine
2005-08-01
Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.
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Geijer, T; Jönsson, E; Neiman, J; Persson, M L; Brené, S; Gyllander, A; Sedvall, G; Rydberg, U; Wasserman, D; Terenius, L
1997-02-01
Associations of polymorphic genetic markers at the tyrosine hydroxylase (TH) and dopamine D4 receptor (DRD4) loci were examined in Scandinavian chronic alcoholics (n = 72) and control subjects (n = 67). Patients were divided into subgroups with regard to the presence of parental alcoholism and age of onset. Neither the TH nor the DRD4 allele distributions were significantly different when alcoholic samples were compared with control subjects. However, a tendency to high prevalence for 1 of the 5 TH alleles assayed (TH-K3) was observed in a subsample of 44 alcoholics characterized by late onset when compared with control subjects (27.3% vs. 10.6%, p = 0.041). Results suggest that no major influence on alcoholism is exerted through genes associated with the DRD4 or TH allelic markers examined.
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Regulation of hepatic 7 alpha-hydroxylase expression by dietary psyllium in the hamster.
Horton, J D; Cuthbert, J A; Spady, D K
1994-01-01
Soluble fiber consistently lowers plasma total and low density lipoprotein (LDL)-cholesterol concentrations in humans and various animal models including the hamster; however, the mechanism of this effect remains incompletely defined. We performed studies to determine the activity of dietary psyllium on hepatic 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and LDL receptor expression in the hamster. In animals fed a cholesterol-free semisynthetic diet containing 7.5% cellulose (avicel) as a fiber source, substitution of psyllium for avicel increased hepatic 7 alpha-hydroxylase activity and mRNA levels by 3-4-fold. Comparable effects on 7 alpha-hydroxylase expression were observed with 1% cholestyramine. Psyllium also increased hepatic 7 alpha-hydroxylase activity and mRNA in animals fed a diet enriched with cholesterol and triglyceride. Activation of 7 alpha-hydroxylase was associated with an increase in hepatic cholesterol synthesis that was apparently not fully compensatory since the cholesterol content of the liver declined. Although dietary psyllium did not increase hepatic LDL receptor expression in animals fed the cholesterol-free, very-low-fat diet, it did increase (or at least restore) receptor expression that had been downregulated by dietary cholesterol and triglyceride. Thus, 7.5% dietary psyllium produced effects on hepatic 7 alpha-hydroxylase and LDL metabolism that were similar to those of 1% cholestyramine. Induction of hepatic 7 alpha-hydroxylase activity by dietary psyllium may account, in large part, for the hypocholesterolemic effect of this soluble fiber. Images PMID:8182140
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Genetics Home Reference: dopamine beta-hydroxylase deficiency
... deficiency Sources for This Page Cubells JF, Zabetian CP. Human genetics of plasma dopamine beta-hydroxylase activity: ... 16. Review. Citation on PubMed Kim CH, Zabetian CP, Cubells JF, Cho S, Biaggioni I, Cohen BM, Robertson ...
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Isoform of castor oleate hydroxylase
Shanklin, John; Whittle, Edward J.
2005-12-13
The present invention relates to oleate hydroxylase genes, proteins, and methods of their use. The present invention also relates to methods of using the oleate hydroxylase genes and proteins, including in their expression in transgenic organisms and in the production of hydroxylated fatty acids.
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Hasegawa, Maki; Tahara, Harunobu; Inoue, Ryo; Kakuni, Masakazu; Tateno, Chise; Ushiki, Junko
2012-03-01
The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.
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Vela-Amieva, M; Abreu-González, M; González-del Angel, A; Ibarra-González, I; Fernández-Lainez, C; Barrientos-Ríos, R; Monroy-Santoyo, S; Guillén-López, S; Alcántara-Ortigoza, M A
2015-07-01
The mutational spectrum of the phenylalanine hydroxylase gene (PAH) in Mexico is unknown, although it has been suggested that PKU variants could have a differential geographical distribution. Genotype-phenotype correlations and genotype-based predictions of responsiveness to tetrahydrobiopterin (BH4 ) have never been performed. We sequenced the PAH gene and determined the geographic origin of each allele, mini-haplotype associated, genotype-phenotype correlations and genotype-based prediction of BH4 responsiveness in 48 Mexican patients. The mutational spectrum included 34 variants with c.60+5G>T being the most frequent (20.8%) and linked to haplotype 4.3 possibly because of a founder effect and/or genetic drift. Two new variants were found c.1A>T and c.969+6T>C. The genotype-phenotype correlation was concordant in 70.8%. The genotype-based prediction to BH4 -responsiveness was 41.7%, this information could be useful for the rational selection of candidates for BH4 testing and therapy. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Jia, Longgang; Dong, Jianzhang; Wang, Ruijie; Mao, Shuhong; Lu, Fuping; Singh, Suren; Wang, Zhengxiang; Liu, Xiaoguang
2017-08-01
Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions.
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Fossbakk, Agnete; Kleppe, Rune; Knappskog, Per M; Martinez, Aurora; Haavik, Jan
2014-01-01
Congenital tyrosine hydroxylase deficiency (THD) is found in autosomal-recessive Dopa-responsive dystonia and related neurological syndromes. The clinical manifestations of THD are variable, ranging from early-onset lethal disease to mild Parkinson disease-like symptoms appearing in adolescence. Until 2014, approximately 70 THD patients with a total of 40 different disease-related missense mutations, five nonsense mutations, and three mutations in the promoter region of the tyrosine hydroxylase (TH) gene have been reported. We collected clinical and biochemical data in the literature for all variants, and also generated mutant forms of TH variants previously not studied (N = 23). We compared the in vitro solubility, thermal stability, and kinetic properties of the TH variants to determine the cause(s) of their impaired enzyme activity, and found great heterogeneity in all these properties among the mutated forms. Some TH variants had specific kinetic anomalies and phenylalanine hydroxylase, and Dopa oxidase activities were measured for variants that showed signs of altered substrate binding. p.Arg233His, p.Gly247Ser, and p.Phe375Leu had shifted substrate specificity from tyrosine to phenylalanine and Dopa, whereas p.Cys359Phe had an impaired activity toward these substrates. The new data about pathogenic mechanisms presented are expected to contribute to develop individualized therapy for THD patients. PMID:24753243
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Zhang, Wenjiang; Ramamoorthy, Yamini; Tyndale, Rachel F; Glick, Stanley D; Maisonneuve, Isabelle M; Kuehne, Martin E; Sellers, Edward M
2002-06-01
18-Methoxycoronaridine, a newly developed ibogaine analog, has been reported to decrease the self-administration of morphine, cocaine, ethanol, and nicotine. It has also been reported to attenuate naltrexone-precipitated signs of morphine withdrawal. In this study, three metabolites of 18-methoxycoronaridine (18-MC) were separated and identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry-mass spectrometry (HPLC-ESI-MS-MS); the major metabolite was 18-hydroxycoronaridine (18-HC). The other two metabolites were elucidated as hydroxylated metabolites on the basis of their MS-MS spectra. Catalytic studies of 18-MC O-demethylase activity in human liver microsomes indicate that one high affinity enzyme is involved in this reaction (K(m) from 2.81 to 7.9 microM; V(max) from 0.045 to 0.29 nmol/mg/min). In cDNA-expressing microsomes, only CYP2C19 displayed significant 18-MC O-demethylase activity (K(m) 1.34 microM; V(max) 0.21 nmol/mg/min). S-Mephenytoin, a selective CYP2C19 inhibitor, inhibited 18-MC O-demethylation by 65% at a concentration of 2 times its K(I), and antibodies against rat 2C (human CYP2C8, 2C9, 2C19) inhibited 18-HC formation by 70%. Studies with other cytochrome P450 (P450)-selective chemical inhibitors and antibodies failed to demonstrate an appreciable role for other P450s in this reaction. In addition, in microsomes from five different human livers, 18-MC O-demethylation correlated with S-mephenytoin 4'hydroxylase activity but not with other P450 probe reactions. These data indicate that 18-HC formation is the predominant pathway of 18-MC metabolism in vitro in human liver microsomes and that this metabolic pathway is primarily catalyzed by the polymorphic CYP2C19. The apparent selectivity of this pathway for CYP2C19 suggests 18-MC as a potentially useful probe of CYP2C19 activity in vitro and in vivo.
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Sircar, Debabrata; Gaid, Mariam M; Chizzali, Cornelia; Reckwell, Dennis; Kaufholdt, David; Beuerle, Till; Broggini, Giovanni A L; Flachowsky, Henryk; Liu, Benye; Hänsch, Robert; Beerhues, Ludger
2015-06-01
Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H. © 2015 American Society of Plant Biologists. All Rights Reserved.
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Casabar, Richard C T; Wallace, Andrew D; Hodgson, Ernest; Rose, Randy L
2006-10-01
Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.
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Petrassi, Mike; Barber, Rob; Be, Celine; Beach, Sarah; Cox, Brian; D’Souza, Anne-Marie; Duggan, Nick; Hussey, Martin; Fox, Roy; Hunt, Peter; Jarai, Gabor; Kosaka, Takatoshi; Oakley, Paul; Patel, Viral; Press, Neil; Rowlands, David; Scheufler, Clemens; Schmidt, Oliver; Srinivas, Honnappa; Turner, Mary; Turner, Rob; Westwick, John; Wolfreys, Alison; Pathan, Nuzhat; Watson, Simon; Thomas, Matthew
2017-01-01
Pulmonary arterial hypertension (PAH) has demonstrated multi-serotonin receptor dependent pathologies, characterized by increased tone (5-HT1B receptor) and complex lesions (SERT, 5-HT1B, 5-HT2B receptors) of the pulmonary vasculature together with right ventricular hypertrophy, ischemia and fibrosis (5-HT2B receptor). Selective inhibitors of individual signaling elements – SERT, 5-HT2A, 5HT2B, and combined 5-HT2A/B receptors, have all been tested clinically and failed. Thus, inhibition of tryptophan hydroxylase 1 (TPH1), the rate limiting step in 5-HT synthesis, has been suggested as a more broad, and thereby more effective, mode of 5-HT inhibition. However, selectivity over non-pathogenic enzyme family members, TPH2, phenylalanine hydroxylase, and tyrosine hydroxylase has hampered therapeutic development. Here we describe the site/sequence, biochemical, and biophysical characterization of a novel allosteric site on TPH1 through which selectivity over TPH2 and related aromatic amino acid hydroxylases is achieved. We demonstrate the mechanism of action by which novel compounds selectively inhibit TPH1 using surface plasma resonance and enzyme competition assays with both tryptophan ligand and BH4 co-factor. We demonstrate 15-fold greater potency within a human carcinoid cell line versus the most potent known TPH1/2 non-specific inhibitor. Lastly, we detail a novel canine in vivo system utilized to determine effective biologic inhibition of newly synthesized 5-HT. These findings are the first to demonstrate TPH1-selective inhibition and may pave the way to a truly effective means to reduce pathologic 5-HT and thereby treat complex remodeling diseases such as PAH. PMID:28529483
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[Prolyl hydroxylase activity in liver specimens in chronic liver diseases (author's transl)].
Langness, U; Clausnitzer, H; Verspohl, M; Grasedyck, K
1978-08-25
100 patients were laparoscopied, liver tissue specimens taken from atypically altered areas. Prolyl hydroxylase was determined in the specimen, in parallel tissue was examined by light microscope. 8 groups of patients could be differentiated: Patients 1. with active, 2, with inactive cirrhosis, 3. with fatty infiltrations, 4. with fatty infiltration and mesenchymal reaction, 5. with aggressive, 6. with persistent, 7. with reactive hepatitis, 8. patients without histological changes. In the case of connective tissue increase in the liver prolyl hydroxylase activities were statistically significant above normal. In addition, there was a statistically significant difference between the enzyme activities of each group. A correlation could be found between prolyl hydroxylase activity and morphologically estimated connective tissue formation, but not the serum enzyme activities usually determined in liver diseases. Therefore, could be concluded that prolyl hydroxylase activity is an index of actual collagen biosynthesis in chronic liver diseases.
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Dawoodji, Amina; Chen, Ji-Li; Shepherd, Dawn; Dalin, Frida; Tarlton, Andrea; Alimohammadi, Mohammad; Penna-Martinez, Marissa; Meyer, Gesine; Mitchell, Anna L; Gan, Earn H; Bratland, Eirik; Bensing, Sophie; Husebye, Eystein S; Pearce, Simon H; Badenhoop, Klaus; Kämpe, Olle; Cerundolo, Vincenzo
2014-09-01
The mechanisms behind destruction of the adrenal glands in autoimmune Addison's disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in >90% of patients, but these autoantibodies are not thought to mediate the disease. In this article, we demonstrate highly frequent 21-hydroxylase-specific T cells detectable in 20 patients with Addison's disease. Using overlapping 18-aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8(+) and CD4(+) T cell responses in a large proportion of Addison's patients both ex vivo and after in vitro culture of PBLs ≤20 y after diagnosis. In a large proportion of patients, CD8(+) and CD4(+) 21-hydroxylase-specific T cells were very abundant and detectable in ex vivo assays. HLA class I tetramer-guided isolation of 21-hydroxylase-specific CD8(+) T cells showed their ability to lyse 21-hydroxylase-positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate that strong CTL responses to 21-hydroxylase often occur in vivo, and that reactive CTLs have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer. Copyright © 2014 by The American Association of Immunologists, Inc.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Bell-Lelong, D.A.; Cusumano, J.C.; Meyer, K.
1997-03-01
Cinnamate-r-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C4H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of C4H was analyzed by RNA blot hybridization analysismore » and in transgenic Arabidopsis carrying a C4H-{beta}-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1-1 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven {beta}-glucuronidase expression accurately reflected the tissue-specificity and wound-inducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis. 77 refs., 5 figs.« less
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Drevytska, T; Gonchar, E; Okhai, I; Lynnyk, O; Mankovska, I; Klionsky, D; Dosenko, V
2018-06-01
The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti-Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψ m ). Furthermore, application of anti-Hif3a siRNA led to an increase in mRNA expression of Epo, Igf1, Slc2a1/Glut-1, and Slc2a4/Glut-4. Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor, Tert, and Pdk1. Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψ m on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.
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2004-01-01
An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive α-subunit (HIF-1α or HIF-2α) and a constitutively expressed β-subunit. In normoxia, HIF-1α is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1α. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1α expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-α. Using a VHL capture assay, we demonstrate that HIF-α prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1α after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1α in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia. PMID:15104534
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Purification and properties of the hydroxylase component of methane monooxygenase
DOE Office of Scientific and Technical Information (OSTI.GOV)
Patel, R.N.; Savas, J.C.
Methane monooxygenase from Methylobacterium sp. strain CRL-26 which catalyzes the oxygenation of hydrocarbons was resolved into two components, a hydroxylase and a flavoprotein. An anaerobic procedure was developed for the purification of the hydroxylase to homogeneity. The molecular weight of the hydroxylase as determined by gel filtration was 220,000, and that determined by sedimentation equilibrium analysis was about 225,000. The purified hydroxylase contained three nonidentical subunits with molecular weights of about 55,000, 40,000, and 20,000, in equal amounts as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is an ..cap alpha../sub 2/..beta gamma.. protein. Optical absorption spectra revealedmore » peaks near 408 and 280 nm, and fluorescence spectra revealed emission peaks at 490 and 630 nm. The purified hydroxylase contained 2.8 +/- 0.2 mol of iron and 0.5 +/- 0.1 mol of zinc per mol of protein but negligible amounts of acid-labile sulfide. The antisera prepared against the hydroxylase showed cross-reactivity with hydroxylase components in soluble extracts from other methanotrophs.« less
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Alkane hydroxylase genes in psychrophile genomes and the potential for cold active catalysis.
Bowman, Jeff S; Deming, Jody W
2014-12-16
Psychrophiles are presumed to play a large role in the catabolism of alkanes and other components of crude oil in natural low temperature environments. In this study we analyzed the functional diversity of genes for alkane hydroxylases, the enzymes responsible for converting alkanes to more labile alcohols, as found in the genomes of nineteen psychrophiles for which alkane degradation has not been reported. To identify possible mechanisms of low temperature optimization we compared putative alkane hydroxylases from these psychrophiles with homologues from nineteen taxonomically related mesophilic strains. Seven of the analyzed psychrophile genomes contained a total of 27 candidate alkane hydroxylase genes, only two of which are currently annotated as alkane hydroxylase. These candidates were mostly related to the AlkB and cytochrome p450 alkane hydroxylases, but several homologues of the LadA and AlmA enzymes, significant for their ability to degrade long-chain alkanes, were also detected. These putative alkane hydroxylases showed significant differences in primary structure from their mesophile homologues, with preferences for specific amino acids and increased flexibility on loops, bends, and α-helices. A focused analysis on psychrophile genomes led to discovery of numerous candidate alkane hydroxylase genes not currently annotated as alkane hydroxylase. Gene products show signs of optimization to low temperature, including regions of increased flexibility and amino acid preferences typical of psychrophilic proteins. These findings are consistent with observations of microbial degradation of crude oil in cold environments and identify proteins that can be targeted in rate studies and in the design of molecular tools for low temperature bioremediation.
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NASA Technical Reports Server (NTRS)
Blee, K.; Choi, J. W.; O'Connell, A. P.; Jupe, S. C.; Schuch, W.; Lewis, N. G.; Bolwell, G. P.
2001-01-01
A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.
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Regulation of embryonic neurotransmitter and tyrosine hydroxylase protein levels by ascorbic acid
Meredith, M. Elizabeth; May, James M.
2013-01-01
Scope: Ascorbic acid (ascorbate) is required to recycle tetrahydrobiopterin, which is necessary for neurotransmitter synthesis by the rate-limiting enzymes tyrosine and tryptophan hydroxylases. We sought to determine whether ascorbate might regulate embryonic brain cortex monoamine synthesis utilizing transgenic mouse models with varying intracellular ascorbate levels. Methods and Results: In embryos lacking the sodium-dependent vitamin C transporter 2 (SVCT2), very low levels of brain ascorbate decreased cortex levels of norepinephrine and dopamine by approximately 33%, but had no effect on cortex serotonin or its metabolite, 5-hydroxyindole acetic acid. This decrease in ascorbate also led to a decrease in protein levels of tyrosine hydroxylase, but not of tryptophan hydroxylase. Increased cortex ascorbate in embryos carrying extra copies of the SVCT2 resulted in increased levels of dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as well as serotonin and 5-hydroxyindole acetic acid. Conclusion: The dependence of embryonic brain cortex neurotransmitter synthesis and tyrosine hydroxylase expression on intracellular ascorbate emphasizes the importance of receiving adequate ascorbate during development. PMID:24095796
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Ziebell, Angela; Gjersing, Erica; Hinchee, Maud; ...
2016-01-20
Lignin reduction through breeding and genetic modification has the potential to reduce costs in biomass processing in pulp and paper, forage, and lignocellulosic ethanol industries. Here, we present detailed characterization of the extractability and lignin structure of Eucalyptus urophylla x Eucalyptus grandis RNAi downregulated in p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) or cinnamate-4-hydroxylase (C4H). Both the C3'H and C4H downregulated lines were found to have significantly higher extractability when exposed to NaOH base extraction, indicating altered cell wall construction. The molecular weight of isolated lignin was measured and lignin structure was determined by HSQC NMR-based lignin subunit analysis for control and themore » C3'H and C4H downregulated lines. The slight reductions in average molecular weights of the lignin isolated from the transgenic lines (C3'H = 7000, C4H = 6500, control = 7300) does not appear to explain the difference in extractability. The HSQC NMR-based lignin subunit analysis showed increases in H lignin content for the C3'H but only slight differences in the lignin subunit structure of the C3'H and C4H downregulated lines when compared to the control. The greatest difference between the C3'H and C4H downregulated lines is the total lignin content; therefore, it appears that overall lowered lignin content contributes greatly to reduced recalcitrance and increased extractability of cell wall biopolymers. Furthermore, studies will be conducted to determine how the reduction in lignin content creates a less rigid cell wall that is more prone to extraction and sugar release.« less
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Kaufholdt, David; Broggini, Giovanni A.L.; Flachowsky, Henryk; Hänsch, Robert
2015-01-01
Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H. PMID:25862456
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Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás
2015-02-01
Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.
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Recommendations for the nutrition management of phenylalanine hydroxylase deficiency
Singh, Rani H.; Rohr, Fran; Frazier, Dianne; Cunningham, Amy; Mofidi, Shideh; Ogata, Beth; Splett, Patricia L.; Moseley, Kathryn; Huntington, Kathleen; Acosta, Phyllis B.; Vockley, Jerry; Van Calcar, Sandra C.
2014-01-01
The effectiveness of a phenylalanine-restricted diet to improve the outcome of individuals with phenylalanine hydroxylase deficiency (OMIM no. 261600) has been recognized since the first patients were treated 60 years ago. However, the treatment regime is complex, costly, and often difficult to maintain for the long term. Improvements and refinements in the diet for phenylalanine hydroxylase deficiency have been made over the years, and adjunctive therapies have proven to be successful for certain patients. Yet evidence-based guidelines for managing phenylalanine hydroxylase deficiency, optimizing outcomes, and addressing all available therapies are lacking. Thus, recommendations for nutrition management were developed using evidence from peer-reviewed publications, gray literature, and consensus surveys. The areas investigated included choice of appropriate medical foods, integration of adjunctive therapies, treatment during pregnancy, monitoring of nutritional and clinical markers, prevention of nutrient deficiencies, providing of access to care, and compliance strategies. This process has not only provided assessment and refinement of current nutrition management and monitoring recommendations but also charted a direction for future studies. This document serves as a companion to the concurrently published American College of Medical Genetics and Genomics guideline for the medical treatment of phenylalanine hydroxylase deficiency. Genet Med 16 2, 121–131. PMID:24385075
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Meadowcroft, Amy M.; Maier, Rayma; Johnson, Brendan M.; Jones, Delyth; Rastogi, Anjay; Zeig, Steven; Lepore, John J.; Cobitz, Alexander R.
2016-01-01
Hypoxia-inducible factor prolyl hydroxylase inhibitors stabilize levels of hypoxia-inducible factor that upregulate transcription of multiple genes associated with the response to hypoxia, including production of erythropoietin. We conducted two phase 2a studies to explore the relationship between the dose of the hypoxia-inducible factor–prolyl hydroxylase inhibitor GSK1278863 and hemoglobin response in patients with anemia of CKD (baseline hemoglobin 8.5–11.0 g/dl) not undergoing dialysis and not receiving recombinant human erythropoietin (nondialysis study) and in patients with anemia of CKD (baseline hemoglobin 9.5–12.0 g/dl) on hemodialysis and being treated with stable doses of recombinant human erythropoietin (hemodialysis study). Participants were randomized 1:1:1:1 to a once-daily oral dose of GSK1278863 (0.5 mg, 2 mg, or 5 mg) or control (placebo for the nondialysis study; continuing on recombinant human erythropoietin for the hemodialysis study) for 4 weeks, with a 2-week follow-up. In the nondialysis study, GSK1278863 produced dose-dependent effects on hemoglobin, with the highest dose resulting in a mean increase of 1 g/dl at week 4. In the hemodialysis study, treatment with GSK1278863 in the 5-mg arm maintained mean hemoglobin concentrations after the switch from recombinant human erythropoietin, whereas mean hemoglobin decreased in the lower-dose arms. In both studies, the effects on hemoglobin occurred with elevations in endogenous erythropoietin within the range usually observed in the respective populations and markedly lower than those in the recombinant human erythropoietin control arm in the hemodialysis study, and without clinically significant elevations in plasma vascular endothelial growth factor concentrations. GSK1278863 was generally safe and well tolerated at the doses and duration studied. GSK1278863 may prove an effective alternative for managing anemia of CKD. PMID:26494831
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Blass, Benjamin E; Iyer, Pravin; Abou-Gharbia, Magid; Childers, Wayne E; Gordon, John C; Ramanjulu, Mercy; Morton, George; Arumugam, Premkumar; Boruwa, Joshodeep; Ellingboe, John; Mitra, Sayan; Nimmareddy, Rajashekar Reddy; Paliwal, Shalini; Rajasekhar, Jamallamudi; Shivakumar, Savithiri; Srivastava, Pratima; Tangirala, Raghuram S; Venkataramanaiah, Konda; Yanamandra, Mahesh
2016-12-01
Metabolic Syndrome, also referred to as 'Syndrome X' or 'Insulin Resistance Syndrome,' remains a major, unmet medical need despite over 30years of intense effort. Recent research suggests that there may be a causal link between this condition and abnormal glucocorticoid processing. Specifically, dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis leads to increased systemic cortisol concentrations. Cushing' syndrome, a disorder that is also typified by a marked elevation in levels of cortisol, produces clinical symptomology that is similar to those observed in MetS, and they can be alleviated by decreasing circulating cortisol concentrations. As a result, it has been suggested that decreasing systemic cortisol concentration might have a positive impact on the progression of MetS. This could be accomplished through inhibition of enzymes in the cortisol synthetic pathway, 11β-hydroxylase (Cyp11B1), 17α-hydroxylase-C17,20-lyase (Cyp17), and 21-hydroxylase (Cyp21). We have identified a series of novel sulfonamide analogs of (2S,4R)-Ketoconazole that are potent inhibitors of these enzymes. In addition, selected members of this class of compounds have pharmacokinetic properties consistent with orally delivered drugs, making them well suited to further investigation as potential therapies for MetS. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Beckmann, Christoph; Rattke, Janine; Sperling, Petra; Heinz, Ernst; Boland, Wilhelm
2003-07-21
The stereochemical course of the dihydroceramide delta 4-(E)-desaturase from Candida albicans, cloned and expressed in the yeast Saccharomyces cerevisiae strain sur2 delta, was determined using stereospecifically labelled (2R,3S)-[2,3,4,4-2H4]-palmitic acid as a metabolic probe. Mass spectrometric analysis of the dinitrophenyl-derivatives of the labelled long-chain bases revealed elimination of a single deuterium atom from C(4) (corresponding to the C(4)-HR) along with a hydrogen atom from C(5) (corresponding to the C(5)-HS). This finding is consistent with an overall syn-elimination of the two vicinal hydrogen atoms. Besides the desaturation product sphingosine (93%) minor amounts of a 4-hydroxylated product (phytosphinganine, 7%) were identified that classify the Candida enzyme as a bifunctional desaturase/hydroxylase. Both processes, desaturation and hydroxylation proceed with loss of C(4)-HR from the chiral precursor. This finding is in agreement with a two-step process involving activation of the substrate by removal of the C(4)-HR to give a C-centred radical or radicaloid followed by either disproportionation into an olefin, water and a reduced diiron complex, or to recombination of the primary reactive intermediate with an active site-bound oxygen to yield a secondary alcohol. This result demonstrates the close mechanistic relationship between desaturation and hydroxylation as two different reaction pathways of a single enzyme and strengthens the mechanistic relationship of desaturases from fatty acid metabolism and sphingolipids.
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Danecka, Marta K; Woidy, Mathias; Zschocke, Johannes; Feillet, François; Muntau, Ania C; Gersting, Søren W
2015-03-01
In phenylketonuria, genetic heterogeneity, frequent compound heterozygosity, and the lack of functional data for phenylalanine hydroxylase genotypes hamper reliable phenotype prediction and individualised treatment. A literature search revealed 690 different phenylalanine hydroxylase genotypes in 3066 phenylketonuria patients from Europe and the Middle East. We determined phenylalanine hydroxylase function of 30 frequent homozygous and compound heterozygous genotypes covering 55% of the study population, generated activity landscapes, and assessed the phenylalanine hydroxylase working range in the metabolic (phenylalanine) and therapeutic (tetrahydrobiopterin) space. Shared patterns in genotype-specific functional landscapes were linked to biochemical and pharmacological phenotypes, where (1) residual activity below 3.5% was associated with classical phenylketonuria unresponsive to pharmacological treatment; (2) lack of defined peak activity induced loss of response to tetrahydrobiopterin; (3) a higher cofactor need was linked to inconsistent clinical phenotypes and low rates of tetrahydrobiopterin response; and (4) residual activity above 5%, a defined peak of activity, and a normal cofactor need were associated with pharmacologically treatable mild phenotypes. In addition, we provide a web application for retrieving country-specific information on genotypes and genotype-specific phenylalanine hydroxylase function that warrants continuous extension, updates, and research on demand. The combination of genotype-specific functional analyses with biochemical, clinical, and therapeutic data of individual patients may serve as a powerful tool to enable phenotype prediction and to establish personalised medicine strategies for dietary regimens and pharmacological treatment in phenylketonuria. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
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Park, Jee Hee; Park, Nam Il; Xu, Hui; Park, Sang Un
2010-08-27
Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) are important enzymes in the phenylpropanoid pathway and also in the accumulation of decursin (1) and decursinol angelate (2), which are major secondary metabolites in Angelica gigas. Using PCR with degenerate primers targeted to conserved regions of available orthologous PAL and C4H sequences, cDNAs encoding PAL and C4H from A. gigas were isolated. Both genes were used to show the comparative developmental and inducible accumulation of mRNAs in different organs and in suspension cells of A. gigas. PAL and C4H were induced most strongly in response to 300 microM methyl jasmonate treatment at 6 and 12 h, respectively, and were highly expressed in the fine roots of A. gigas. Similarly, the production of 1 and 2 was most prolific in the fine roots of the plant.
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Granvil, C P; Madan, A; Sharkawi, M; Parkinson, A; Wainer, I W
1999-04-01
The central nervous system toxicity of ifosfamide (IFF), a chiral antineoplastic agent, is thought to be dependent on its N-dechloroethylation by hepatic cytochrome P-450 (CYP) enzymes. The purpose of this study was to identify the human CYPs responsible for IFF-N-dechloroethylation and their corresponding regio- and enantioselectivities. IFF exists in two enantiomeric forms, (R) - and (S)-IFF, which can be dechloroethylated at either the N2 or N3 positions, producing the corresponding (R,S)-2-dechloroethyl-IFF [(R, S)-2-DCE-IFF] and (R,S)-3-dechloroethyl-IFF [(R,S)-3-DCE-IFF]. The results of the present study suggest that the production of (R)-2-DCE-IFF and (S)-3-DCE-IFF from (R)-IFF is catalyzed by different CYPs as is the production of (S)-2-DCE-IFF and (R)-3-DCE-IFF from (S)-IFF. In vitro studies with a bank of human liver microsomes revealed that the sample-to-sample variation in the production of (S)-3-DCE-IFF from (R)-IFF and (S)-2-DCE-IFF from (S)-IFF was highly correlated with the levels of (S)-mephenytoin N-demethylation (CYP2B6), whereas (R)-2-DCE-IFF production from (R)-IFF and (R)-3-DCE-IFF production from (S)-IFF were both correlated with the activity of testosterone 6beta-hydroxylation (CYP3A4/5). Experiments with cDNA-expressed P-450 and antibody and chemical inhibition studies supported the conclusion that the formation of (S)-3-DCE-IFF and (S)-2-DCE-IFF is catalyzed primarily by CYP2B6, whereas (R)-2-DCE-IFF and (R)-3-DCE-IFF are primarily the result of CYP3A4/5 activity.
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24-Hydroxylase: potential key regulator in hypervitaminosis D3 in growing dogs.
Tryfonidou, M A; Oosterlaken-Dijksterhuis, M A; Mol, J A; van den Ingh, T S G A M; van den Brom, W E; Hazewinkel, H A W
2003-03-01
A group of growing dogs supplemented with cholecalciferol (vitamin D(3); HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D(3)/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of P(i) and growth-regulating hormones between groups and no signs of vitamin D(3) intoxication in HVitD. For the duration of the study in HVitD vs. CVitD, plasma 25-hydroxycholecalciferol levels increased 30- to 75-fold; plasma 24,25-dihydroxycholecalciferol levels increased 12- to 16-fold and were accompanied by increased renal 24-hydroxylase gene expression, indicating increased renal 24-hydroxylase activity. Although the synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] was increased in HVitD vs. CVitD (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased renal 1alpha-hydroxylase gene expression), plasma 1,25(OH)(2)D(3) levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)(2)D(3) (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased gene expression of intestinal and renal 24-hydroxylase). A shift of the Ca set point for parathyroid hormone to the left indicated increased sensitivity of the chief cells. Effective counterbalance was provided by hypoparathyroidism, hypercalcitoninism, and the key regulator 24-hydroxylase, preventing the development of vitamin D(3) toxicosis.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yoshida, Mariko; Hiromoto, Takeshi; Hosokawa, Keiichi
2007-10-19
MobR from Comamonas testosteroni KH122-3s is a member of the MarR family of transcriptional regulators and functions as a repressor for the mobA gene that encodes a 3-hydroxybenzoate 4-hydroxylase. 3-Hydroxybenzoate binds to MobR as a ligand, resulting in an efficient induction of mobA. Various 3-hydroxybenzoate analogues were examined for their inducibilities using the mobA::lacZ transcriptional fusion system. {beta}-Galactosidase was induced by the addition of 2,3-dihydroxybenzoate or 3,5-dihydroxybenzoate besides 3-hydroxybenzoate, suggesting that the hydroxyl group at position 3 is critical in addition to the carboxyl group on the aromatic ring. A gel mobility-shift assay also showed that MobR was released frommore » the target DNA in the presence of these compounds. Circular dichroism studies demonstrated that MobR adopted two conformational states corresponding to the 3-hydroxybenzoate-bound and unbound forms. Other ligands also induced the structural change as well; however, the tertiary structures of converted forms were different from those by 3-hydroxybenzoate.« less
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Electronic structure description of the cis-MoOS unit in models for molybdenum hydroxylases.
Doonan, Christian J; Rubie, Nick D; Peariso, Katrina; Harris, Hugh H; Knottenbelt, Sushilla Z; George, Graham N; Young, Charles G; Kirk, Martin L
2008-01-09
The molybdenum hydroxylases catalyze the oxidation of numerous aromatic heterocycles and simple organics and, unlike other hydroxylases, utilize water as the source of oxygen incorporated into the product. The electronic structures of the cis-MoOS units in CoCp2[TpiPrMoVOS(OPh)] and TpiPrMoVIOS(OPh) (TpiPr = hydrotris(3-isopropylpyrazol-1-yl)borate), new models for molybdenum hydroxylases, have been studied in detail using S K-edge X-ray absorption spectroscopy, vibrational spectroscopy, and detailed bonding calculations. The results show a highly delocalized Mo=S pi* LUMO redox orbital that is formally Mo(dxy) with approximately 35% sulfido ligand character. Vibrational spectroscopy has been used to quantitate Mo-Ssulfido bond order changes in the cis-MoOS units as a function of redox state. Results support a redox active molecular orbital that has a profound influence on MoOS bonding through changes to the relative electro/nucleophilicity of the terminal sulfido ligand accompanying oxidation state changes. The bonding description for these model cis-MoOS systems supports enzyme mechanisms that are under orbital control and dominantly influenced by the unique electronic structure of the cis-MoOS site. The electronic structure of the oxidized enzyme site is postulated to play a role in polarizing a substrate carbon center for nucleophilic attack by metal activated water and acting as an electron sink in the two-electron oxidation of substrates.
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Falcioni, Francesco; Blank, Lars M.; Frick, Oliver; Karau, Andreas; Schmid, Andreas
2013-01-01
Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio- and enantio-selective proline hydroxylation with recombinant Escherichia coli expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors. PMID:23455348
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Kramer, A C; Mirto, A J; Austin, K J; Roselli, C E; Alexander, B M
2017-12-01
Dopamine synthesis in the ventral tegmental area (VTA) is necessary for the reinforcement of sexual behavior. The objective of this study determined if sexual stimuli initiates reward, and whether reward is attenuated in sexually inactive rams. Sexually active rams were exposed to urine from estrous (n=4) or ovariectomized (n=3) ewes with inactive rams (n=3) exposed to urine from estrous ewes. Following exposure, rams were exsanguinated and brains perfused. Alternating sections of the VTA were stained for Fos related antigens (FRA), tyrosine hydroxylase, and dopamine beta-hydroxylase activity. Forebrain tissue, mid-sagittal ventral to the anterior corpus callosum, was stained for dopamine D 2 receptors. Concentrations of cortisol was determined prior to and following exposure. Exposure to ovariectomized-ewe urine in sexually active rams did not influence (P=0.6) FRA expression, but fewer (P<0.05) neurons were positive for tyrosine hydroxylase in the VTA. Sexually inactive rams had fewer (P<0.05) FRA and tyrosine hydroxylase positive neurons in the VTA than sexually active rams following exposure to estrous ewe urine. VTA neurons staining positive for dopamine beta-hydroxylase did not differ by sexual activity (P=0.44) or urine exposure (P=0.07). Exposure to stimulus did not influence (P=0.46) numbers of forebrain neurons staining positive for dopamine D2 receptors in sexually active rams, but fewer (P=0.04) neurons stain positive in inactive rams. Serum concentrations of cortisol did not differ (P≥0.52) among rams prior to or following stimulus. In conclusion sexual inactivity is unlikely due to stress, but may be partially a result of decreased tyrosine hydroxylase and/or the response to dopamine. Copyright © 2017 Elsevier B.V. All rights reserved.
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An, Jeung Hee; Oh, Byung-Keun; Choi, Jeong Woo
2013-04-01
Tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosysthesis, is predominantly expressed in several cell groups within the brain, including the dopaminergic neurons of the substantia nigra and ventral tegmental area. We evaluated the efficacy of this protein-detection method in detecting tyrosine hydroxylase in normal and oxidative stress damaged dopaminergic cells. In this study, a coupling of DNA barcode and bead-based immnunoassay for detecting tyrosine hydroxylaser with PCR-like sensitivity is reported. The method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes were identified by PCR analysis. The concentration of tyrosine hydroxylase in dopaminergic cell can be easily and rapidly detected using bio-barcode assay. The bio-barcode assay is a rapid and high-throughput screening tool to detect of neurotransmitter such as dopamine.
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Yu, Bingwu; Ruppert, Martin; Stöckigt, Joachim
2002-08-01
Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for >3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme.
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Struntz, Katelyn H; Siegel, Jessica A
2018-08-01
Methamphetamine (MA) is a psychomotor stimulant drug that can alter behavior, the stress response system, and the dopaminergic system. The effects of MA can be modulated by age, however relatively little research has examined the acute effects of MA in adolescents and how the effects compare to those found in adults. The hippocampal dopamine system is altered by MA exposure and can modulate anxiety-like behavior, but the effects of MA on the hippocampal dopamine system have not been well studied, especially in adolescent animals. In order to assess potential age differences in the effects of MA exposure, this research examined the effects of acute MA exposure on locomotor and anxiety-like behavior in the open field test, plasma corticosterone levels, and hippocampal total tyrosine hydroxylase and phosphorylated tyrosine hydroxylase levels in adolescent and adult male C57BL/6 J mice. Tyrosine hydroxylase is the rate limiting enzyme in the synthesis of dopamine and was used as a marker of the hippocampal dopaminergic system. Mice were exposed to saline or 4 mg/kg MA and locomotor and anxiety-like behavior were measured in the open field test. Serum and brains were collected immediately after testing and plasma corticosterone and hippocampal total tyrosine hydroxylase and phosphorylated tyrosine hydroxylase levels measured. MA-exposed mice showed increased locomotor activity and anxiety-like behavior in the open field test compared with saline controls, regardless of age. There was no effect of MA on plasma corticosterone levels or hippocampal total tyrosine hydroxylase or phosphorylated tyrosine hydroxylase levels in either adolescent or adult mice. These data suggest that acute MA exposure during adolescence and adulthood increases locomotor activity and anxiety-like behavior but does not alter plasma corticosterone levels or hippocampal total tyrosine hydroxylase or phosphorylated tyrosine hydroxylase levels, and that these effects are not modulated by age
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Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain chol-4.
Guevara, Govinda; Heras, Laura Fernández de Las; Perera, Julián; Llorens, Juana María Navarro
2017-09-01
The 3-Ketosteroid-9α-Hydroxylase, also known as KshAB [androsta-1,4-diene-3,17-dione, NADH:oxygen oxidoreductase (9α-hydroxylating); EC 1.14.13.142)], is a key enzyme in the general scheme of the bacterial steroid catabolism in combination with a 3-ketosteroid-Δ 1 -dehydrogenase activity (KstD), being both responsible of the steroid nucleus (rings A/B) breakage. KshAB initiates the opening of the steroid ring by the 9α-hydroxylation of the C9 carbon of 4-ene-3-oxosteroids (e.g. AD) or 1,4-diene-3-oxosteroids (e.g. ADD), transforming them into 9α-hydroxy-4-androsten-3,17-dione (9OHAD) or 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD), respectively. The redundancy of these enzymes in the actinobacterial genomes results in a serious difficulty for metabolic engineering this catabolic pathway to obtain intermediates of industrial interest. In this work, we have identified three homologous kshA genes and one kshB gen in different genomic regions of R. ruber strain Chol-4. We present a set of data that helps to understand their specific roles in this strain, including: i) description of the KshAB enzymes ii) construction and characterization of ΔkshB and single, double and triple ΔkshA mutants in R. ruber iii) growth studies of the above strains on different substrates and iv) genetic complementation and biotransformation assays with those strains. Our results show that KshA2 isoform is needed for the degradation of steroid substrates with short side chain, while KshA3 works on those molecules with longer side chains. KshA1 is a more versatile enzyme related to the cholic acid catabolism, although it also collaborates with KshA2 or KshA3 activities in the catabolism of steroids. Accordingly to what it is described for other Rhodococcus strains, our results also suggest that the side chain degradation is KshAB-independent. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Involvement of CYP2D6 but not CYP2C19 in nicergoline metabolism in humans.
Böttiger, Y; Dostert, P; Benedetti, M S; Bani, M; Fiorentini, F; Casati, M; Poggesti, I; Alm, C; Alvan, G; Bertilsson, L
1996-12-01
1. Nicergoline, an ergot derivative previously used as a vasodilator, has gained a new indication in treating the symptoms of senile dementia. 2. Nicergoline is rapidly hydrolysed to an alcohol derivative, 1-methyl-10-alpha-methoxy-9,10-dihydrolysergol (MMDL), which is further N-demethylated to form 10-alpha-methoxy-9,10-dihydrolysergol (MDL). A few individuals display aberrant metabolism of this drug, as shown by their diminished capacity to form the MDL metabolite. The aim of this study was to determine whether defective nicergoline metabolism is associated with the debrisoquine and/or the S-mephenytoin hydroxylation polymorphisms. 3. After a single, oral 30 mg dose of nicergoline, the plasma concentrations of its two metabolites were studied in 15 subjects, divided into three groups with respect to their debrisoquine and S-mephenytoin hydroxylation phenotypes. 4. The pharmacokinetic parameters of MMDL and MDL were similar in the ten subjects who were extensive metabolisers of debrisoquine (five of whom were poor metabolisers of S-mephenytoin) (mean MMDL Cmax 59 nmol l-1 and AUC (0, th) 144 nmol l-1h, mean MDL Cmax 183 nmol l-1 and AUC 2627 nmol l-1h) but were markedly different from the five subjects who were poor metabolisers of debrisoquine (mean MMDL Cmax 356 nmol l-1 and AUC 10512 nmol l-1h, MDL concentrations below limit of quantitation). 5. We conclude that the formation of MDL from MMDL in the metabolism of nicergoline is catalysed to a major extent by CYP2D6 and that the observed interindividual variation in the metabolic pattern of the drug is related to the debrisoquine hydroxylation polymorphism.
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[Steroid 21-hydroxylase deficiencies and female infertility: pathophysiology and management].
Robin, G; Decanter, C; Baffet, H; Catteau-Jonard, S; Dewailly, D
2014-06-01
Steroid 21-hydroxylase deficiency is the most common adrenal genetic disease and is also named congenital adrenal hyperplasia. Depending on the severity of CYP21A2 gene mutations, there are severe or "classical" forms and moderate or "nonclassical" forms of 21-hydroxylase deficiency. The enzyme deficiency causes a disruption of adrenal steroidogenesis, which induces hyperandrogenism and elevated plasma levels of progesterone and 17-hydroxyprogesterone, the two substrates of 21-hydroxylase. These endocrine abnormalities will disrupt gonadal axis, endometrial growth and maturation and finally secretion of cervical mucus. All these phenomena contribute to a female hypofertility. Infertility is more severe in classical forms. When to become pregnant, treatment with hydrocortisone or dexamethasone can limit the production of adrenal androgens and progesterone and improves spontaneous pregnancy rates while minimizing the risk of miscarriage, which is usually relatively high in this disease. When planning pregnancy in patients with a 21-hydroxylase deficiency, genotyping the partner is required to screen for heterozygozity (1/50) and to assess the risk of transmission of a classical form in the progeny. Copyright © 2014 Elsevier Masson SAS. All rights reserved.
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Tryptophan Hydroxylase 2 Gene Polymorphism in Anxiety and Depressive Disorder in Kashmiri Population
Shoib, Sheikh; Shah, Tabindah; Mushtaq, Sahil
2014-01-01
Background: The gene of tryptophan hydroxylase is widely recognized as a major candidate gene in many psychiatric disorders. However, no study has been done which investigates tryptophan hydroxylase 2 gene polymorphism in anxiety and depressive disorders in Kashmiri population (India). Objectives: To study tryptophan hydroxylase 2 (TPH2) C 11993 A gene polymorphism in anxiety and depressive disorders. Method: Sixty patients of depression disorder, 60 patients of anxiety disorder and 40 unrelated healthy volunteers (control) were studied in a case control design. Polymorphism was determined using polymerase chain reaction (PCR) and agarose gel electrophoresis after digestion with HAP II enzyme. Genotypes and allele frequencies were compared using Chi-square tests, Fischer’s exact test, odds ratio, 95% confidence interval (C.I) and p-value of <0.05 was considered to be statistical significant. Results: The mean age ± SD of anxiety, depression and control group was 32.73±10.99, 32.20±10 and 29.75±10.12 respectively and the difference was found to be statistically non significant (p=0.349).The mean HAM-A (Hamilton rating scale for anxiety) score and HAM-D (Hamilton rating scale for depression) score was high in both groups (anxiety and depression) and found to be statistically significant (p=0.001).Depression group had AA genotype (55.2%) than control (37.5%) and was found to be statistically non significant (p=0.890).Comparison of allelic frequency revealed no association of A allele in anxiety group (76.67%) compared with control (75.5%) and was found to be statistically non significant (p= 0.866), OR 1.09 (0.56-2.11). Conclusion: TPH2C 11993 A gene was not found to be associated with major depressive disorder (MDD) and anxiety disorder in Kashmiri population. PMID:25121048
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Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Ferrando, Sara; Gallus, Lorenzo; Giovine, Marco
2015-08-01
Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and β. Here, we report the cDNA cloning, characterization, and expression analysis of the α and β subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the β subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and βP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2β2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.
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CINNAMIC ACID HYDROXYLASE IN SPINACH,
An acetone precipitate from an extract of spinach leaves catalysed the hydroxylation of trans- cinnamic acid to p-coumaric acid . The enzyme was...and addition of L-phenylalanine inhibited cinnamic acid hydroxylase activity. (Author)...Tetrahydrofolic acid and a reduced pyridine nucleotide coenzyme were necessary for maximum activity. Aminopterin was a potent inhibitor of the hydroxylating
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An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog
DOE Office of Scientific and Technical Information (OSTI.GOV)
Van De Loo, F.J.; Broun, P.; Turner, S.
1995-07-18
Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 andmore » with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.« less
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2016-01-01
Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467
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Lindgren, N; Xu, Z Q; Lindskog, M; Herrera-Marschitz, M; Goiny, M; Haycock, J; Goldstein, M; Hökfelt, T; Fisone, G
2000-06-01
The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.
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Chan, F C; Potter, G A; Barrie, S E; Haynes, B P; Rowlands, M G; Houghton, J; Jarman, M
1996-08-16
Various 3- and 4-pyridylalkyl 1-adamantanecarboxylates have been synthesized and tested for inhibitory activity toward the 17 alpha-hydroxylase and C17,20-lyase activities of human testicular cytochrome P450(17 alpha). The 4-pyridylalkyl esters were much more inhibitory than their 3-pyridylalkyl counterparts. The most potent was (S)-1-(4-pyridyl)ethyl 1-adamantanecarboxylate (3b; IC50 for lyase, 1.8 nM), whereas the (R)-enantiomer 3a was much less inhibitory (IC50 74 nM). Nearly as potent as 3b was the dimethylated counterpart, the 2-(4-pyridylpropan-2-yl) ester 5 (IC50 2.7 nM), which was also more resistant to degradation by esterases. In contrast to their 4-pyridyl analogs, the enantiomers of the 1-(3-pyridyl)ethyl ester were similarly inhibitory (IC50 for lyase; (R)-isomer 8a 150 nM, (S)-isomer 8b 230 nM). Amides corresponding to the 4-pyridylmethyl ester 1 and the (S)-1-(4-pyridyl)ethyl ester 3b, respectively 11 and 15b, were much less inhibitory than their ester counterparts. On the basis of a combination of inhibitory potency and resistance to esterases, the ester 5 was the best candidate for further development as a potential nonsteroidal inhibitor of cytochrome P450(17 alpha) for the treatment of prostate cancer.
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A Cell surface β-Hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma
Aihara, Arihiro; Huang, Chiung-Kuei; Olsen, Mark J.; Lin, Qiushi; Chung, Waihong; Tang, Qi; Dong, Xiaoqun; Wands, Jack R.
2014-01-01
Hepatocellular carcinoma (HCC) has a poor prognosis due to widespread intrahepatic and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(Asparaginyl)-β-hydroxylase (ASPH) is a cell surface enzyme that generates enhanced cell motility, migration, invasion and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer assisted drug design. Candidate compounds were tested for inhibition of β-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biologic effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor MO-I-1100 was selected since it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion and anchorage independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. Conclusions These studies suggest that the enzymatic activity of ASPH was important for hepatic oncogenesis. Reduced β-hydroxylase activity generated by the SMI MO-I-1100 led to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease. PMID:24954865
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Involvement of CYP2D6 but not CYP2C19 in nicergoline metabolism in humans
BÖTTIGER, Y.; DOSTERT, P.; STROLIN BENEDETTI, M.; BANI, M.; FIORENTINI, F.; CASATI, M.; POGGESTI, I.; ALM, C.; ALVAN, G.; BERTILSSON, L.
1996-01-01
1Nicergoline, an ergot derivative previously used as a vasodilator, has gained a new indication in treating the symptoms of senile dementia. 2Nicergoline is rapidly hydrolysed to an alcohol derivative, 1-methyl-10-α-methoxy-9,10-dihydrolysergol (MMDL), which is further N-demethylated to form 10-α-methoxy-9,10-dihydrolysergol (MDL). A few individuals display aberrant metabolism of this drug, as shown by their diminished capacity to form the MDL metabolite. The aim of this study was to determine whether defective nicergoline metabolism is associated with the debrisoquine and/or the S-mephenytoin hydroxylation polymorphisms. 3After a single, oral 30 mg dose of nicergoline, the plasma concentrations of its two metabolites were studied in 15 subjects, divided into three groups with respect to their debrisoquine and S-mephenytoin hydroxylation phenotypes. 4The pharmacokinetic parameters of MMDL and MDL were similar in the ten subjects who were extensive metabolisers of debrisoquine (five of whom were poor metabolisers of S-mephenytoin) (mean MMDL Cmax 59 nmol l−1 and AUC (0, th) 144 nmol l−1h, mean MDL Cmax 183 nmol l−1 and AUC 2627 nmol l−1h) but were markedly different from the five subjects who were poor metabolisers of debrisoquine (mean MMDL Cmax 356 nmol l−1 and AUC 10512 nmol l−1h, MDL concentrations below limit of quantitation). 5We conclude that the formation of MDL from MMDL in the metabolism of nicergoline is catalysed to a major extent by CYP2D6 and that the observed interindividual variation in the metabolic pattern of the drug is related to the debrisoquine hydroxylation polymorphism. PMID:8971425
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Lourenco-Jaramillo, Diana Lelidett; Sifuentes-Rincón, Ana María; Parra-Bracamonte, Gaspar Manuel; de la Rosa-Reyna, Xochitl Fabiola; Segura-Cabrera, Aldo; Arellano-Vera, Williams
2012-01-01
DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH) gene and 97% and 13% of the bovine dopamine β-hydroxylase (DBH) coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs) and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs) that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs) were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia) were interesting from a behavioural point of view because of the differences in temperament between these breeds. PMID:22888292
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[Tyrosine hydroxylase in telencephalon and diencephalon of Rhodeus sericeus (Cyprinidae)].
Pushchina, E V
2009-01-01
Immunohistochemical labeling of tyrosine hydroxylase was used to demonstrate catecholaminergic neuronal populations in the telencephalon and diencephalonof adult cypryniform fish Rhodeus sericeus. Various immunoreactive cell populations have been found in the telencephalon (ventral, central and lateral nuclei of ventral telencephalic area). Immunoreactive cells and fibers were discovered in dorsal nucleus of ventral telencephalic area and supracomissural nucleus in the caudal part of the telencephalon. In the diencephalon, periventricular nuclei (preoptic, periventricular nucleus of posterior tuberculum and periventricular organ) contained considerable TH-ergic cells. High activity of tyrosine hydroxylase was revealed in the pretectal, ventro-medial, ventro-lateral and suprachiasmatic nuclei. Periventricular hypothalamic nuclei also displayed high activity of tyrosine hydroxylase. Pseudounipolar neurons prevailed in all TH-immunereactive structures of the telencephalon and diencephalon: numerous bipolar liquor-contacting cells were discovered in the periventricular nuclei. Large pear-shaped cells and bipolar TH-ergic cells were found in posterior tuberculum. These cells may be functionally related to the dopamine-acquiring system.
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The Role of Oxygen Sensors, Hydroxylases, and HIF in Cardiac Function and Disease.
Townley-Tilson, W H Davin; Pi, Xinchun; Xie, Liang
2015-01-01
Ischemic heart disease is the leading cause of death worldwide. Oxygen-sensing proteins are critical components of the physiological response to hypoxia and reperfusion injury, but the role of oxygen and oxygen-mediated effects is complex in that they can be cardioprotective or deleterious to the cardiac tissue. Over 200 oxygen-sensing proteins mediate the effects of oxygen tension and use oxygen as a substrate for posttranslational modification of other proteins. Hydroxylases are an essential component of these oxygen-sensing proteins. While a major role of hydroxylases is regulating the transcription factor HIF, we investigate the increasing scope of hydroxylase substrates. This review discusses the importance of oxygen-mediated effects in the heart as well as how the field of oxygen-sensing proteins is expanding, providing a more complete picture into how these enzymes play a multifaceted role in cardiac function and disease. We also review how oxygen-sensing proteins and hydroxylase function could prove to be invaluable in drug design and therapeutic targets for heart disease.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Toerroenen, R.; Pelkonen, K.; Kaerenlampi, S.
1991-01-01
Extracts of several rodent diets were studied for their cytotoxic and aryl hydrocarbon hydroxylase-inducing properties by an in vitro method. The cell culture system based on a mouse hepatoma cell line (Hepa-1) was shown to be convenient and sensitive method for screening of diets for these parameters implying the presence of compounds potentially harmful in vivo. Considerable differences among diets and batches were detected. Smallest effects were observed with a semipurified diet and with the unrefined diet which - contrary to other four unrefined diets - contained no fish.
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Guidelines for diagnosis and treatment of 21-hydroxylase deficiency (2014 revision)
Ishii, Tomohiro; Anzo, Makoto; Adachi, Masanori; Onigata, Kazumichi; Kusuda, Satoshi; Nagasaki, Keisuke; Harada, Shohei; Horikawa, Reiko; Minagawa, Masanori; Minamitani, Kanshi; Mizuno, Haruo; Yamakami, Yuji; Fukushi, Masaru; Tajima, Toshihiro
2015-01-01
Purpose of developing the guidelines: The first guidelines for diagnosis and treatment of 21-hydroxylase deficiency (21-OHD) were published as a diagnostic handbook in Japan in 1989, with a focus on patients with severe disease. The “Guidelines for Treatment of Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency) Found in Neonatal Mass Screening (1999 revision)” published in 1999 were revised to include 21-OHD patients with very mild or no clinical symptoms. Accumulation of cases and experience has subsequently improved diagnosis and treatment of the disease. Based on these findings, the Mass Screening Committee of the Japanese Society for Pediatric Endocrinology further revised the guidelines for diagnosis and treatment. Target disease/conditions: 21-hydroxylase deficiency. Users of the guidelines: Physician specialists in pediatric endocrinology, pediatric specialists, referring pediatric practitioners, general physicians; and patients. PMID:26594092
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XAFS of human tyrosine hydroxylase
NASA Astrophysics Data System (ADS)
Meyer, W.; Haavik, J.; Winkler, H.; Trautwein, A. X.; Nolting, H.-F.
1995-02-01
Tyrosine hydroxylase (TH) catalyses the rate-limiting step (hydroxylation of tyrosine to form dihydroxyphenylalanine) in the biosynthetic pathway leading to the catecholamines dopamine, noradrenaline and adrenaline. The human enzyme (hTH) is present in four isoforms, generated by splicing of pre-mRNA. The purified apoenzyme (metal free) binds stoichiometric amounts of iron. The incorporation of Fe(II) results in a rapid and up to 40-fold increase of activity [1]. Besides the coordination of the metal centers in native enzyme we studied the purported inhibition of TH by its immediate products. So we analysed Fe-hTH isoform 1 native as well as oxidized with dopamine and Co-hTH isoform 2.
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Carlson, G P; Dziezak, J D; Johnson, K M
1979-07-01
1,2,4-Trichlorobenzene, 1,3,5-trichlorobenzene, hexachlorobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene and hexabromobenzene were compared for their abilities to induce acetanilide esterase, acentailide hydroxylase and procaine esterase. Except for hexabromobenzene all induced acetanilide esterase whereas the hydroxylation of acetanilide was seen only with the fully halogenated benzenes and with 1,3,5-tribromobenzene. Hepatic procaine esterase activity was increased by the three chlorinated benzenes and 1,2,4-tribromobenzene.
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Nagatsu, Toshiharu; Nagatsu, Ikuko
2016-11-01
Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic L-amino acid decarboxylase (AADC); dopamine β-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as L-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.
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Molitor, Christian; Mauracher, Stephan Gerhard
2016-01-01
Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571
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Pacary, Emilie; Petit, Edwige; Bernaudin, Myriam
2008-12-12
This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.
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Abramova, M A; Calas, A; Maiily, P; Thibault, J; Ugriumov, M V
1999-06-01
This study has evaluated the dynamic of intracellular vasopressin and tyrosine hydroxylase contents in the neuron cell bodies in the supraoptic nucleus and in the axons of the posterior lobe in rats drinking 2% NaCl for 1, 2, and 3 weeks. The number of vasopressin-immunoreactive neurons increased by the end of the second week of osmotic stimulation that might be explained by the onset of vasopressin synthesis in the neurons which do not synthesize this neurohormone under normal physiological conditions. The concentration of vasopressin fell down continuously during the first two weeks of salt-loading, apparently, due to predominance of the vasopressin release over its synthesis. Over the third week of salt-loading, the intracellular concentration of vasopressin was not changed significantly suggesting the establishment of the dynamic equilibrium between the vasopressin synthesis and release. The number of tyrosine hydroxylase-immunoreactive neurons and the amount of tyrosine hydroxylase in cell bodies and the large axonal swellings, Herring bodies, increased gradually showing that the rate of tyrosine hydroxylase synthesis prevailed over that of its enzymatic degradation. Thus, the chronic stimulation of vasopressin neurons is accompanied by a number of the adaptive reactions; the most important is related to the onset of vasopressin and tyrosine hydroxylase synthesis in the neurons which do not synthetize both of them under normal conditions.
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Bajpai, Anurag; Kabra, Madhulika; Menon, P S N
2006-06-01
Diagnosis of 11beta-hydroxylase deficiency was made in a boy at the age of 2 1/2 years on the basis of peripheral precocious puberty, growth acceleration (height standard deviation score +4.4) with advanced skeletal maturation (bone age 8.4 years) and elevated deoxycortisol levels. Glucocorticoid supplementation led to normalization of blood pressure but was associated with progression to central precocious puberty and increase in bone age resulting in decrease in predicted adult height to 133.7 cm (target height 163 cm). The child was started on GnRH analog (triptorelin 3.75 mg every 28 days), which led to improvement in predicted adult height by 3.1 cm over 15 months. Addition of growth hormone (0.1 IU/kg/day) resulted in improvement in predicted adult height (151 cm) and height deficit (12 cm) over the next 3.6 years. Final height (151 cm) exceeded predicted height at the initiation of GnRH analog treatment by 17.3 cm. This report suggests that combination GH and GnRH analog treatment may be useful in improving height outcome in children with 11beta-hydroxylase deficiency and compromised final height.
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Prolyl hydroxylase activity in serum and rectal mucosa in inflammatory bowel disease.
Farthing, M F; Dick, A P; Heslop, G; Levene, C I
1978-01-01
Prolyl hydroxylase activity in rectal mucosa was found to be significantly greater in 11 patients with Crohn's disease than in 11 control subjects with the irritable bowel syndrome and 16 patients with ulcerative colitis (P less than 0.005). Seven of the patients with Crohn's disease had a histologically normal rectum. This abnormality in apparently normal mucosa supports the concept that Crohn's disease is a 'continuous' disease of the gastrointestinal tract. Although there was no significant difference in prolyl hydroxylase activity between control subjects and patients with ulcerative colitis, those patients with quiescent disease tended to have lower values than those with active mucosal inflammation. Prolyl hydroxylase activity could not, however, be detected in the sera of either healthy control subjects or patients with inflammatory bowel disease. PMID:210089
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Bellward, G D; Gontovnick, L S; Otten, M
1977-01-01
Methadone-HCl added to the drinking water of adult female Wistar rats for 4 weeks produced an increase in the aryl hydrocarbon hydroxylase activity of the hepatic microsomal fraction to 222% of control levels. No change was seen in epoxide hydrase activity. In contrast, when male rats were treated similarly, there was an increase in epoxide hydrase activity to 212% of controls with no change in aryl hydrocarbon hydroxylase activity. No such changes were observed when the subcutaneous route of administration or chronic, low-dose, intraperitoneal injections were used. There were no differences in hepatic cytochrome P-450 or protein concentrations in treated animals as compared to their respective control groups. Control studies were carried out with quinine sulfate in the drinking water to decrease water intake to the level of the methadone-treated group. No elevation in either enzyme activity occurred in this control group. Similarly, paired-feeding studies showed the elevation of enzyme activity to be due to the methadone, not food deprivation. The effects of concurrent therapy of methadone with phenobarbital sodium or 3-methylcholanthrene were compared.
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Cheniany, Monireh; Ganjeali, Ali
2016-12-01
Phenylalanine-ammonia-lyase and cinnamate-4-hydroxylase play important role in the phenylpropanoid pathway, which produces many biologically important secondary metabolites participating in normal plant development. Flavonol quercetin is the main representant of these compounds that has been identified in numerous Juglans spp. In this survey, the developmental expression patterns of PAL and C4H genes during in vitro rooting of two walnut cultivars 'Sunland' and 'Howard' was examined by RT-PCR. To understand the potential role in rooting, the changing pattern of endogenous content of quercetin was also analyzed by HPLC. The 'Sunland' with better capacity to root had more quercetin content during the "inductive phase" of rooting than 'Howard'. In each cultivar, the level of PAL transcripts showed the same behavior with the changing patterns of quercetin during root formation of microshoots. The positive correlation between the changes of quercetin and PAL-mRNA indicated that PAL gene may have an immediate effect on flavonoid pathway metabolites including quercetin. Although the behavioral change of C4H expression was similar in both cultivars during root formation (with significantly more level for 'Howard'), it was not coincide with the changes of quercerin concentrations. Our results showed that C4H function is important for the normal development, but its transcriptional regulation does not correlate with quercetin as an efficient phenolic compound for walnut rhizogenesis.
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Schlangen, Karin; Miosic, Silvija; Thill, Jana; Halbwirth, Heidi
2010-07-01
A chalcone 3-hydroxylase (CH3H) cDNA clone was isolated and characterized from Cosmos sulphureus petals accumulating butein (2',3,4,4'-tetrahydroxychalcone) derivatives as yellow flower pigments. The recombinant protein catalyses the introduction of an additional hydroxyl group in the B-ring of chalcones, a reaction with high similarity to the hydroxylation of flavonoids catalysed by the well-studied flavonoid 3'-hydroxylase (F3'H). CH3H shows high specificity for chalcones, but a low F3'H activity was also detected. By contrast, the common F3'H from C. sulphureus does not accept chalcones as substrates and is therefore unlikely to be involved in the creation of the B-ring hydroxylation pattern of the yellow flower pigments. CH3H was primarily expressed in young buds, the main tissue for chalcone pigment formation. Expression levels in open flowers and 3-d-old seedlings were lower and almost no CH3H expression was observed in leaves. F3'H, in contrast, showed the highest expression also in buds, but comparable expression rates in all other tissues tested. Recombinant hybrid proteins constructed from CH3H and F3'H fragments demonstrated that amino acid residues at a substrate recognition site and an insertion of four amino acid residues in a putative loop region have an impact on chalcone acceptance. This is the first identification of a CH3H cDNA from any plant species.
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Turcu, Adina F; Nanba, Aya T; Chomic, Robert; Upadhyay, Sunil K; Giordano, Thomas J; Shields, James J; Merke, Deborah P; Rainey, William E; Auchus, Richard J
2016-05-01
To comprehensively characterize androgens and androgen precursors in classic 21-hydroxylase deficiency (21OHD) and to gain insights into the mechanisms of their formation. Serum samples were obtained from 38 patients (19 men) with classic 21OHD, aged 3-59, and 38 sex- and age-matched controls; 3 patients with 11β-hydroxylase deficiency; 4 patients with adrenal insufficiency; and 16 patients (8 men) undergoing adrenal vein sampling. Paraffin-embedded normal (n = 5) and 21OHD adrenal tissues (n = 3) were used for immunohistochemical studies. We measured 11 steroids in all sera by liquid chromatography-tandem mass spectrometry. Immunofluroescence localized 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) within the normal and 21OHD adrenals. Four 11-oxygenated 19-carbon (11oxC19) steroids were significantly higher in male and female 21OHD patients than in controls: 11β-hydroxyandrostenedione, 11-ketoandrostenedione 11β-hydroxytestosterone, and 11-ketotestosterone (3-4-fold, P < 0.0001). For 21OHD patients, testosterone and 11-ketotestosterone were positively correlated in females, but inversely correlated in males. All 11oxC19 steroids were higher in the adrenal vein than in the inferior vena cava samples from men and women and rose with cosyntropin stimulation. Only trace amounts of 11oxC19 steroids were found in the sera of patients with 11β-hydroxylase deficiency and adrenal insufficiency, confirming their adrenal origin. HSD3B2 and CYB5A immunoreactivities were sharply segregated in the normal adrenal glands, whereas areas of overlapping expression were identified in the 21OHD adrenals. All four 11oxC19 steroids are elevated in both men and women with classic 21OHD. Our data suggest that 11oxC19 steroids are specific biomarkers of adrenal-derived androgen excess. © 2016 European Society of Endocrinology.
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Oyekan, A O; Youseff, T; Fulton, D; Quilley, J; McGiff, J C
1999-10-01
Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of omega-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [(14)C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes. Sodium nitroprusside (10-100 microM), an NO donor, inhibited renal microsomal conversion of [(14)C]AA to HETEs and EETs in a dose-dependent manner. 8-bromo cGMP (100 microM), the cell-permeable analogue of cGMP, did not affect conversion of [(14)C]AA. Inhibition of NOS with N(omega)-nitro-L-arginine-methyl ester (L-NAME) significantly increased conversion of [(14)C]AA to HETE and greatly increased the expression of omega-hydroxylase protein, but this treatment had only a modest effect on epoxygenase activity. L-NAME induced a 4-fold increase in renal efflux of 20-HETE, as did L-nitroarginine. Oral treatment with 2% sodium chloride (NaCl) for 7 days increased renal epoxygenase activity, both in the cortex and the medulla. In contrast, cortical omega-hydroxylase activity was reduced by treatment with 2% NaCl. Coadministration of L-NAME and 2% NaCl decreased conversion of [(14)C]AA to HETEs without affecting epoxygenase activity. Thus, inhibition of NOS increased omega-hydroxylase activity, CYP4A expression, and renal efflux of 20-HETE, whereas 2% NaCl stimulated epoxygenase activity.
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Ma, Gang; Zhang, Lancui; Yungyuen, Witchulada; Tsukamoto, Issei; Iijima, Natsumi; Oikawa, Michiru; Yamawaki, Kazuki; Yahata, Masaki; Kato, Masaya
2016-06-29
Xanthophylls are oxygenated carotenoids and fulfill critical roles in plant growth and development. In plants, two different types of carotene hydroxylases, non-heme di-iron and heme-containing cytochrome P450, were reported to be involved in the biosynthesis of xanthophyll. Citrus fruits accumulate a high amount of xanthophylls, especially β,β-xanthophylls. To date, however, the roles of carotene hydroxylases in regulating xanthophyll content and composition have not been elucidated. In the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C) in the biosynthesis of xanthophyll in citrus fruits were investigated. Phylogenetic analysis showed that the four citrus carotene hydroxylases presented in four distinct clusters which have been identified in higher plants. CitHYb was a non-heme di-iron carotene hydroxylase, while CitCYP97A, CitCYP97B, and CitCYP97C were heme-containing cytochrome P450-type carotene hydroxylases. Gene expression results showed that the expression of CitHYb increased in the flavedo and juice sacs during the ripening process, which was well consistent with the accumulation of β,β-xanthophyll in citrus fruits. The expression of CitCYP97A and CitCYP97C increased with a peak in November, which might lead to an increase of lutein in the juice sacs during the ripening process. The expression level of CitCYP97B was much lower than that of CitHYb, CitCYP97A, and CitCYP97C in the juice sacs during the ripening process. Functional analysis showed that the CitHYb was able to catalyze the hydroxylation of the β-rings of β-carotene and α-carotene in Escherichia coli BL21 (DE3) cells. Meanwhile, when CitHYb was co-expressed with CitCYP97C, α-carotene was hydroxylated on the β-ring and ε-ring sequentially to produce lutein. CitHYb was a key gene for β,β-xanthophyll biosynthesis in citrus fruits. CitCYP97C functioned as an ε-ring hydroxylase to produce lutein using zeinoxanthin as a substrate
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lin, J.P.; Hanis, C.L.; Boerwinkle, E.
1994-09-01
Among Mexican Americans the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this study no formal genetic analysis of gallbladder disease had been carried out nor had any contributing gene been identified. The results of complex segregation analysis in a sample of 232 Mexican Americans with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will bemore » affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes {open_quotes}AA{close_quotes}, {open_quotes}Aa{close_quotes}, and {open_quotes}aa{close_quotes}, respectively, and 0.68, 0.30, and 0.00 for males, respectively. Human cholesterol 7a-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7a-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. For loci in the 5{prime}-end of the cholesterol 7a-hydroxylase gene, the frequency of the susceptibility alleles was twice as high in gallbladder disease patients compared to controls. The results of a linkage analysis provide evidence that the cholesterol 7a-hydroxylase gene and the inferred gallbladder disease gene are genetically linked.« less
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Korner, Germaine; Noain, Daniela; Ying, Ming; Hole, Magnus; Flydal, Marte I; Scherer, Tanja; Allegri, Gabriella; Rassi, Anahita; Fingerhut, Ralph; Becu-Villalobos, Damasia; Pillai, Samyuktha; Wueest, Stephan; Konrad, Daniel; Lauber-Biason, Anna; Baumann, Christian R; Bindoff, Laurence A; Martinez, Aurora; Thöny, Beat
2015-10-01
Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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de Kraker, Jan-Willem; Franssen, Maurice C. R.; Dalm, Marcella C. F.; de Groot, Aede; Bouwmeester, Harro J.
2001-01-01
Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity. PMID:11299372
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Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer
Johnson, Amanda L.; Edson, Katheryne Z.; Totah, Rheem A.; Rettie, Allan E.
2015-01-01
Cytochrome P450-dependent ω-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ω-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ω-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression. PMID:26233909
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Zhang, Nan; Han, Zhentai; Sun, Guiling; Hoffman, Angela; Wilson, Iain W; Yang, Yanfang; Gao, Qian; Wu, Jianqiang; Xie, Dan; Dai, Jungui; Qiu, Deyou
2014-01-17
Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp. Copyright © 2013 Elsevier Inc. All rights reserved.
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Pakala, Suresh B; Gorla, Purushotham; Pinjari, Aleem Basha; Krovidi, Ravi Kumar; Baru, Rajasekhar; Yanamandra, Mahesh; Merrick, Mike; Siddavattam, Dayananda
2007-01-01
A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component "A" were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component "A", typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia.
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Kharrat, M; Trabelsi, S; Chaabouni, M; Maazoul, F; Kraoua, L; Ben Jemaa, L; Gandoura, N; Barsaoui, S; Morel, Y; M'rad, R; Chaabouni, H
2010-10-01
Steroid 11β-hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, resulting in virilization, glucocorticoid deficiency and hypertension. The 11β-hydroxylase enzyme is encoded by the CYP11B1 gene and mutations in this gene are responsible for this disease. The aim of this study was to characterize mutations in the CYP11B1 gene and to determine their frequencies in a cohort of Tunisian patients. The molecular genetic analysis was performed by direct nucleotide sequencing of the CYP11B1 gene in 15 unrelated Tunisian patients suffering from classical 11β-hydroxylase deficiency. Only two mutations were detected in homozygous state in the CYP11B1 gene of all patients, the p.Q356X in exon 6 (26.6%) and the novel p.G379V in exon 7 with large prevalence (73.3%). This is the first report of screening for mutations of CYP11B1 gene in the Tunisian population and even in the Arab population. © 2010 John Wiley & Sons A/S.
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Pavon, Jorge Alex; Fitzpatrick, Paul F
2006-09-12
Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results
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Pavon, Jorge Alex; Fitzpatrick, Paul F.
2006-01-01
Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-2H]-, [3,5-2H2]-, and 2H5-phenylalanine as substrates. All Dkcat values are normal with Δ117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12–1.41. In contrast, for Δ117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the Dkcat value with [4-2H]-phenylalanine is 0.92 but is normal with [3,5-2H2]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Δ117PheH V379D shows a similar inverse isotope effect with [4-2H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-2H]-and [3,52H2]-phenylalanine, are identical for Δ117PheH and Δ117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the Dkcat value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight (∼34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With 2H5-indole-tryptophan as a substrate for Δ117PheH, the Dkcat value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the Dkcat values are within error of those for Δ117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same
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Sadiku, Pranvera; Willson, Joseph A; Dickinson, Rebecca S; Murphy, Fiona; Harris, Alison J; Lewis, Amy; Sammut, David; Mirchandani, Ananda S; Ryan, Eilise; Watts, Emily R; Thompson, A A Roger; Marriott, Helen M; Dockrell, David H; Taylor, Cormac T; Schneider, Martin; Maxwell, Patrick H; Chilvers, Edwin R; Mazzone, Massimilliano; Moral, Veronica; Pugh, Chris W; Ratcliffe, Peter J; Schofield, Christopher J; Ghesquiere, Bart; Carmeliet, Peter; Whyte, Moira Kb; Walmsley, Sarah R
2017-09-01
Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease.
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Lack of tryptophan hydroxylase-1 in mice results in gait abnormalities.
Suidan, Georgette L; Duerschmied, Daniel; Dillon, Gregory M; Vanderhorst, Veronique; Hampton, Thomas G; Wong, Siu Ling; Voorhees, Jaymie R; Wagner, Denisa D
2013-01-01
The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (-/-) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system.
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Soriano, M A; Justicia, C; Ferrer, I; Rodríguez-Farré, E; Planas, A M
1997-01-01
Dopaminergic neurons of the substantia nigra pars compacta were examined in the rat brain following striatal infarction subsequent to transient focal cerebral ischemia. Rats had the middle cerebral artery occluded for 2 h or were sham-operated, and tyrosine hydroxylase immunoreactivity was evaluated by Western blot and immunohistochemistry at different times ranging from 1 to 60 days after ischemia. The number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta was counted under the light microscope and compared to that in the contralateral side and controls. No changes of tyrosine hydroxylase immunoreactivity were detected in the ipsilateral versus the contralateral substantia nigra of sham-operated rats or 1 day after ischemia. However, a statistically significant reduction of tyrosine hydroxylase-immunoreactive cells became apparent in the ipsilateral compared with the contralateral substantia nigra at 7 and 14 days after ischemia. This reduction showed a clear recovery at 30 days after ischemia, and no signs of difference between the ipsilateral and the contralateral side were apparent by 60 days. Therefore, the reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra was only transiently seen from 1 to 2 weeks following ischemia. The observed loss of tyrosine hydroxylase was not accompanied by signs of cell death or gliosis in the ipsilateral pars compacta. The present results show a transitory reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra pars compacta after focal ischemia and suggest that striatal infarction causes a transient deficit of dopaminergic function.
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Sun, Yi; Huang, He; Meng, Li; Hu, Ke; Dai, Si-Lan
2013-10-01
As the key enzyme in the biosynthesis of blue flower color pigments, flavonoid 3',5'-hydroxylase (F3'5'H) can catalyze the conversion of its major substrates, 2-S naringenin and dihydrokaempferol, into 3',4',5'-hydroxylated pentahydroxyflavanone and dihydromyricetin, respectively. Unlike other F3'5'Hs belonging to the CYP75A subfamily, Asteraceae-specific F3'5'Hs belong to the CYP75B subfamily. Furthermore, cineraria F3'5'H expressed in yeast exhibited not only F3'H (flavonoid 3'-hydroxylase) activity but also F3'5'H activity in vitro. In this study, Southern blotting showed that there was only one copy of a homolog of the F3'5'H gene PCFH in the Pericallis × hybrida genome. This gene could be detected by Northern blot in the primary developmental stages of ligulate florets of the purple- and blue-flowered cultivars, and its transcripts also accumulated in the leaves. Heterologous expression of PCFH could produce new delphinidin derivatives in the corollas of transgenic tobacco plants, increased the content of cyanidin derivatives and lead to the blue- and red-shifting of flower color in T₀ generation plants. These results indicate that cineraria F3'5'H exhibited both F3'5'H- and F3'H-activity in vivo. The types and contents of anthocyanins and flower color phenotypes of the T₁ generation were similar to those of T₀ generation plants. PCFH exhibited stable inheritance and normal functions between generations. This study supplies new evidence to understand Asteraceae-specific F3'5'Hs and provides important references for the further study of molecular breeding of blue-flowered chrysanthemums using the PCFH gene. © 2013 Scandinavian Plant Physiology Society.
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Molin, Arnaud; Wiedemann, Arnaud; Demers, Nick; Kaufmann, Martin; Do Cao, Jérémy; Mainard, Laurent; Dousset, Brigitte; Journeau, Pierre; Abeguile, Geneviève; Coudray, Nadia; Mittre, Hervé; Richard, Nicolas; Weryha, Georges; Sorlin, Arthur; Jones, Glenville; Kottler, Marie-Laure; Feillet, Francois
2017-09-01
Vitamin D requires a two-step activation by hydroxylation: The first step is catalyzed by hepatic 25-hydroxylase (CYP2R1, 11p15.2) and the second one is catalyzed by renal 1α-hydroxylase (CYP27B1, 12q13.1), which produces the active hormonal form of 1,25-(OH) 2 D. Mutations of CYP2R1 have been associated with vitamin D-dependent rickets type 1B (VDDR1B), a very rare condition that has only been reported to affect 4 families to date. We describe 7 patients from 2 unrelated families who presented with homozygous loss-of-function mutations of CYP2R1. Heterozygous mutations were present in their normal parents. We identified a new c.124_138delinsCGG (p.Gly42_Leu46delinsArg) variation and the previously published c.296T>C (p.Leu99Pro) mutation. Functional in vitro studies confirmed loss-of-function enzymatic activity in both cases. We discuss the difficulties in establishing the correct diagnosis and the specific biochemical pattern, namely, very low 25-OH-D suggestive of classical vitamin D deficiency, in the face of normal/high concentrations of 1,25-(OH) 2 D. Siblings exhibited the three stages of rickets based on biochemical and radiographic findings. Interestingly, adult patients were able to maintain normal mineral metabolism without vitamin D supplementation. One index case presented with a partial improvement with 1alfa-hydroxyvitamin D 3 or alfacalcidol (1α-OH-D 3 ) treatment, and we observed a dramatic increase in the 1,25-(OH) 2 D serum concentration, which indicated the role of accessory 25-hydroxylase enzymes. Lastly, in patients who received calcifediol (25-OH-D 3 ), we documented normal 24-hydroxylase activity (CYP24A1). For the first time, and according to the concept of personalized medicine, we demonstrate dramatic improvements in patients who were given 25-OH-D therapy (clinical symptoms, biochemical data, and bone densitometry). In conclusion, the current study further expands the CYP2R1 mutation spectrum. We note that VDDR1B could be easily
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Ban, Jun-Gyu; Woo, Min-Woo; Lee, Bo-Ram; Lee, Mi-Jin; Choi, Si-Sun; Kim, Eung-Soo
2014-05-01
The regio-specific hydroxylation at the 4th N-methyl leucine of the immunosuppressive agent cyclosporin A (CsA) was previously proposed to be mediated by a unique cytochrome P450 hydroxylase (CYP), CYP-sb21 from the rare actinomycetes Sebekia benihana. Interestingly, a different rare actinomycetes species, Pseudonocardia autotrophica, was found to possess a different regio-selectivity, the preferential hydroxylation at the 9th N-methyl leucine of CsA. Through an in silico analysis of the whole genome of P. autotrophica, we describe here the classification of 31 total CYPs in P. autotrophica. Three putative CsA CYP genes, showing the highest sequence homologies with CYPsb21, were successfully inactivated using PCR-targeted gene disruption. Only one knock-out mutant, ΔCYP-pa1, failed to convert CsA to its hydroxylated forms. The hydroxylation activity of CsA by CYP-pa1 was confirmed by CYP-pa1 gene complementation as well as heterologous expression in the CsA non-hydroxylating Streptomyces coelicolor. Moreover, the cyclosporine regio-selectivity of CYP-pa1 expressed in the ΔCYP-sb21 S. benihana mutant strain was also confirmed unchanged through cross complementation. These results show that preferential regio-specific hydroxylation at the 9th N-methyl leucine of CsA is carried out by a specific P450 hydroxylase gene in P. autotrophica, CYP-pa1, setting the stage for the biotechnological application of CsA regioselective hydroxylation.
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Schoch, Guillaume A.; Nikov, Georgi N.; Alworth, William L.; Werck-Reichhart, Danièle
2002-01-01
The cinnamate (CA) 4-hydroxylase (C4H) is a cytochrome P450 that catalyzes the second step of the main phenylpropanoid pathway, leading to the synthesis of lignin, pigments, and many defense molecules. Salicylic acid (SA) is an essential trigger of plant disease resistance. Some plant species can synthesize SA from CA by a mechanism not yet understood. A set of specific inhibitors of the C4H, including competitive, tight-binding, mechanism-based irreversible, and quasi-irreversible inhibitors have been developed with the main objective to redirect cinnamic acid to the synthesis of SA. Competitive inhibitors such as 2-hydroxy-1-naphthoic acid and the heme-coordinating compound 3-(4-pyridyl)-acrylic acid allowed strong inhibition of C4H activity in a tobacco (Nicotiana tabacum cv Bright Yellow [BY]) cell suspension culture. This inhibition was however rapidly relieved either because of substrate accumulation or because of inhibitor metabolism. Substrate analogs bearing a methylenedioxo function such as piperonylic acid (PIP) or a terminal acetylene such as 4-propynyloxybenzoic acid (4PB), 3-propynyloxybenzoic acid, and 4-propynyloxymethylbenzoic acid are potent mechanism-based inactivators of the C4H. PIP and 4PB, the best inactivators in vitro, were also efficient inhibitors of the enzyme in BY cells. Inhibition was not reversed 46 h after cell treatment. Cotreatment of BY cells with the fungal elicitor β-megaspermin and PIP or 4PB led to a dramatic increase in SA accumulation. PIP and 4PB do not trigger SA accumulation in nonelicited cells in which the SA biosynthetic pathway is not activated. Mechanism-based C4H inactivators, thus, are promising tools for the elucidation of the CA-derived SA biosynthetic pathway and for the potentiation of plant defense. PMID:12376665
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Mahmoud, Soheil S; Williams, Matthew; Croteau, Rodney
2004-03-01
cDNA clones encoding limonene synthase and limonene-3-hydroxylase, both driven by the CaMV 35S promoter, were independently transformed into peppermint (Menthaxpiperita) to alter the production and disposition of (-)-limonene, the first committed intermediate of essential oil biosynthesis in this species. Although both genes were constitutively expressed in leaves of transformed plants, the corresponding enzyme activities were not significantly increased in the glandular trichome sites of essential oil biosynthesis; thus, there was no effect on oil yield or composition in the regenerated plants. Cosuppression of the hydroxylase gene, however, resulted in the accumulation of limonene (up to 80% of the essential oil compared to about 2% of the oil in wild type plants), without influence on oil yield. These results indicate that limonene does not impose negative feedback on the synthase, or apparently influence other enzymes of monoterpene biosynthesis in peppermint, and suggests that pathway engineering can be employed to significantly alter essential oil composition without adverse metabolic consequences.
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Retinoic Acid 4-Hydroxylase Inducibility and Clinical Response to Isotretinoin in Acne Patients
Wang, Frank; Kwak, Heh Shin R.; Elbuluk, Nada; Kaczmarek, Anya L.; Hamilton, Ted; Voorhees, John J.; Fisher, Gary J.; Kang, Sewon
2011-01-01
Background The cytochrome P450 enzyme CYP26 (retinoic acid 4-hydroxylase) initiates the catabolism of all-trans retinoic acid (tRA) and limits the effects of tRA. The CYP26 enzyme acts on tRA, but not 13-cis RA (isotretinoin), a retinoid used to treat severe acne. However, 13-cis RA can isomerize to tRA, which can then be metabolized by CYP26. Objective In healthy subjects, we assessed the variability of CYP26 enzymatic activity. We then investigated whether response to oral 13-cis RA among acne patients correlates with variability in CYP26 expression. Methods In healthy subjects, we isolated microsomal fractions from the epidermis of keratome biopsies and measured CYP26 enzymatic activity in untreated skin and skin treated with tRA. Enzymatic activity was determined based on rate of formation of 4-hydroxy RA (pg/min) per mg microsomal protein. Using real-time PCR we quantified CYP26 mRNA induction after tRA application in acne patients who responded or did not respond to one course of 13-cis RA. Results In normal skin (N=118), CYP26 enzymatic activity was widely variable (1–180 pg/min per mg microsomal fraction; mean 42.7 ± 3.5). Furthermore, CYP26 enzymatic activity was inducible in a dose-dependent manner in normal skin following tRA application, but not correlated with age or sex (N=29). In acne patients, CYP26 mRNA induction following 0.1% tRA application did not differ (P>0.05) between subjects who responded (N=8, 587±325 fold) or did not respond (N=8, 657±227 fold) to one course of 13-cis RA. Limitations The small number of acne patients treated with 13-cis RA was a major limitation. Conclusion Factors other than CYP26 activity may determine response to isotretinoin in acne. PMID:19525031
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Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.
Eachus, Helen; Zaucker, Andreas; Oakes, James A; Griffin, Aliesha; Weger, Meltem; Güran, Tülay; Taylor, Angela; Harris, Abigail; Greenfield, Andy; Quanson, Jonathan L; Storbeck, Karl-Heinz; Cunliffe, Vincent T; Müller, Ferenc; Krone, Nils
2017-12-01
Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.
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Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase
Pallan, Pradeep S.; Wang, Chunxue; Lei, Li; Yoshimoto, Francis K.; Auchus, Richard J.; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin
2015-01-01
Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. PMID:25855791
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An Unusual Case: The Comorbidity of Mood Disorder and 17-α-Hydroxylase Deficiency
TUNÇ, Serhat; YİĞİTER, Sera; ALTINBAŞ, Kürşat; KURT, Erhan; ORAL, Timuçin
2013-01-01
17-α-hydroxylase enzyme has a crucial role in the steroid biosynthesis and, deficiency of this enzyme is an autosomal recessive monogenic disorder which is one of the two hypertensive form of congenital adrenal hyperplasia. It is characterized with the deficiency in glucocorticoid, adrenal androgen, and sex steroid synthesis with concomitant mineralocorticoid excess due to genetic defect in steroid biosynthesis. The relationship of hormone system physiology with psychiatric signs and syndromes are complex. Any problem in the hypothalamo-pituitary axis may cause psychiatric syndromes. On the other hand, many psychiatric disorders, such as mood-anxiety symptoms, depression, mania, psychosis, and delirium can be seen secondary to the treatment of hormone deficiency. We present the case of a male patient with pseudohermaphroditism who has been followed and treated in Raşit Tahsin Mood Clinic with the diagnosis of mood disorder not otherwise specified and was diagnosed with 46, XY karyotype and 17-α-hydroxylase deficiency after referring to a hospital with delayed puberty. Considering the medical literature, 17-α-hydroxylase deficiency has been evaluated from the aspects of gender-related behavioral disorders, psychological developmental and anxiety disorders. To the best of our knowledge, in the medical literature, this is the first case of 17-α-hydroxylase deficiency associated with mood disorder. Here, the relationship between mood disorders and hypothalamo-pituitary axis is discussed in the light of the literature PMID:28360556
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Zhang, Dongmei; van der Wel, Hanke; Johnson, Jennifer M; West, Christopher M
2012-01-13
Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.
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Volpato, M; Prentice, L; Chen, S; Betterle, C; Rees Smith, B; Furmaniak, J
1998-01-01
Steroid 21-hydroxylase (21-OH) autoantibodies are found in patients with autoimmune Addison's disease (AAD), either isolated or associated with autoimmune polyglandular syndrome (APS) type I and II and in adrenal-cortex autoantibody (ACA)-positive patients without AAD. In order to assess any differences in the 21-OH autoantibodies in these different patient groups, we have studied their reactivity with different epitopes on 21-OH using full length and modified 35S-labelled 21-OH proteins produced in an in vitro transcription/translation system. There were no major differences in the pattern of autoantibody reactivity with the different modified 21-OH proteins in patients with isolated AAD or with APS types I and II, and in 21-OH autoantibody-positive patients with clinical AAD, subclinical AAD and those maintaining a normal adrenal function. Our studies also indicate that the main epitopes for 21-OH autoantibodies in patients with different forms of autoimmune adrenal disease are located in the C-terminal end and in a central region of 21-OH. PMID:9486414
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Feng, Guoxing; Shi, Hui; Li, Jiong
Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 atmore » the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells
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Gozal, Evelyne; Shah, Zahoor A; Pequignot, Jean-Marc; Pequignot, Jacqueline; Sachleben, Leroy R; Czyzyk-Krzeska, Maria F; Li, Richard C; Guo, Shang-Z; Gozal, David
2005-08-01
Tyrosine hydroxylase, a hypoxia-regulated gene, may be involved in tissue adaptation to hypoxia. Intermittent hypoxia, a characteristic feature of sleep apnea, leads to significant memory deficits, as well as to cortex and hippocampal apoptosis that are absent after sustained hypoxia. To examine the hypothesis that sustained and intermittent hypoxia induce different catecholaminergic responses, changes in tyrosine hydroxylase mRNA, protein expression, and activity were compared in various brain regions of male rats exposed for 6 h, 1 day, 3 days, and 7 days to sustained hypoxia (10% O(2)), intermittent hypoxia (alternating room air and 10% O(2)), or normoxia. Tyrosine hydroxylase activity, measured at 7 days, increased in the cortex as follows: sustained > intermittent > normoxia. Furthermore, activity decreased in the brain stem and was unchanged in other brain regions of sustained hypoxia-exposed rats, as well as in all regions from animals exposed to intermittent hypoxia, suggesting stimulus-specific and heterotopic catecholamine regulation. In the cortex, tyrosine hydroxylase mRNA expression was increased, whereas protein expression remained unchanged. In addition, significant differences in the time course of cortical Ser(40) tyrosine hydroxylase phosphorylation were present in the cortex, suggesting that intermittent and sustained hypoxia-induced enzymatic activity differences are related to different phosphorylation patterns. We conclude that long-term hypoxia induces site-specific changes in tyrosine hydroxylase activity and that intermittent hypoxia elicits reduced tyrosine hydroxylase recruitment and phosphorylation compared with sustained hypoxia. Such changes may not only account for differences in enzyme activity but also suggest that, with differential regional brain susceptibility to hypoxia, recruitment of different mechanisms in response to hypoxia will elicit region-specific modulation of catecholamine response.
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Ginsberg, S D; Hof, P R; Young, W G; Morrison, J H
1993-01-22
the paraventricular nucleus of the hypothalamus. The methodology employed in this study allowed for the high resolution of immunoreactive profiles through the volume of tissue being analyzed, and was more accurate than conventional light microscopy in terms of varicosity quantification. Quantitatively, a significant difference in the density of dopamine-beta-hydroxylase-immunoreactive varicosities was found between magnocellular and parvicellular regions, suggesting that parvicellular neurons received a denser noradrenergic input. These differential patterns may reflect an important functional role for norepinephrine in the regulation of anterior pituitary secretion through the hypothalamic-pituitary-adrenal stress axis.
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Lack of Tryptophan Hydroxylase-1 in Mice Results in Gait Abnormalities
Suidan, Georgette L.; Vanderhorst, Veronique; Hampton, Thomas G.; Wong, Siu Ling; Voorhees, Jaymie R.; Wagner, Denisa D.
2013-01-01
The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (−/−) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system. PMID:23516593
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Chen, Wuyan; Xu, Zhi; Nishitani, Miki; Van Ryzin, Carol; McDonnell, Nazli B; Merke, Deborah P
2012-12-01
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of cortisol biosynthesis caused by CYP21A2 mutations. An increase in gene copy number variation (CNV) exists at the CYP21A2 locus. CNV of C4, a neighboring gene that encodes complement component 4, is associated with autoimmune disease susceptibility. In this study, we performed comprehensive genetic analysis of the RP-C4-CYP21-TNX (RCCX) region in 127 unrelated 21-OHD patients (100 classic, 27 nonclassic). C4 copy number was determined by Southern blot. C4 CNV and serum C4 levels were evaluated in relation to CYP21A2 mutations and relevant phenotypes. We found that the most common CYP21A2 mutation associated with the nonclassic form of CAH, V281L, was associated with high C4 copy number (p = 7.13 × 10(-16)). Large CYP21A2 deletion, a common mutation associated with the classic form of CAH, was associated with low C4 copy number (p = 1.61 × 10(-14)). Monomodular RCCX with a short C4 gene, a risk factor for autoimmune disease, was significantly less frequent in CAH patients compared to population estimates (2.8 vs. 10.6 %; p = 1.08 × 10(-4)). In conclusion, CAH patients have increased C4 CNV, with mutation-specific associations that may be protective for autoimmune disease. The study of CYP21A2 in relation to neighboring genes provides insight into the genetics of CNV hotspots, an important determinant of human health.
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Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site*
Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H. J.
2014-01-01
Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner. PMID:25172507
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Anti-dopamine beta-hydroxylase immunotoxin-induced sympathectomy in adult rats
NASA Technical Reports Server (NTRS)
Picklo, M. J.; Wiley, R. G.; Lonce, S.; Lappi, D. A.; Robertson, D.
1995-01-01
Anti-dopamine beta-hydroxylase immunotoxin (DHIT) is an antibody-targeted noradrenergic lesioning tool comprised of a monoclonal antibody against the noradrenergic enzyme, dopamine beta-hydroxylase, conjugated to saporin, a ribosome-inactivating protein. Noradrenergic-neuron specificity and completeness and functionality of sympathectomy were assessed. Adult, male Sprague-Dawley rats were given 28.5, 85.7, 142 or 285 micrograms/kg DHIT i.v. Three days after injection, a 6% to 73% decrease in the neurons was found in the superior cervical ganglia of the animals. No loss of sensory, nodose and dorsal root ganglia, neurons was observed at the highest dose of DHIT. In contrast, the immunotoxin, 192-saporin (142 micrograms/kg), lesioned all three ganglia. To assess the sympathectomy, 2 wk after treatment (285 micrograms/kg), rats were anesthetized with urethane (1 g/kg) and cannulated in the femoral artery and vein. DHIT-treated animals' basal systolic blood pressure and heart rate were significantly lower than controls. Basal plasma norepinephrine levels were 41% lower in DHIT-treated animals than controls. Tyramine-stimulated release of norepinephrine in DHIT-treated rats was 27% of controls. Plasma epinephrine levels of DHIT animals were not reduced. DHIT-treated animals exhibited a 2-fold hypersensitivity to the alpha-adrenergic agonist phenylephrine. We conclude that DHIT selectively delivered saporin to noradrenergic neurons resulting in destruction of these neurons. Anti-dopamine beta-hydroxylase immunotoxin administration produces a rapid, irreversible sympathectomy.
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Mechanism-based inactivation of benzo(a)pyrene hydroxylase by aryl acetylenes and aryl olefins
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gan, L.S.; Lu, J.Y.L.; Alworth, W.L.
A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxgenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, 3-ethynylperylene, 2-ethynylfluorene, methyl 1-pyrenyl acetylene, cis- and trans-1-(2-bromovinyl)pyrene, and 1-allylpyrene serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo(a)pyrene hydroxylase, while 1-vinylpyrene and phenyl 1-pyrenyl acetylene do not cause a detectable suicide inhibition of benzo(a)pyrene hydroxylase. The mechanism-based loss of benzo(a)pyrene hydroxylase caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes (suicide destruction). The suicide inhibition by these aryl acetylenesmore » therefore does not involve covalent binding to the heme moiety of the monooxygenase. Nevertheless, in the presence of NADPH, /sup 3/H-labeled 1-ethynylpyrene becomes covalently attached to the cytochrome P-450 protein; the measured stoichiometry of binding is one 1-ethynylpyrene per P-450 heme unit. The authors conclude that the inhibition of benzo(a)pyrene hydroxylase produced by 1-ethynylpyrene may be related to the mechanism of suicide inhibition of P-450 activity by chloramphenicol rather than the mechanism of suicide destruction of P-450 previously described for acetylene and propyne.« less
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Characterization of carotenoid hydroxylase gene promoter in Haematococcus pluvialis.
Meng, C X; Wei, W; Su, Z- L; Qin, S
2006-10-01
Astaxanthin, a high-value ketocarotenoid is mainly used in fish aquaculture. It also has potential in human health due to its higher antioxidant capacity than beta-carotene and vitamin E. The unicellular green alga Haematococcus pluvialis is known to accumulate astaxanthin in response to environmental stresses, such as high light intensity and salt stress. Carotenoid hydroxylase plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, we report the characterization of a promoter-like region (-378 to -22 bp) of carotenoid hydroxylase gene by cloning, sequence analysis and functional verification of its 919 bp 5'-flanking region in H. pluvialis. The 5'-flanking region was characterized using micro-particle bombardment method and transient expression of LacZ reporter gene. Results of sequence analysis showed that the 5'-flanking region might have putative cis-acting elements, such as ABA (abscisic acid)-responsive element (ABRE), C-repeat/dehydration responsive element (C-repeat/DRE), ethylene-responsive element (ERE), heat-shock element (HSE), wound-responsive element (WUN-motif), gibberellin-responsive element (P-box), MYB-binding site (MBS) etc., except for typical TATA and CCAAT boxes. Results of 5' deletions construct and beta-galactosidase assays revealed that a highest promoter-like region might exist from -378 to -22 bp and some negative regulatory elements might lie in the region from -919 to -378 bp. Results of site-directed mutagenesis of a putative C-repeat/DRE and an ABRE-like motif in the promoter-like region (-378 to -22 bp) indicated that the putative C-repeat/DRE and ABRE-like motif might be important for expression of carotenoid hydroxylase gene.
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Cao, Yue-De; He, Yu-Cai; Li, Hao; Kai, Guo-Yin; Xu, Jian-He; Yu, Hui-Lei
2015-10-10
Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6β-hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus (AaH6H). A double mutant, AaH6HM1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34h cultivation of Escherichia coli cells harboring Aah6hm1 in a 5-L bioreactor with a working volume of 3L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500mgL(-1) (-)-hyoscyamine in 97% conversion, and 1.068g of the product were isolated, corresponding to a space-time yield of 251mgL(-1)d(-1). This study shows that the protein engineering of some key enzymes is a promising and effective way for improving the production of rare natural products such as scopolamine. Copyright © 2015 Elsevier B.V. All rights reserved.
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Genetics Home Reference: 17 alpha-hydroxylase/17,20-lyase deficiency
... Center Frequency 17α-hydroxylase/17,20-lyase deficiency accounts for about 1 percent of congenital adrenal hyperplasia cases. It is estimated to occur in 1 in 1 million people worldwide. Related Information What information about a genetic condition can statistics ...
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24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics
Luo, Wei; Hershberger, Pamela A.; Trump, Donald L.; Johnson, Candace S.
2013-01-01
The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linking the incidence and prognosis of certain cancers to low serum 25 (OH)D3 levels and high expression of vitamin D 24-hydroxylase supporting the idea that elevated CYP24A1 expression may stimulate degradation of vitamin D metabolites including 25-(OH)D3 and 1,25(OH)2D3. The over expression of CYP24A1 in cancer cells may be a factor affecting 1,25(OH)2D3 bioavailability and anti-proliferative activity pre-clinically and clinically. The combination of 1,25(OH)2D3 with CYP24A1 inhibitors enhances 1,25(OH)2D3 mediated signaling and anti-proliferative effects and may be useful in overcoming effects of aberrant CYP24 expression. PMID:23059474
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Evidence for the Slow Reaction of Hypoxia-Inducible Factor Prolyl Hydroxylase 2 with Oxygen
Flashman, Emily; Hoffart, Lee M.; Hamed, Refaat B.; Bollinger, J. Martin; Krebs, Carsten; Schofield, Christopher J.
2010-01-01
SUMMARY The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor (HIF). Human HIF is regulated by four Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases: Prolyl hydroxylase domain enzymes (PHDs or EGLNs) 1–3 catalyse hydroxylation of two prolyl-residues in HIF, triggering its degradation by the proteasome. Factor inhibiting HIF (FIH) catalyses hydroxylation of an asparagine-residue in HIF, inhibiting its transcriptional activity. Collectively, the HIF hydroxylases negatively regulate HIF in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 (PHD2) is the most important oxygen sensor in human cells; however the underlying kinetic basis of the oxygen sensing function of PHD2 is unclear. We report analyses of the reaction of PHD2 with oxygen. Chemical quench/mass spectrometry experiments showed that reaction of a complex of PHD2, Fe(II), 2OG and the C-terminal oxygen-dependent degradation domain of HIF-α (CODD) with oxygen to form hydroxylated CODD and succinate is much slower (~100 fold) than for other similarly studied 2OG oxygenases. Stopped flow/UV-visible spectroscopy experiments showed that the reaction produces a relatively stable species absorbing at 320nm; Mössbauer spectroscopic experiments implied that this species is likely not a Fe(IV)=O intermediate, as observed for other 2OG oxygenases. Overall the results suggest that, at least compared to other studied 2OG oxygenases, PHD2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor. PMID:20840591
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Topp, E; Xun, L Y; Orser, C S
1992-01-01
A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative. PMID:1610174
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Fu, Jian; Taubman, Mark B.
2010-01-01
The egg-laying abnormal-9 (EGLN) prolyl hydroxylases have been shown to regulate the stability and thereby the activity of the α subunits of hypoxia-inducible factor (HIF) through its ability to catalyze their hydroxylation. We have previously shown that EGLN3 promotes differentiation of C2C12 skeletal myoblasts. However, the mechanism underlying this effect remains to be fully elucidated. Here, we report that exposure of C2C12 cells to dimethyl oxalylglycine (DMOG), desferrioxamine, and hypoxia, all inhibitors of prolyl hydroxylase activity, led to repression of C2C12 myogenic differentiation. Inactivation of HIF by expression of a HIF dominant-negative mutant or deletion of HIF-1α by RNA interference did not affect the inhibitory effect of DMOG, suggesting that the effect of DMOG is HIF-independent. Pharmacologic inactivation of EGLN3 hydroxylase resulted in activation of the canonical NF-κB pathway. The inhibitory effect of DMOG on myogenic differentiation was markedly impaired in C2C12 cells expressing a dominant-negative mutant of IκBα. Exogenous expression of wild-type EGLN3, but not its catalytically inactive mutant, significantly inhibited NF-κB activation induced by overexpressed TRAF2 or IκB kinase 2. In contrast, deletion of EGLN3 by small interfering RNAs led to activation of NF-κB. These data suggest that EGLN3 is a negative regulator of NF-κB, and its prolyl hydroxylase activity is required for this effect. Furthermore, wild-type EGLN3, but not its catalytically inactive mutant, potentiated myogenic differentiation. This study demonstrates a novel role for EGLN3 in the regulation of NF-κB and suggests that it is involved in mediating myogenic differentiation, which is HIF-independent. PMID:20089853
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A Connective Tissue Disorder Caused by Mutations of the Lysyl Hydroxylase 3 Gene
Salo, Antti M.; Cox, Helen; Farndon, Peter; Moss, Celia; Grindulis, Helen; Risteli, Maija; Robins, Simon P.; Myllylä, Raili
2008-01-01
Lysyl hydroxylase 3 (LH3, encoded by PLOD3) is a multifunctional enzyme capable of catalyzing hydroxylation of lysyl residues and O-glycosylation of hydroxylysyl residues producing either monosaccharide (Gal) or disaccharide (Glc-Gal) derivatives, reactions that form part of the many posttranslational modifications required during collagen biosynthesis. Animal studies have confirmed the importance of LH3, particularly in biosynthesis of the highly glycosylated type IV and VI collagens, but to date, the functional significance in vivo of this enzyme in man is predominantly unknown. We report here a human disorder of LH3 presenting as a compound heterozygote with recessive inheritance. One mutation dramatically reduced the sugar-transfer activity of LH3, whereas another abrogated lysyl hydroxylase activity; these changes were accompanied by reduced LH3 protein levels in cells. The disorder has a unique phenotype causing severe morbidity as a result of features that overlap with a number of known collagen disorders. PMID:18834968
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Monteleone, Palmiero; Tortorella, Alfonso; Martiadis, Vassilis; Serino, Ismene; Di Filippo, Carmela; Maj, Mario
2007-06-21
Genes involved in serotonin transmission are likely involved in the biological predisposition to bulimia nervosa. We investigated whether the A218C polymorphism of the tryptophan-hydroxylase-1 gene was associated to bulimia nervosa and/or to some phenotypic aspects of the disorder. One hundred eighty Caucasian women (91 patients with bulimia nervosa and 89 healthy controls) were enrolled into the study. They underwent a blood sample collection for A218C polymorphism of the tryptophan-hydroxylase-1 genotyping and a clinical evaluation assessing comorbidity for Axis I and II psychiatric disorders, harm avoidance personality dimension and bulimic symptoms. The distribution of both tryptophan-hydroxylase-1 A218C genotypes and alleles did not significantly differ between patients and controls. Bulimic women with the AA genotype exhibited a more severe binge eating behavior and higher harm avoidance scores than those with CC genotype. These findings support the idea that tryptophan-hydroxylase-1 A218C polymorphism does not play a part in the genetic susceptibility to bulimia nervosa, but it seems to be involved in predisposing bulimic patients to a more disturbed eating behavior and higher harm avoidance.
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Flachowsky, Henryk; Halbwirth, Heidi; Treutter, Dieter; Richter, Klaus; Hanke, Magda-Viola; Szankowski, Iris; Gosch, Christian; Stich, Karl; Fischer, Thilo C
2012-02-01
Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants. Copyright © 2011 Elsevier Masson SAS. All rights reserved.
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Prosser, David E.; Kaufmann, Martin; O'Leary, Brendan; Byford, Valarie; Jones, Glenville
2007-01-01
Studies of 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1α,25-(OH)2D3, leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1α,25-(OH)2D3-26,23-lactone. The degree to which CYP24A1 performs either 23- or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design. PMID:17646648
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Fatty acid ω-hydroxylases from Solanum tuberosum.
Bjelica, Anica; Haggitt, Meghan L; Woolfson, Kathlyn N; Lee, Daniel P N; Makhzoum, Abdullah B; Bernards, Mark A
2016-12-01
Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1. Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.
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Espinosa, Pedro; Silva, Roxana A.; Sanguinetti, Nicole K.; Venegas, Francisca C.; Riquelme, Raul; González, Luis F.; Cruz, Gonzalo; Renard, Georgina M.; Moya, Pablo R.; Sotomayor-Zárate, Ramón
2016-01-01
We sought to determine the long-term changes produced by neonatal sex hormone administration on the functioning of midbrain dopaminergic neurons in adult male rats. Sprague-Dawley rats were injected subcutaneously at postnatal day 1 and were assigned to the following experimental groups: TP (testosterone propionate of 1.0 mg/50 μL); DHT (dihydrotestosterone of 1.0 mg/50 μL); EV (estradiol valerate of 0.1 mg/50 μL); and control (sesame oil of 50 μL). At postnatal day 60, neurochemical studies were performed to determine dopamine content in substantia nigra-ventral tegmental area and dopamine release in nucleus accumbens. Molecular (mRNA expression of tyrosine hydroxylase) and cellular (tyrosine hydroxylase immunoreactivity) studies were also performed. We found increased dopamine content in substantia nigra-ventral tegmental area of TP and EV rats, in addition to increased dopamine release in nucleus accumbens. However, neonatal exposure to DHT, a nonaromatizable androgen, did not affect midbrain dopaminergic neurons. Correspondingly, compared to control rats, levels of tyrosine hydroxylase mRNA and protein were significantly increased in TP and EV rats but not in DHT rats, as determined by qPCR and immunohistochemistry, respectively. Our results suggest an estrogenic mechanism involving increased tyrosine hydroxylase expression, either by direct estrogenic action or by aromatization of testosterone to estradiol in substantia nigra-ventral tegmental area. PMID:26904299
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Espinosa, Pedro; Silva, Roxana A; Sanguinetti, Nicole K; Venegas, Francisca C; Riquelme, Raul; González, Luis F; Cruz, Gonzalo; Renard, Georgina M; Moya, Pablo R; Sotomayor-Zárate, Ramón
2016-01-01
We sought to determine the long-term changes produced by neonatal sex hormone administration on the functioning of midbrain dopaminergic neurons in adult male rats. Sprague-Dawley rats were injected subcutaneously at postnatal day 1 and were assigned to the following experimental groups: TP (testosterone propionate of 1.0 mg/50 μL); DHT (dihydrotestosterone of 1.0 mg/50 μL); EV (estradiol valerate of 0.1 mg/50 μL); and control (sesame oil of 50 μL). At postnatal day 60, neurochemical studies were performed to determine dopamine content in substantia nigra-ventral tegmental area and dopamine release in nucleus accumbens. Molecular (mRNA expression of tyrosine hydroxylase) and cellular (tyrosine hydroxylase immunoreactivity) studies were also performed. We found increased dopamine content in substantia nigra-ventral tegmental area of TP and EV rats, in addition to increased dopamine release in nucleus accumbens. However, neonatal exposure to DHT, a nonaromatizable androgen, did not affect midbrain dopaminergic neurons. Correspondingly, compared to control rats, levels of tyrosine hydroxylase mRNA and protein were significantly increased in TP and EV rats but not in DHT rats, as determined by qPCR and immunohistochemistry, respectively. Our results suggest an estrogenic mechanism involving increased tyrosine hydroxylase expression, either by direct estrogenic action or by aromatization of testosterone to estradiol in substantia nigra-ventral tegmental area.
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Vadasz, Csaba; Smiley, John F.; Figarsky, Khadija; Saito, Mariko; Toth, Reka; Gyetvai, Beatrix M.; Oros, Melinda; Kovacs, Krisztina K.; Mohan, Panaiyur; Wang, Ray
2007-01-01
The mesotelencephalic dopamine system shows substantial genetic variation which fundamentally affects normal and pathological behaviors related to motor function, motivation, and learning. Our earlier radioenzyme assay studies demonstrated significantly higher activity of tyrosine hydroxylase (TH), the first and rate limiting enzyme in the biosynthesis of catecholamine neurotransmitters, in the substantia nigra – ventral tegmental area of BALB/cJ mice in comparison with that of C57BL/6ByJ mice. Here, using quantitative immunoblotting and immunocytochemistry, we tested the hypothesis that mesencephalic TH protein content and number of nigral TH-positive neurons show strain-dependent differences in C57BL/6ByJ and BALB/cJ parallel to those observed in the TH activity studies. Immunoblotting experiments detected significantly higher mesencephalic TH protein content in BALB/cJ in comparison to C57BL/6ByJ (p<0.05). Immunocytochemical studies demonstrated that the number of TH-positive cells in substantia nigra was 31.3% higher in BALB/cJ than that in C57BL/6ByJ (p<0.01), while the average dopamine neuron volume was not significantly different. In a search for candidate genes that modulate TH content and the size of mesencephalic dopamine neuron populations we also studied near-isogenic mouse sublines derived from the C57BL/6ByJ and BALB/cJ progenitor strains. A whole-genome scan with 768 single nucleotide polymorphism markers indicated that two sublines, C4A6/N and C4A6/B, were genetically very similar (98.3%). We found significantly higher mesencephalic tyrosine hydroxylase (TH) protein content in C4A6/B in comparison to C4A6/N (p=0.01), and a tendency for higher number of dopamine neurons in the substantia nigra in C4A6/B in comparison to C4A6/N, which, however, did not reach statistical significance. To identify the genetic source of the TH content difference we analyzed the SNP genotype data of the whole-genome scan, and detected two small differential chromosome
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Capyk, Jenna K; D'Angelo, Igor; Strynadka, Natalie C; Eltis, Lindsay D
2009-04-10
KshAB (3-Ketosteroid 9alpha-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O(2). Nevertheless, the reactivity of KshAB with O(2) was low in the presence of 1,4-androstadiene-3,17-dione, with a k(cat)/K(m)(O(2)) of 2450 +/- 80 m(-1) s(-1). The crystallographic structure of KshA, determined to 2.3A(,) revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance.
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High temperature neutron powder diffraction study of the Cu12Sb4S13 and Cu4Sn7S16 phases
NASA Astrophysics Data System (ADS)
Lemoine, Pierric; Bourgès, Cédric; Barbier, Tristan; Nassif, Vivian; Cordier, Stéphane; Guilmeau, Emmanuel
2017-03-01
Ternary copper-containing sulfides Cu12Sb4S13 and Cu4Sn7S16 have attracted considerable interest since few years due to their high-efficiency conversion as absorbers for solar energy and promising thermoelectric materials. We report therein on the decomposition study of Cu12Sb4S13 and Cu4Sn7S16 phases using high temperature in situ neutron powder diffraction. Our results obtained at a heating rate of 2.5 K/min indicate that: (i) Cu12Sb4S13 decomposes above ≈792 K into Cu3SbS3, and (ii) Cu4Sn7S16 decomposes above ≈891 K into Sn2S3 and a copper-rich sulfide phase of sphalerite ZnS-type structure with an assumed Cu3SnS4 stoichiometry. Both phase decompositions are associated to a sulfur volatilization. While the results on Cu12Sb4S13 are in fair agreement with recent published data, the decomposition behavior of Cu4Sn7S16 differs from other studies in terms of decomposition temperature, thermal stability and products of reaction. Finally, the crystal structure refinements from neutron powder diffraction data are reported and discussed for the Cu4Sn7S16 and tetrahedrite Cu12Sb4S13 phases at 300 K, and for the high temperature form of skinnerite Cu3SbS3 at 843 K.
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Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*
Penfield, Jonathan S.; Worrall, Liam J.; Strynadka, Natalie C.; Eltis, Lindsay D.
2014-01-01
KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >105 s−1 m−1 for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (KiS ∼100 μm). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450. PMID:25049233
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Sadeghi, Mahnaz; Dehghan, Sara; Fischer, Rainer; Wenzel, Uwe; Vilcinskas, Andreas; Kavousi, Hamid Reza; Rahnamaeian, Mohammad
2013-01-01
Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3–6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6–24 h and 3–6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance. PMID:24309561
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Kojima, Misaki; Sekikawa, Kenji; Nemoto, Kiyomitsu; Degawa, Masakuni
2005-10-01
We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor-alpha (TNF-alpha), downregulated gene expression of cholesterol 7alpha-hydroxylase. Herein, to clarify the role of TNF-alpha in LN-induced downregulation of cholesterol 7alpha-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) in TNF-alpha-knockout (KO) and TNF-alpha-wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6-12 h after treatment with LN (100 mumol/kg body weight, iv). Levels of hepatic TNF-alpha protein in either WT or KO mice were below the detection limit, although expression levels of the TNF-alpha gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1beta protein, which is known to be a suppressor of the cholesterol 7alpha-hydroxylase gene in hamsters, were significantly increased 3-6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor alpha, retinoid X receptor alpha, fetoprotein transcription factor, and hepatocyte nuclear factor 4alpha) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-alpha-dependent pathway and might occur mainly through an IL-1beta-dependent pathway.
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Li, Xinliang; Zhang, Xuwang; Zhou, Jiti
2012-01-01
Background Indigoids, as popular dyes, can be produced by microbial strains or enzymes catalysis. However, the new valuable products with their transformation mechanisms, especially inter-conversion among the intermediates and products have not been clearly identified yet. Therefore, it is necessary to investigate novel microbial catalytic processes for indigoids production systematically. Findings A phenol hydroxylase gene cluster (4,606 bp) from Arthrobacter sp. W1 (PHw1) was obtained. This cluster contains six components in the order of KLMNOP, which exhibit relatively low sequence identities (37–72%) with known genes. It was suggested that indole and all the tested indole derivatives except for 3-methylindole were transformed to various substituted indigoid pigments, and the predominant color products derived from indoles were identified by spectrum analysis. One new purple product from indole, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one, should be proposed as the dimerization of isatin and 7-hydroxylindole at the C-2 and C-6 positions. Tunnel entrance and docking studies were used to predict the important amino acids for indoles biotransformation, which were further proved by site-directed mutagenesis. Conclusions/Significance We showed that the phenol hydroxylase from genus Arthrobacter could transform indoles to indigoids with new chemical compounds being produced. Our work should show high insights into understanding the mechanism of indigoids bio-production. PMID:23028517
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Vuica, Ana; Ferhatović Hamzić, Lejla; Vukojević, Katarina; Jerić, Milka; Puljak, Livia; Grković, Ivica; Filipović, Natalija
2015-12-01
Diabetes mellitus (DM) is a metabolic disorder associated with serious liver complications. As a metabolic chronic disease, DM is very common in the elderly. Recent studies suggest ameliorating effects of vitamin D on metabolic and oxidative stress in the liver tissue in an experimental model of DM. The aim of this study was to investigate the expression of vitamin D receptors (VDRs) and 1α-hydroxylase, the key enzyme for the production of active vitamin D form (calcitriol) in the liver during long-term diabetes mellitus type 1 (DM1) in aging rats. We performed immunohistochemical analysis of liver expression of 1α-hydroxylase and VDRs during aging in long-term streptozotocin-induced DM1. 1α-Hydroxylase was identified in the monocyte/macrophage system of the liver. In addition to the nuclear expression, we also observed the expression of VDR in membranes of lipid droplets within hepatocytes. Aging and long-term DM1 resulted in significant increases in the number of 1α-hydroxylase immunoreactive cells, as well as the percentage of strongly positive VDR hepatocytes. In conclusion, the liver has the capacity for active vitamin D synthesis in its monocyte/macrophage system that is substantially increased in aging and long-term diabetes mellitus. These conditions are also characterized by significant increases in vitamin D receptor expression in hepatocytes. The present study suggests that VDR signaling system could be a potential target in prevention of liver complications caused by diabetes and aging. Copyright © 2015 Elsevier Inc. All rights reserved.
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Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu
2015-01-01
The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity. PMID:26030746
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Meesapyodsuk, Dauenpen; Qiu, Xiao
2008-07-01
Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Delta(12) oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.
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Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani; ...
2015-05-20
Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C 2’H) from sweet potato. By comparing these twomore » structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani
Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C 2’H) from sweet potato. By comparing these twomore » structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.« less
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Cui, Zhen; Henderson, Richard A
2002-08-12
Kinetic studies, using stopped-flow spectrophotometry, on the reactions of [M(4)(SPh)(10)](2)(-) (M = Fe or Co) with PhS(-) to form [M(SPh)(4)](2)(-) are described, as are the reactions between [M(4)(SPh)(10)](2)(-) and [MoS(4)](2)(-) to form [S(2)MoS(2)Fe(SPh)(2)](2)(-) or [S(2)MoS(2)CoS(2)MoS(2)](2)(-). The kinetics of the reactions with PhS(-) are consistent with an initial associative substitution mechanism involving attack of PhS(-) at one of the tetrahedral M sites of [M(4)(SPh)(10)](2)(-) to form [M(4)(SPh)(11)](3)(-). Subsequent or concomitant cleavage of a micro-SPh ligand, at the same M, initiates a cascade of rapid reactions which result ultimately in the complete rupture of the cluster and formation of [M(SPh)(4)](2)(-). The kinetics of the reaction between [M(4)(SPh)(10)](2)(-) and [MoS(4)](2)(-) indicate an initial dissociative substitution mechanism at low concentrations of [MoS(4)](2)(-), in which rate-limiting dissociation of a terminal thiolate from [M(4)(SPh)(10)](2)(-) produces [M(4)(SPh)(9)](-) and the coordinatively unsaturated M site is rapidly attacked by a sulfido group of [MoS(4)](2)(-). It is proposed that subsequent chelation of the MoS(4) ligand results in cleavage of an M-micro-SPh bond, initiating a cascade of reactions which lead to the ultimate break-up of the cluster and formation of the products, [S(2)MoS(2)Fe(SPh)(2)](2)(-) or [S(2)MoS(2)CoS(2)MoS(2)](2)(-). With [Co(4)(SPh)(10)](2)(-), at higher concentrations of [MoS(4)](2)(-), a further substitution pathway is evident which exhibits a second order dependence on the concentration of [MoS(4)](2)(-). The mechanistic picture of cluster disruption which emerges from these studies rationalizes the "all or nothing" reactivity of [M(4)(SPh)(10)](2)(-).
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Sakowski, Stacey A; Geddes, Timothy J; Thomas, David M; Levi, Edi; Hatfield, James S; Kuhn, Donald M
2006-04-26
Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.
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Park, Eun-Bee; Jeon, Joo-Yeong; Jeon, Chang-Jin
2018-05-09
A growing number of studies have revealed the functional neuroarchitecture of the microbat retina and suggested that microbats can see using their eyes. To better understand the organization of the microbat retina, quantitative analysis of protein kinase C alpha (PKCα)- and tyrosine hydroxylase (TH)-immunoreactive (IR) cells was conducted on the greater horseshoe bat (Rhinolophus ferrumequinum) retina. As a result, PKCα immunoreactivity was observed in rod bipolar cells, consistent with previous studies on other mammalian retinas. PKCα-IR cell distribution in the inner nuclear layer showed regional differences in density, with the highest density found in the nasal retina. The average density of PKCα-IR cells was 10,487±441 cells/mm2 (mean ± S.D.; n=4), with a total of 43,077±1,843 cells/retina. TH-IR cells in the Rhinolophus ferrumequinum retina could be classified into four types based on soma location and ramification in the inner plexiform layer: conventional amacrine, displaced amacrine, interplexiform, and intercalated cells. The majority of TH-IR cells were conventional amacrine cells. TH-IR cells were nonrandomly distributed at low density over the retina. The average density was 29.7±3.1 cells/mm2 (mean ± S.D.; n=3), with a total of 124.0±11.3 cells/retina. TH-IR processes showed varicosities and formed ring-like structures encircling AII amacrine cells. Our study provides the foundation for understanding the neurochemical architecture of the microbat retina and supports the notion that the eyes do play a role in the visual system of microbats.
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Immunological detection of phenylalanine hydroxylase protein in Drosophila melanogaster.
Silva, F J; Bel, Y; Botella, L M; Cotton, R G; Ferré, J
1992-01-01
A monoclonal antibody raised against monkey liver phenylalanine hydroxylase (PAH) has been used to detect this protein in Drosophila melanogaster. A cross-reacting material (CRM) band of apparent molecular mass 50-52 kDa, equivalent to that deduced for the Drosophila melanogaster PAH protein based on the pah gene cDNA sequence, has been detected. This CRM was analysed throughout development and showed an equivalent pattern to that reported for PAH activity in this insect, with maxima at pupariation and at pharate adult formation. Distribution of this CRM in larval tissues, the haemolymph and the adult body is mainly restricted to the larval fat body and the adult head. Demonstration of this CRM as the PAH protein comes from the correlation between the decreased PAH enzyme activities of two mutant strains and their decreased amounts of CRM by Western blotting. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1417795
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p53-mediated inhibition of angiogenesis through up-regulation of a collagen prolyl hydroxylase.
Teodoro, Jose G; Parker, Albert E; Zhu, Xiaochun; Green, Michael R
2006-08-18
Recent evidence suggests that antiangiogenic therapy is sensitive to p53 status in tumors, implicating a role for p53 in the regulation of angiogenesis. Here we show that p53 transcriptionally activates the alpha(II) collagen prolyl-4-hydroxylase [alpha(II)PH] gene, resulting in the extracellular release of antiangiogenic fragments of collagen type 4 and 18. Conditioned media from cells ectopically expressing either p53 or alpha(II)PH selectively inhibited growth of primary human endothelial cells. When expressed intracellularly or exogenously delivered, alpha(II)PH significantly inhibited tumor growth in mice. Our results reveal a genetic and biochemical linkage between the p53 tumor suppressor pathway and the synthesis of antiangiogenic collagen fragments.
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Nilsson, G E; Tottmar, O
1987-04-21
The effect of indole-3-acetaldehyde, 5-hydroxyindole-3-acetaldehyde, disulfiram, diethyldithiocarbamate, coprine, and 1-amino-cyclopropanol on tryptophan hydroxylase activity was studied in vitro using high performance liquid chromatography with electro-chemical detection. With the analytical method developed, 5-hydroxytryptophan, serotonin, and 5-hydroxyindole-3-acetic acid could be measured simultaneously. Indole-3-acetaldehyde (12-1200 microM) was found to cause a 6-33% inhibition of the enzyme. Dependent upon the nature of the sulfhydryl- or reducing-agent (dithiotreitol, glutathione, or ascorbate) present in the incubates, the degree of inhibition by disulfiram varied, probably due to the formation of various mixed disulfides. Also the presence of diethyldithiocarbamate (160-1600 microM) was found to inhibit tryptophan hydroxylase (28-91%), while 5-hydroxyindole-3-acetaldehyde, coprine, or 1-aminocyclopropanol appeared to have no effect on the enzyme activity.
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Tschank, G; Raghunath, M; Günzler, V; Hanauske-Abel, H M
1987-01-01
Two pyridinedicarboxylates, predicted [Hanauske-Abel (1983) M.D.-Ph.D. Thesis, Philipps Universität Marburg] and later found to be potent reversible inhibitors of purified prolyl 4-hydroxylase [Majaama, Hanauske-Abel, Günzler & Kivirikko (1984) Eur. J. Biochem. 138, 239-245] were investigated with respect to their effect on hydroxyprolyl biosynthesis in the fibroblast/collagen and the macrophage/Clq systems, and the effect was compared with that of the iron chelator 2,2'-dipyridyl, the compound usually employed to inhibit cellular hydroxyprolyl formation. Only the enzyme-mechanism-derived pyridinedicarboxylates were highly selective inhibitors, and only they lacked overt cytotoxicity. Morphologically, their effect was restricted to the site of cellular hydroxyprolyl biosynthesis, i.e. the cisternae of the rough-surfaced endoplasmic reticulum. They were equally effective in the different cell types studied, and human and guinea-pig fibroblasts showed the same sensitivity. The minimal lipophilicity of the pyridinedicarboxylates necessitated high concentrations to achieve suppression of cellular hydroxyprolyl formation, but lipophilic bio-activatable pro-inhibitors may overcome this disadvantage. For the first time, experimental evidence is presented suggesting that, in cell culture, the biosynthesis of interstitial collagens and Clq can be suppressed selectively, identifying the pyridinedicarboxylates as promising pilot compounds for experiments in vivo. Images Fig. 3. Fig. 4. PMID:2829835
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Latrech, Hanane; Gaouzi, Ahmed
2015-01-01
L'hyperplasie congénitale des surrénales par déficit en 11ß hydroxylase se voit dans 5 à 10% des cas et est diagnostiquée, habituellement, devant des signes de virilisation d'un nouveau né ou d'un fœtus 46 XX et plus tard devant une HTA avec une hypokaliémie. Le pronostic de taille chez ces patients est très souvent compromis. Nous rapportons le cas d'un déficit en 11ß hydroxylase chez une enfant âgée de 7 ans révélé par une pseudo- puberté précoce traitée par hydrocortisone. La survenue d'une puberté précoce centrale complique encore plus la situation par la mise en jeu du pronostic de taille finale et nécessitant une prise en charge thérapeutique particulière. PMID:26113905
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Association between Tryptophan Hydroxylase 2 Gene Polymorphism and Completed Suicide
ERIC Educational Resources Information Center
Fudalej, Sylwia; Ilgen, Mark; Fudalej, Marcin; Kostrzewa, Grazyna; Barry, Kristen; Wojnar, Marcin; Krajewski, Pawel; Blow, Frederic; Ploski, Rafal
2010-01-01
The association between suicide and a single nucleotide polymorphism (rs1386483) was examined in the recently identified tryptophan hydroxylase 2 (TPH2) gene. Blood samples of 143 suicide victims and 162 age- and sex-matched controls were examined. The frequency of the TT genotype in the TPH2 polymorphism was higher in suicide victims than in…
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Perez, Dale M.; Richards, Mark P.; Parker, Robert S.; Berres, Mark E.; Wright, Aaron T.; Sifri, Mamduh; Sadler, Natalie C; Tatiyaborworntham, Nantawat; Li, Na
2016-01-01
Turkeys and chickens reared to 5 weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). Alpha tocopherol hydroxylase activity was greater in liver microsomes prepared from turkeys compared to that from chickens (p < 0.01). Alpha and gamma tocopherol metabolites were higher in turkey bile than in chicken when assessing the RRR AcT diet and the all-racemic AcT diet at 58 mg/kg feed (p < 0.01). Turkey cytochrome P450 2C29 was increased relative to its chicken ortholog on the basis of RNA-Seq transcript abundance (p < 0.001) and activity-based protein profiling (p < 0.01) of liver tissue. Alpha tocopherol concentrations in plasma, liver, and muscle from turkey were lower than the respective tissues from chicken (p < 0.05). Lipid oxidation was greater in turkey thigh than in chicken (p < 0.05). These results suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylase(s) in turkeys contributes to the decreased accumulation of alpha tocopherol in turkey tissues compared to that of chickens. PMID:26653675
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Rodríguez, Amparo; Ezquieta, Begoña; Labarta, José Igancio; Clemente, María; Espino, Rafael; Rodriguez, Amaia; Escribano, Aranzazu
2017-08-01
Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is an autosomal recessive disorder caused by mutations in the CYP21A2 gene. Cortisol and aldosterone synthesis are impaired in the classic forms (adrenal insufficiency and salt-wasting crisis). Females affected are virilised at birth, and are at risk for genital ambiguity. In this article we give recommendations for an early as possible diagnosis and an appropriate and individualised treatment. A patient and family genetic study is essential for the diagnosis of the patient, and allows genetic counselling, as well as a prenatal diagnosis and treatment for future pregnancy. Copyright © 2016 Asociación Española de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.
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Van de Wouwer, Dorien; Decou, Raphaël; Audenaert, Dominique; Nguyen, Long
2016-01-01
Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881
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Aro, Ellinoora; Salo, Antti M; Khatri, Richa; Finnilä, Mikko; Miinalainen, Ilkka; Sormunen, Raija; Pakkanen, Outi; Holster, Tiina; Soininen, Raija; Prein, Carina; Clausen-Schaumann, Hauke; Aszódi, Attila; Tuukkanen, Juha; Kivirikko, Kari I; Schipani, Ernestina; Myllyharju, Johanna
2015-07-03
Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Aro, Ellinoora; Salo, Antti M.; Khatri, Richa; Finnilä, Mikko; Miinalainen, Ilkka; Sormunen, Raija; Pakkanen, Outi; Holster, Tiina; Soininen, Raija; Prein, Carina; Clausen-Schaumann, Hauke; Aszódi, Attila; Tuukkanen, Juha; Kivirikko, Kari I.; Schipani, Ernestina; Myllyharju, Johanna
2015-01-01
Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1−/−) leads to embryonic lethality in mouse, whereas P4ha1+/− mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2−/− mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1+/−;P4ha2−/− mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1+/−;P4ha2−/− mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2−/− mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype. PMID:26001784
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Pavon, Jorge Alex; Eser, Bekir; Huynh, Michaela T.; Fitzpatrick, Paul F.
2010-01-01
Tryptophan hydroxylase (TrpH) uses a non-heme mononuclear iron center to catalyze the tetrahydropterin-dependent hydroxylation of tryptophan to 5-hydroxytryptophan. The reactions of the TrpH·Fe(II), TrpH·Fe(II)·tryptophan, TrpH·Fe(II)·6MePH4·tryptophan, and TrpH·Fe(II)·6MePH4·phenylalanine complexes with O2 were monitored by stopped-flow absorbance spectroscopy and rapid quench methods. The second-order rate constant for the oxidation of TrpH·Fe(II) has a value of 104 M−1s−1 irrespective of the presence of tryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH·Fe(II)·6MePH4·tryptophan complex with oxygen are consistent with the initial step being reversible binding of oxygen, followed by the formation with a rate constant of 65 s−1 of an intermediate I that has maximal absorbance at 420 nm. The rate constant for decay of I, 4.4 s−1, matches that for formation of the 4a-hydroxypterin product monitored at 248 nm. Chemical-quench analyses show that 5-hydroxytryptophan forms with a rate constant of 1.3 s−1, and that overall turnover is limited by a subsequent slow step, presumably product release, with a rate constant of 0.2 s−1. All of the data with tryptophan as substrate can be described by a five-step mechanism. In contrast, with phenylalanine as substrate, the reaction can be described by three steps: a second-order reaction with oxygen to form I, decay of I as tyrosine forms, and slow product release. PMID:20687613
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Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A; Nagata, Kazuhiro; Bächinger, Hans Peter
2009-06-26
The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.
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Placenta-specific Methylation of the Vitamin D 24-Hydroxylase Gene
Novakovic, Boris; Sibson, Mandy; Ng, Hong Kiat; Manuelpillai, Ursula; Rakyan, Vardhman; Down, Thomas; Beck, Stephan; Fournier, Thierry; Evain-Brion, Danielle; Dimitriadis, Eva; Craig, Jeffrey M.; Morley, Ruth; Saffery, Richard
2009-01-01
Plasma concentrations of biologically active vitamin D (1,25-(OH)2D) are tightly controlled via feedback regulation of renal 1α-hydroxylase (CYP27B1; positive) and 24-hydroxylase (CYP24A1; catabolic) enzymes. In pregnancy, this regulation is uncoupled, and 1,25-(OH)2D levels are significantly elevated, suggesting a role in pregnancy progression. Epigenetic regulation of CYP27B1 and CYP24A1 has previously been described in cell and animal models, and despite emerging evidence for a critical role of epigenetics in placentation generally, little is known about the regulation of enzymes modulating vitamin D homeostasis at the fetomaternal interface. In this study, we investigated the methylation status of genes regulating vitamin D bioavailability and activity in the placenta. No methylation of the VDR (vitamin D receptor) and CYP27B1 genes was found in any placental tissues. In contrast, the CYP24A1 gene is methylated in human placenta, purified cytotrophoblasts, and primary and cultured chorionic villus sampling tissue. No methylation was detected in any somatic human tissue tested. Methylation was also evident in marmoset and mouse placental tissue. All three genes were hypermethylated in choriocarcinoma cell lines, highlighting the role of vitamin D deregulation in this cancer. Gene expression analysis confirmed a reduced capacity for CYP24A1 induction with promoter methylation in primary cells and in vitro reporter analysis demonstrated that promoter methylation directly down-regulates basal promoter activity and abolishes vitamin D-mediated feedback activation. This study strongly suggests that epigenetic decoupling of vitamin D feedback catabolism plays an important role in maximizing active vitamin D bioavailability at the fetomaternal interface. PMID:19237542
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Wang, Xiao-Qin; Liu, Yan; Cai, Huan-Huan; Peng, Yu-Ping; Qiu, Yi-Hua
2016-12-01
Tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of catecholamines, is expressed in T lymphocytes. However, the role of T cell-expressed TH in rheumatoid arthritis (RA) is less clear. Herein, we aimed to show the contribution of TH expression by CD4 + T cells to alleviation of helper T (Th)17/regulatory T (Treg) imbalance in collagen-induced arthritis (CIA), a mouse model of RA. CIA was prepared by intradermal injection of collagen type II (CII) at tail base of DBA1/J mice. Expression of TH in the spleen and the ankle joints was measured by real-time polymerase chain reaction and Western blot analysis. Percentages of TH-expressing Th17 and Treg cells in splenic CD4 + T cells were determined by flow cytometry. Overexpression and knockdown of TH gene in CD4 + T cells were taken to evaluate effects of TH on Th17 and Treg cells in CIA. TH expression was upregulated in both the inflamed tissues (spleen and ankle joints) and the CD4 + T cells of CIA mice. In splenic CD4 + T cells, the cells expressing TH were increased during CIA. These cells that expressed more TH in CIA were mainly Th17 cells rather than Treg cells. TH gene overexpression in CD4 + T cells from CIA mice reduced Th17 cell percentage as well as Th17-related transcription factor and cytokine expression and secretion, whereas TH gene knockdown enhanced the Th17 cell activity. In contrast, TH gene overexpression increased Treg-related cytokine expression and secretion in CD4 + T cells of CIA mice, while TH gene knockdown decreased the Treg cell changes. Collectively, these findings show that CIA induces TH expression in CD4 + T cells, particularly in Th17 cells, and suggest that the increased TH expression during CIA represents an anti-inflammatory mechanism.
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Ishiguro, Kanako; Taniguchi, Masumi; Tanaka, Yoshikazu
2012-05-01
The enzymes flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) play an important role in flower color by determining the B-ring hydroxylation pattern of anthocyanins, the major floral pigments. F3'5'H is necessary for biosynthesis of the delphinidin-based anthocyanins that confer a violet or blue color to most plants. Antirrhinum majus does not produce delphinidin and lacks violet flower colour while A. kelloggii produces violet flowers containing delphinidin. To understand the cause of this inter-specific difference in the Antirrhinum genus, we isolated one F3'H and two F3'5'H homologues from the A. kelloggii petal cDNA library. Their amino acid sequences showed high identities to F3'Hs and F3'5'Hs of closely related species. Transgenic petunia expressing these genes had elevated amounts of cyanidin and delphinidin respectively, and flower color changes in the transgenics reflected the type of accumulated anthocyanidins. The results indicate that the homologs encode F3'H and F3'5'H, respectively, and that the ancestor of A. majus lost F3'5'H activity after its speciation from the ancestor of A. kelloggii.
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Capyk, Jenna K.; D'Angelo, Igor; Strynadka, Natalie C.; Eltis, Lindsay D.
2009-01-01
KshAB (3-Ketosteroid 9α-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O2. Nevertheless, the reactivity of KshAB with O2 was low in the presence of 1,4-androstadiene-3,17-dione, with a kcat/KmO2 of 2450 ± 80 m–1 s–1. The crystallographic structure of KshA, determined to 2.3Å, revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance. PMID:19234303
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Stability studies of As4S4 nanosuspension prepared by wet milling in Poloxamer 407.
Bujňáková, Zdenka; Dutková, Erika; Baláž, Matej; Turianicová, Erika; Baláž, Peter
2015-01-15
In this paper the stability of the arsenic sulfide (As4S4) nanosuspension prepared by wet milling in a circulation mill in the environment of copolymer Poloxamer 407 was studied. The obtained As4S4 particles in nanosuspension were of ∼ 100 nm in size. The influence of temperature and UV irradiation on the changes in physical and/or chemical properties was followed. Long-term stability was observed via particle size distribution and zeta potential measurements. Influence of UV irradiation was studied via UV-vis spectroscopy (UV-vis), photoluminicsence (PL) technique and Fourier transform infrared spectroscopy (FTIR) measurements. The best stability of the nanosuspension (24 weeks) was achieved when stored at 4°C and in the dark. Copyright © 2014 Elsevier B.V. All rights reserved.
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3-[4-Bromo-α( R*)-methoxybenzyl]-6-chloro-3( S*),4( S*)-dihydroxychroman: X-ray and DFT Studies
NASA Astrophysics Data System (ADS)
Sepay, Nayim; Mondal, Rina; Guha, Chayan; Mallik, Asok K.
2018-05-01
Sodium borohydride reduction of E-3-benzylidenechromanone epoxides in dry methanol has afforded 3( S*), 4( S*)-dihydroxy-3-[α( R*)-methoxybenzyl]chromans as an interesting class of products, the structures of which have been assigned mainly from spectral data and consideration of the mechanistic aspects. X-ray diffraction study of one of them, 3-[4-bromo-α( R*)-methoxybenzyl]-6-chloro-3( S*),4( S*)- dihydroxychroman, is performed. The title compound crystallizes in the monoclinic sp. gr. P21/ n, with a = 13.336(6) Å, b = 10.866(5) Å, c = 27.166(11) Å, β = 95.193(6)°, V = 3920(3) Å3, and Z = 8. Supramolecular construction of the compound involves O-H···O intermolecular hydrogen bonds as well as three other types of non-covalent interactions which are responsible for crystal packing. Density functional theory was applied for geometry optimization, molecular orbital calculations, and prediction of UV spectral features. The geometric parameters (bond lengths, bond angles, and dihedral angles) for the representative compound obtained from density functional theory with B3LY6-31G basis set were in good agreement with experimental values.
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Rietschel, M; Schorr, A; Albus, M; Franzek, E; Kreiner, R; Held, T; Knapp, M; Müller, D J; Schulze, T G; Propping, P; Maier, W; Nöthen, M M
2000-06-12
The tryptophan hydroxylase (TPH) gene encodes for the rate-limiting enzyme of the serotonin metabolism and, therefore, has to be considered a major candidate for association studies in affective disorders. Recently, an association between this gene and bipolar affective disorder has been reported in a French population. We sought to replicate this finding in a German sample. Allele frequencies of a biallelic polymorphism (A218C) of the TPH gene were determined in 95 bipolar I patients and their parents. Preferential transmission of alleles from heterozygous parents to bipolar offspring was tested with the "transmission disequilibrium test" (TDT), which eliminates the contribution of population stratification to an association finding. Our sample yielded a power >90% to detect the originally reported effect. Neither allele 218A nor allele 218C were preferentially transmitted from heterozygous parents to bipolar offspring. Our results, therefore, do not support the hypothesis that the TPH gene is involved in the etiology of bipolar disorder.
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Pozzolini, Marina; Scarfì, Sonia; Mussino, Francesca; Salis, Annalisa; Damonte, Gianluca; Benatti, Umberto; Giovine, Marco
2015-08-20
Prolyl 4-hydroxylase (P4H) is a α2β2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens. Here we report on the production and selection, by enzymatic and biomolecular analyses, of a triple transformed Pichia pastoris strain expressing a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recombinant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by mass spectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeast cell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets. In conclusion this Pichia system seems ideal for high-level production of hydroxylated sponge- or marine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharmacological interest needing this particular post-translational modification. Copyright © 2015 Elsevier B.V. All rights reserved.
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Molecular characterization of ferulate 5-hydroxylase gene from kenaf (Hibiscus cannabinus L.)
USDA-ARS?s Scientific Manuscript database
The purpose of this research was to clone and characterize the expression pattern of a kenaf (Hibiscus cannabinus L.) F5H gene that encodes ferulate 5-hydroxylase in the phenylpropanoid pathway. Kenaf is well known as a fast growing dicotyledonous plant, which makes it a valuable biomass plant. The ...
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Pribat, Anne; Noiriel, Alexandre; Morse, Alison M.; Davis, John M.; Fouquet, Romain; Loizeau, Karen; Ravanel, Stéphane; Frank, Wolfgang; Haas, Richard; Reski, Ralf; Bedair, Mohamed; Sumner, Lloyd W.; Hanson, Andrew D.
2010-01-01
Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism. PMID:20959559
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NASA Astrophysics Data System (ADS)
Unelius, C. R.; Park, K.-C.; McNeill, M.; Wee, S. L.; Bohman, B.; Suckling, D. M.
2013-02-01
An investigation to identify a sex or aggregation pheromone of Sitona discoideus Gyllenhål (Coleoptera: Curculionidae) is presented. Antenna flicking and attraction behaviors evoked by conspecifics of both sexes were recorded in arena bioassays, where attraction of females to males was observed. Air entrainment of both males and females was conducted in separate chambers. Gas chromatographic-mass spectrometric analysis of headspace volatiles revealed that two male-specific compounds, 4-methyl-3,5-heptanedione (major) and (4 S,5 S)-5-hydroxy-4-methyl-3-heptanone (minor), were emitted during the autumnal post-aestivatory flight period. The stereoisomers of the minor component were separated by enantioselective gas chromatography and their absolute configurations assigned by NMR (diastereomers) and the known preference of enantioselective transesterification reactions catalyzed by Candida antarctica lipase B. Electroantennogram and single sensillum recording studies indicate that 4-methyl-3,5-heptanedione as well as all individual stereoisomers of 5-hydroxy-4-methyl-3-heptanone are detected by the antennae of male and female S. discoideus. Further, single sensillum recordings suggest that both sexes of S. discoideus have specialized olfactory receptor neurons (ORNs) for detecting 4-methyl-3,5-heptanedione and different populations of stereoselective ORNs for detecting the stereoisomers of 5-hydroxy-4-methyl-3-heptanone. Some of these stereoselective ORNs appear to be sex-specific in S. discoideus.
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Matallana-Rhoades, Audrey Mary; Corredor-Castro, Juan David; Bonilla-Escobar, Francisco Javier; Mecias-Cruz, Bony Valentina; Mejia de Beldjena, Liliana
2016-09-30
It is presented the phenotype of a new compound heterozygous mutation of the genes R384X and Q356X encoding the enzyme of 11-beta-hydroxylase. Severe virilization, peripheral hypertension, and early puberty. Managed with hormone replacement therapy (corticosteroid) and antihypertensive therapy (beta-blocker), resulting in the control of physical changes and levels of arterial tension. According to the phenotypic characteristics of the patient, it is inferred that the R384X mutation carries an additional burden on the Q356X mutation, with the latter previously described as a cause of 11-beta-hydroxylase deficiency. The description of a new genotype, as in this case, expands the understanding of the hereditary burden and deciphers the various factors that lead to this pathology as well as the other forms of congenital adrenal hyperplasia (CAH), presenting with a broad spectrum of clinical presentations. This study highlights the importance of a complete description of the patient's CAH genetic profile as well as their parents' genetic profile.
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Adachi, Takahiro; Tomita, Masahiro; Yoshizato, Katsutoshi
2005-04-01
The present study shows that hemocytic granular cells synthesize and secrete type IV collagen (ColIV) in the silkworm Bombyx mori (B. mori) and suggests that these cells play roles in the formation of basement membrane, the encapsulation of foreign bodies, and the metamorphic remodeling of the gut. The full- and partial-length cDNA of B. mori prolyl 4-hydroxylase alpha subunit (BmP4Halpha) and B. mori ColIV (BmColIV) were cloned, respectively. In situ hybridization and immunocytochemistry on larval tissues and cells identified hemocytic granular cells as the cells that express mRNAs and proteins of both BmP4Halpha and BmColIV. Immunohistochemistry and immunocytochemistry demonstrated that BmColIV was present in the basement membrane and in the secretory granules of granular cells, respectively. Granular cells in culture secreted BmColIV without accompanying the degranulation and discharged it from the granules when the cells were degranulated. Nylon threads were inserted into the hemocoel of larvae. Granular cells concentrated around the nylon threads and encapsulated them as a self-defense reaction. BmColIV was found to be a component of the capsules. Furthermore, the present study showed that actively BmColIV-expressing granular cells accumulated around the midgut epithelium and formed BmColIV-rich thick basal lamina-like structures there in larval to pupal metamorphosis.
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Hollis, B W
1990-01-01
In vitro studies were performed to assess the ability of hepatic homogenates, mitochondria, and microsomes to 1 alpha-hydroxylate 25-hydroxyvitamin D3 [25(OH)D3]. Addition of 25(OH)D3 to either hepatic mitochondria or microsomes caused a concentration-dependent increase in the production of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Hepatic homogenates also produced purported 1,25(OH)2D3, although at a much reduced efficiency as compared with hepatic mitochondria or microsomes. Purported 1,25(OH)2D3 synthesized by hepatic mitochondria or microsomes was identified by its mobility on several high-performance liquid chromatographic systems and, ultimately, by its ability to interact with the bovine thymus 1,25(OH)2D3 receptor protein. Production of 1,25(OH)2D3 by hepatic mitochondria and microsomes was dependent on time of incubation, protein content, and pH of incubation medium, and it required an adequate source of reducing equivalents. Generation of 1,25(OH)2D3 by these organelles could be totally blocked by the cytochrome P-450 inhibitor ketoconazole. The microsomal 1 alpha-hydroxylase could not be saturated even at the highest concentration (240 microM) of 25(OH)D3 used. The mitochondrial 1 alpha-hydroxylase, however, displayed saturation at approximately 40 microM 25(OH)D3. Eadie-Hofstee reciprocal plot analysis of the hepatic mitochondrial 1 alpha-hydroxylase gave a Km of 17 microM 25(OH)D3 and a Vmax of 481 pg of 1,25(OH)2D3 per min per mg of protein. Because of its inability to achieve substrate saturation, meaningful kinetic parameters could not be calculated for the hepatic microsomal 1 alpha-hydroxylase. These data demonstrate the liver to be an even more dynamic organ than was previously believed with respect to vitamin D metabolism in that the liver has the potential to produce 1,25(OH)2D3 in situ by at least two separate mechanisms. PMID:2385581
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NASA Astrophysics Data System (ADS)
Zhang, Kaikai; Mai, Kangsen; Xu, Wei; Zhou, Huihui; Liufu, Zhiguo; Zhang, Yanjiao; Peng, Mo; Ai, Qinghui
2015-06-01
This study was conducted to investigate the effect of dietary proline (Pro), and Pro and hydroxyproline (Hyp) in combination on the growth performance, total Hyp and collagen concentrations of tissues, and prolyl 4-hydroxylase α(I) (P4H α(I)) gene expression in juvenile turbot feeding high plant protein diets. A diet containing 50% crude protein and 12% crude lipid was formulated as the basal and control, on which other two protein and lipid contents identical experimental diets were formulated by supplementing the basal with either 0.75% Pro (Pro-0.75) or 0.75% Pro and 0.75% Hyp (Pro+Hyp). Four groups of fish in indoor seawater recirculating systems, 35 individuals each, were fed twice a day to apparent satiation for 10 weeks. The results showed that dietary Pro and Hyp supplementation had no significant effect on growth performance and feed utilization of juvenile turbot (P > 0.05). Total Hyp and collagen concentrations in muscle were significantly increased when dietary Pro and Hyp increased (P <0.05), and fish fed diet Pro+Hyp showed significantly higher free Hyp content in plasma than those fed other diets (P <0.05). The expression of P4H a(I) gene in liver and muscle was significantly up regulated in fish fed diet Pro-0.75 in comparison with control (P <0.05); however the gene was significantly down regulated in fish fed diet Pro+Hyp in muscle in comparison with fish fed diet Pro-0.75 (P <0.05). It can be concluded that supplement of crystal L-Pro and L-Hyp to high plant protein diets did not show positive effects on growth performance of juvenile turbot, but enhanced total collagen concentrations in muscle.
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Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A.; Nagata, Kazuhiro; Bächinger, Hans Peter
2009-01-01
The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. PMID:19419969
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Day, D.J.; Barany, F.; Speiser, P.W.
Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and themore » remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems. 30 refs., 5 figs.« less
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Non-classic 21-hydroxylase deficiency in an 18-year-old female athlete. A case report
Silverman; Coupey; Muram
2000-05-01
Background: In non-classic 21-hydroxylase deficiency, age at presentation and genital findings are variable. Late diagnosis with dramatic signs of virilization precludes early treatment and thus prevention of anatomic and psychosocial consequences. The following case illustrates the complexity of late diagnosis.Case: An 18-year-old West Indian female was seen for evaluation of clitoromegaly and hirsutism discovered in the emergency department when she presented after sexual assault. She had allegedly been drugged and raped in her dorm room. She was a college student with an athletic scholarship and had a striking masculinized, broad-shouldered appearance. She denied any use of anabolic steroids or other drugs. Menarche was at age 16 with infrequent menses. She was sexually active with 4 life-time partners, all male. On physical exam, her height was 152 cm, weight 50 kg, blood pressure 110/70 mm Hg. Breasts were hypoplastic with hyperpigmented nipples. She was hirsute with a Ferriman-Gallwey score of 14. Genitalia were abnormal with clitoris measuring 5.5 x 1.5 cm and posterior labial fusion. Initial non-fasting serum 17-hydroxyprogesterone level was 2890 ng/dL, testosterone was 274 ng/dL, and cortisol was 9 &mgr;g/dL. Chromosome analysis was 46, XX and ACTH stimulation test confirmed the diagnosis of 21-hydroxylase deficiency. The patient was initially reluctant to begin glucocorticoid treatment because of concern that it would decrease her muscle mass and negatively impact on her athletic performance and scholarship support. One year after diagnosis and 10 months after beginning treatment, she elected surgical correction of her clitoromegaly because of extreme embarrassment over having erections during sex. She underwent excision of most of the corpus cavernosum with repositioning of the glans. The neurovascular elements were preserved. The patient is pleased with the cosmetic result and reports no change in achieving orgasm. She has not notices any change in
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Zhang, Lin; Li, Ming-Ming; Corcoran, Marie; Zhang, Shaoping; Cooper, Garth J S
2015-01-05
Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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Savaş-Erdeve, Şenay; Çetinkaya, Semra; Abalı, Zehra Yavaş; Poyrazoğlu, Şükran; Baş, Firdevs; Berberoğlu, Merih; Sıklar, Zeynep; Korkmaz, Özlem; Buluş, Derya; Akbaş, Emine Demet; Güran, Tülay; Böber, Ece; Akın, Onur; Yılmaz, Gülay Can; Aycan, Zehra
2017-07-26
The clinical, laboratory, genetic properties and final height of a large cohort of patients with nonclassical 21-hydroxylase deficiency (NC21OHD) in Turkey were analyzed. This multicenter, nationwide web-based study collected data. The mean age was 9.79±4.35 years (229 girls, 29 boys). The most common symptoms were premature pubarche (54.6%) and hirsutism (28.6%). The peak cortisol was found below 18 μg/dL in three (15.45%) patients. A mutation was detected in the CYP21A2 gene of 182 (87.5%) patients. The most common mutation was V281L. Final height in female patients who were diagnosed and treated before attaining final height or near final height was found to be shorter than the final height in female patients who were diagnosed after attaining final height or near final height. The final height of the patients who were treated during childhood was found to be shorter than the final height of patients during the adolescent period.
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Bardowell, Sabrina A.; Duan, Faping; Manor, Danny; Swanson, Joy E.; Parker, Robert S.
2012-01-01
Vitamin E is a family of naturally occurring and structurally related lipophilic antioxidants, one of which, α-tocopherol (α-TOH), selectively accumulates in vertebrate tissues. The ω-hydroxylase cytochrome P450–4F2 (CYP4F2) is the only human enzyme shown to metabolize vitamin E. Using cDNA cloning, cell culture expression, and activity assays, we identified Cyp4f14 as a functional murine ortholog of CYP4F2. We then investigated the effect of Cyp4f14 deletion on vitamin E metabolism and status in vivo. Cyp4f14-null mice exhibited substrate-specific reductions in liver microsomal vitamin E-ω-hydroxylase activity ranging from 93% (γ-TOH) to 48% (γ-tocotrienol). In vivo data obtained from metabolic cage studies showed whole-body reductions in metabolism of γ-TOH of 90% and of 68% for δ- and α-TOH. This metabolic deficit in Cyp4f14−/− mice was partially offset by increased fecal excretion of nonmetabolized tocopherols and of novel ω-1- and ω-2-hydroxytocopherols. 12′-OH-γ-TOH represented 41% of whole-body production of γ-TOH metabolites in Cyp4f14−/− mice fed a soybean oil diet. Despite these counterbalancing mechanisms, Cyp4f14-null mice fed this diet for 6 weeks hyper-accumulated γ-TOH (2-fold increase over wild-type littermates) in all tissues and appeared normal. We conclude that CYP4F14 is the major but not the only vitamin E-ω-hydroxylase in mice. Its disruption significantly impairs whole-body vitamin E metabolism and alters the widely conserved phenotype of preferential tissue deposition of α-TOH. This model animal and its derivatives will be valuable in determining the biological actions of specific tocopherols and tocotrienols in vivo. PMID:22665481
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Noradrenergic lesioning with an anti-dopamine beta-hydroxylase immunotoxin
NASA Technical Reports Server (NTRS)
Picklo, M. J.; Wiley, R. G.; Lappi, D. A.; Robertson, D.
1994-01-01
Sympathectomy has been achieved by a variety of methods but each has its limitations. These include lack of tissue specificity, incomplete lesioning, and the age range of susceptibility to the lesioning. To circumvent these drawbacks, an immunotoxin was constructed using a monoclonal antibody against the noradrenergic specific enzyme dopamine beta-hydroxylase (D beta H) coupled via a disulfide bond to saporin, a ribosomal inactivating protein. Three days after intravenous injection of the anti-D beta H immunotoxin (50 micrograms) into adult Sprague-Dawley rats, 66% of neurons in the superior cervical ganglia were chromatolytic. Superior cervical ganglia neurons were poisoned in 1 day old and 1 week old (86% of neurons) neonatal rats following subcutaneous injection of 3.75 and 15 micrograms, respectively. The anti-D beta H immunotoxin will be a useful tool in the study of the peripheral noradrenergic system in adult and neonatal animals.
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[Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix].
Li, Jun; Long, Deng-Kai; Zhou, Tao; Ding, Ling; Zheng, Wei; Jiang, Wei-Ke
2016-07-01
Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress. Copyright© by the Chinese Pharmaceutical Association.
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Point mutation of Arg440 to his in cytochrome P450c17 causes severe 17{alpha}-hydroxylase deficiency
DOE Office of Scientific and Technical Information (OSTI.GOV)
Fardella, C.E.; Hum, D.W.; Miller, W.L.
Genetic disorders in the gene encoding P450c17 cause 17{alpha}-hydroxylase deficiency. The consequent defects in the synthesis of cortisol and sex steroids cause sexual infantilism and a female phenotype in both genetic sexes as well as mineralorcorticoid excess and hypertension. A 15-yr-old patient from Germany was seen for absent pubertal development and mild hypertension with hypokalemia, high concentrations of 17-deoxysteroids, and hypergonadotropic hypogonadism. Analysis of her P450c17 gene by polymerase chain reaction amplification and direct sequencing showed mutation of codon 440 from CGC (Arg) to CAC (His). Expression of a vector encoding this mutated form of P450c17 in transfected nonsteroidogenic COS-1more » cells showed that the mutant P450c17 protein was produced, but it lacked both 17{alpha}-hydroxylase and 17,20-lyase activities. To date, 15 different P450c17 mutations have been described in 23 patients with 17{alpha}-hydroxylase deficiency, indicating that mutations in this gene are due to random events. 36 refs., 3 figs., 2 tabs.« less
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Karuppagounder, Saravanan S.; Alim, Ishraq; Khim, Soah J.; ...
2016-03-02
Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. Here we show that the hypoxia-inducible factor prolyl-hydroxylase (HIF- PHD) family of iron-dependent oxygen sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in mouse striatum improved functional recovery following ICH. A low molecular weight hydroxyquinoline inhibitor of the HIF-PHDs, adaptaquin, reduced neuronal death and behavioral deficitsmore » following ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of expression of the prodeath factor ATF4 rather than activation of a HIF-dependent prosurvival pathway. In conclusion, together these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier permeable inhibitor adaptaquin can improve functional outcomes following ICH in multiple rodent species.« less
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Karuppagounder, Saravanan S.; Alim, Ishraq; Khim, Soah J.; Bourassa, Megan W.; Sleiman, Sama F.; John, Roseleen; Thinnes, Cyrille C.; Yeh, Tzu-Lan; Demetriades, Marina; Neitemeier, Sandra; Cruz, Dana; Gazaryan, Irina; Killilea, David W.; Morgenstern, Lewis; Xi, Guohua; Keep, Richard F.; Schallert, Timothy; Tappero, Ryan V.; Zhong, Jian; Cho, Sunghee; Maxfield, Frederick R.; Holman, Theodore R.; Culmsee, Carsten; Fong, Guo-Hua; Su, Yijing; Ming, Guo-li; Song, Hongjun; Cave, John W.; Schofield, Christopher J.; Colbourne, Frederick; Coppola, Giovanni; Ratan, Rajiv R.
2017-01-01
Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier-permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models. PMID:26936506
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Karuppagounder, Saravanan S.; Alim, Ishraq; Khim, Soah J.
Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. Here we show that the hypoxia-inducible factor prolyl-hydroxylase (HIF- PHD) family of iron-dependent oxygen sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in mouse striatum improved functional recovery following ICH. A low molecular weight hydroxyquinoline inhibitor of the HIF-PHDs, adaptaquin, reduced neuronal death and behavioral deficitsmore » following ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of expression of the prodeath factor ATF4 rather than activation of a HIF-dependent prosurvival pathway. In conclusion, together these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier permeable inhibitor adaptaquin can improve functional outcomes following ICH in multiple rodent species.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sanderson, J.T.; Giesy, J.P.; Janz, D.M.
In a continuing effort to evaluate biomarkers of exposure of great blue herons (Ardea herodias) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons, the authors examined the effect of TCDD on hepatic microsomal testosterone hydroxylase activities. Heron embryos were exposed in ovo to 2 {micro}g TCDD/kg egg (or corn oil vehicle) and sacrificed at hatch or 7 d posthatch. Adult herons were exposed intraperitoneally to 20 {micro}g TCDD/kg and sacrificed 2 weeks later. The sex of the birds was known for the adults only. Hepatic microsomes of herons of each age group were able to hydroxylate testosterone at the 2{beta},more » 6{beta}, 15{alpha}, 16{alpha}, or 16{beta} positions. In 7-d-old chicks, an additional unidentified compound was formed. The age of the untreated herons had a strong influence on the activities of the five hydroxylases, with changes of up to 17-fold. The TCDD significantly induced 2{beta}-, 6{beta}, and 15{alpha}-testosterone hydroxylase activities in the adult females, 15{alpha} in the adult males, and 6{beta}-testosterone hydroxylase activity in the hatchlings. In the 7-d-old chicks, induction was no longer apparent. A significant correlation existed between hepatic microsomal ethoxyresorufin O-deethylase (EROD) and 6{beta}-testosterone hydroxylase activity in hatchlings and adult female herons. The TCDD-induced changes in testosterone hydroxylase activities occurred at doses that resulted in tissue concentrations and levels of EROD induction that were environmentally relevant, but did not result in overt toxicities.« less
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Sheu, Bor-Shyang; Cheng, Hsiu-Chi; Yeh, Yi-Chun; Chang, Wei-Lun
2012-01-01
The present study determined whether the genotypes of S-mephenytoin 4'-hydroxylase (CYP2C19) could serve as an indicator to assess the success of long-term on-demand therapy (ODT) with pantoprazole for the patients with severe reflux esophagitis as Los Angles grade C or D (RE-CD). A total of 240 patients with RE-CD were prospectively enrolled to receive continuous pantoprazole, 40 mg daily for 6 months. The patients, who achieved complete healing and were free from acid reflux-related symptoms during follow up, were included to receive ODT with a 40 mg pantoprazole tablet up to 1 year. Each patient was followed to assess the monthly tablet number of 40 mg pantoprazole and the cumulative rate of failure of ODT. The CYP2C19 genotype of each included patient was defined as homologous extensive metabolizer (HomoEM), heterologous extensive metabolizer (HeteroEM), and poor metabolizer (PM). Two-hundred patients were included to receive ODT, including 51 as HomoEM, 108 as HeteroEM, and 41 as PM. There were no differences in demographic and endoscopic features among patients with different CYP2C19 genotypes (P > 0.05). The 1-year cumulative failure rate of ODT was significantly higher in HomoEM than in HeteroEM and PM (P < 0.05, by log-rank test). For those with successful ODT during the 1-year follow up, the mean monthly tablet number of pantoprazole was lower in PM than in HeteroEM and HomoEM (11.5 vs 16.3 and 18.6, P < 0.05). For RE-CD with complete healing after continuous pantoprazole, the successful shift to ODT is determined by the CYP2C19 genotypes of the patients. © 2011 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.
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Wood, Ruth I; Armstrong, Abigail; Fridkin, Vlad; Shah, Vivek; Najafi, Allison; Jakowec, Michael
2013-02-17
In humans and animals, anabolic-androgenic steroids (AAS) increase aggression, but the underlying behavioral mechanisms are unclear. AAS may increase the motivation to fight. Alternatively, AAS may increase impulsive behavior, consistent with the popular image of 'roid rage. To test this, adolescent male rats were treated chronically with testosterone (7.5mg/kg) or vehicle and tested for aggressive motivation and impulsivity. Rats were trained to respond on a nose-poke on a 10 min fixed-interval schedule for the opportunity to fight in their home cage with an unfamiliar rat. Although testosterone increased aggression (6.3±1.3 fights/5 min vs 2.4±0.8 for controls, p<0.05), there was no difference in operant responding (28.4±1.6 nose-pokes/10 min for testosterone, 32.4±7.0 for vehicle). This suggests that testosterone does not enhance motivation for aggression. To test for impulsivity, rats were trained to respond for food in a delay-discounting procedure. In an operant chamber, one lever delivered one food pellet immediately, the other lever gave 4 pellets after a delay (0, 15, 30 or 45 s). In testosterone- and vehicle-treated rats, body weights and food intake did not differ. However, testosterone-treated rats chose the larger, delayed reward more often (4.5±0.7 times in 10 trials with 45 s delay) than vehicle controls (2.5±0.5 times, p<0.05), consistent with a reduction in impulsive choice. Thus, although chronic high-dose testosterone enhances aggression, this does not include an increase in impulsive behavior or motivation to fight. This is further supported by measurement of tyrosine hydroxylase (TH) by Western immunoblot analysis in brain regions important for motivation (nucleus accumbens, Acb) and executive function (medial prefrontal cortex, PFC). There were no differences in TH between testosterone- and vehicle-treated rats in Acb or PFC. However, testosterone significantly reduced TH (to 76.9±3.1% of controls, p<0.05) in the caudate-putamen, a
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Kasajima, Ichiro; Sasaki, Katsutomo
2016-05-03
The development of new phenotypes is key to the commercial development of the main floricultural species and cultivars. Important new phenotypes include features such as multiple-flowers, color variations, increased flower size, new petal shapes, variegation and distinctive petal margin colourations. Although their commercial use is not yet common, the transgenic technologies provide a potentially rapid means of generating interesting new phenotypes. In this report, we construct 5 vectors which we expected to change the color of the flower anthocyanins, from purple to blue, regulating vacuolar pH. When these constructs were transformed into purple torenia, we unexpectedly recovered some genotypes having slightly margined petals. These transgenic lines expressed a chimeric repressor of the petunia PhPH4 gene under the control of Cauliflower mosaic virus 35 S RNA promoter. PhPH4 is an R2R3-type MYB transcription factor. The transgenic lines lacked pigmentation in the petal margin cells both on the adaxial and abaxial surfaces. Expressions of Flavanone 3-hydroxylase (F3H), Flavonoid 3'-hydroxylase (F3'H) and Flavonoid 3'5'-hydroxylase (F3'5'H) genes were reduced in the margins of these transgenic lines, suggesting an inhibitory effect of PhPH4 repressor on anthocyanin synthesis.
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Todoroki, Yasushi; Kobayashi, Kyotaro; Shirakura, Minaho; Aoyama, Hikaru; Takatori, Kokichi; Nimitkeatkai, Hataitip; Jin, Mei-Hong; Hiramatsu, Saori; Ueno, Kotomi; Kondo, Satoru; Mizutani, Masaharu; Hirai, Nobuhiro
2009-09-15
To develop a specific inhibitor of abscisic acid (ABA) 8'-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, seed dormancy, and other various physiological events, we designed and synthesized conformationally restricted analogues of uniconazole (UNI), a well-known plant growth retardant, which inhibits a biosynthetic enzyme (ent-kaurene oxidase) of gibberellin as well as ABA 8'-hydroxylase. Although most of these analogues were less effective than UNI in inhibition of ABA 8'-hydroxylase and rice seedling growth, we found that a lactol-bridged analogue with an imidazole is a potent inhibitor of ABA 8'-hydroxylase but not of plant growth. This compound, abscinazole-F1, induced drought tolerance in apple seedlings upon spray treatment with a 10 microM solution.
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Xu, Yuechi; Brown, Kevin M.; Wang, Zhuo A.; van der Wel, Hanke; Teygong, Crystal; Zhang, Dongmei; Blader, Ira J.; West, Christopher M.
2012-01-01
In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O2 and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O2 regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3SCF ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O2 sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O2 levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O2 requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O2. The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O2 by detecting the levels of O2-regulated metabolites such as α-ketoglutarate. PMID:22648409
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Upregulation of cholesterol 24-hydroxylase following hypoxia-ischemia in neonatal mouse brain.
Lu, Fuxin; Zhu, Jun; Guo, Selena; Wong, Brandon J; Chehab, Farid F; Ferriero, Donna M; Jiang, Xiangning
2018-06-01
BackgroundMaintenance of cholesterol homeostasis is crucial for brain development. Brain cholesterol relies on de novo synthesis and is cleared primarily by conversion to 24S-hydroxycholesterol (24S-HC) with brain-specific cholesterol 24-hydroxylase (CYP46A1). We aimed to investigate the impact of hypoxia-ischemia (HI) on brain cholesterol metabolism in the neonatal mice.MethodsPostnatal day 9 C57BL/6 pups were subjected to HI using the Vannucci model. CYP46A1 expression was assessed with western blotting and its cellular localization was determined using immunofluorescence staining. The amount of brain cholesterol, 24S-HC in the cortex and in the serum, was measured with enzyme-linked immunosorbent assay (ELISA).ResultsThere was a transient cholesterol loss at 6 h after HI. CYP46A1 was significantly upregulated at 6 and 24 h following HI with a concomitant increase of 24S-HC in the ipsilateral cortex and in the serum. The serum levels of 24S-HC correlated with those in the brain, as well as with necrotic and apoptotic cell death evaluated by the expression of spectrin breakdown products and cleaved caspase-3 at 6 and 24 h after HI.ConclusionEnhanced cholesterol turnover by activation of CYP46A1 represents disrupted brain cholesterol homeostasis early after neonatal HI. 24S-HC might be a novel blood biomarker for severity of hypoxic-ischemic encephalopathy with potential clinical application.
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Saitoh, T; Fukushima, Y; Otsuka, H; Hirakawa, J; Mori, H; Asano, T; Ishikawa, T; Katsube, T; Ogawa, K; Ohkawa, S
2002-10-01
To investigate the inhibitory effects on gastric acid secretion of three proton pump inhibitors, omeprazole, lansoprazole and rabeprazole, using a three-way crossover design in healthy Helicobacter pylori-negative,S-mephenytoin 4'-hydroxylase (CYP2C19) homo- and hetero-extensive metabolizers. Eight healthy Japanese male volunteers were enrolled. After the administration of rabeprazole (10 mg/day), lansoprazole (30 mg/day) or omeprazole (20 mg/day), intragastric pH monitoring was commenced from 24 h before the first proton pump inhibitor dose, and continued for days 1-3 after proton pump inhibitor administration. The pH electrode was used for 48 h and changed just before pH monitoring on day 2. For the administration of 10 mg/day rabeprazole, the mean ratios of the 24-h pH > or = 3 holding time were 5.7 +/- 1.1%,13.6 +/- 2.2%, 35.3 +/- 2.7% and 62.8 +/- 3.1% for the pre-treatment day and days 1, 2 and 3, respectively. The same ratios for lansoprazole (30 mg/day) were 5.7 +/- 0.7%, 7.4 +/- 1.5%, 13.6 +/- 3.4% and 26.6 +/- 4.9%; the same ratios for 20 mg/day omeprazole were 5.9 +/- 0.9%, 6.1 +/- 1.2%, 11.4 +/- 2.8% and 16.4 +/- 4.6%. The mean ratio of the 24-h pH > or = 3 holding time of days 1-3 increased significantly compared to the pre-treatment day (P < 0.01) with the administration of rabeprazole and lansoprazole. The magnitude of inhibition of gastric acid secretion after rabeprazole administration was stronger than that after lansoprazole. A significant elevation of the mean ratio of the 24-h pH > or = 3 holding time was demonstrated on days 2 and 3 with omeprazole (P < 0.01). In H. pylori-negative CYP2C19 extensive metabolizers, rabeprazole (10 mg/day) shows a faster onset of rising intragastric pH and a stronger inhibition of gastric acid secretion than do lansoprazole (30 mg/day) or omeprazole (20 mg/day).
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Pierson, Tyler Mark; Simeonov, Dimitre R; Sincan, Murat; Adams, David A; Markello, Thomas; Golas, Gretchen; Fuentes-Fajardo, Karin; Hansen, Nancy F; Cherukuri, Praveen F; Cruz, Pedro; Blackstone, Craig; Tifft, Cynthia; Boerkoel, Cornelius F; Gahl, William A
2012-01-01
Fatty acid hydroxylase-associated neurodegeneration due to fatty acid 2-hydroxylase deficiency presents with a wide range of phenotypes including spastic paraplegia, leukodystrophy, and/or brain iron deposition. All previously described families with this disorder were consanguineous, with homozygous mutations in the probands. We describe a 10-year-old male, from a non-consanguineous family, with progressive spastic paraplegia, dystonia, ataxia, and cognitive decline associated with a sural axonal neuropathy. The use of high-throughput sequencing techniques combined with SNP array analyses revealed a novel paternally derived missense mutation and an overlapping novel maternally derived ∼28-kb genomic deletion in FA2H. This patient provides further insight into the consistent features of this disorder and expands our understanding of its phenotypic presentation. The presence of a sural nerve axonal neuropathy had not been previously associated with this disorder and so may extend the phenotype. PMID:22146942
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Liu, Hailiang; Pathak, Preeti; Boehme, Shannon; Chiang, John Y. L.
2016-01-01
Cholesterol 7α-hydroxylase (CYP7A1) plays a critical role in control of bile acid and cholesterol homeostasis. Bile acids activate farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5) to regulate lipid, glucose, and energy metabolism. However, the role of bile acids in hepatic inflammation and fibrosis remains unclear. In this study, we showed that adenovirus-mediated overexpression of Cyp7a1 ameliorated lipopolysaccharide (LPS)-induced inflammatory cell infiltration and pro-inflammatory cytokine production in WT and TGR5-deficient (Tgr5−/−) mice, but not in FXR-deficient (Fxr−/−) mice, suggesting that bile acid signaling through FXR protects against hepatic inflammation. Nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-luciferase reporter assay showed that FXR agonists significantly inhibited TNF-α-induced NF-κB activity. Furthermore, chromatin immunoprecipitation and mammalian two-hybrid assays showed that ligand-activated FXR interacted with NF-κB and blocked recruitment of steroid receptor coactivator-1 to cytokine promoter and resulted in inhibition of NF-κB activity. Methionine/choline-deficient (MCD) diet increased hepatic inflammation, free cholesterol, oxidative stress, apoptosis, and fibrosis in CYP7A1-deficient (Cyp7a1−/−) mice compared with WT mice. Remarkably, adenovirus-mediated overexpression of Cyp7a1 effectively reduced hepatic free cholesterol and oxidative stress and reversed hepatic inflammation and fibrosis in MCD diet-fed Cyp7a1−/− mice. Current studies suggest that increased Cyp7a1 expression and bile acid synthesis ameliorate hepatic inflammation through activation of FXR, whereas reduced bile acid synthesis aggravates MCD diet-induced hepatic inflammation and fibrosis. Maintaining bile acid and cholesterol homeostasis is important for protecting against liver injury and nonalcoholic fatty liver disease. PMID:27534992
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Mahesh, Venkataramaiah; Million-Rousseau, Rachel; Ullmann, Pascaline; Chabrillange, Nathalie; Bustamante, José; Mondolot, Laurence; Morant, Marc; Noirot, Michel; Hamon, Serge; de Kochko, Alexandre; Werck-Reichhart, Danièle; Campa, Claudine
2007-05-01
Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Gan, L.S.
A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxygenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene (EP), 3-ethynylperylene (EPL), cis- and trans-1-(2-bromo-vinyl)pyrene (c-BVP and t-BVP), and 1-allylpyrene (AP) serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo(a)pyrene (BP) hydroxylase, while 1-vinyl-pyrene (VP) and phenyl 1-pyrenyl acetylene (PPA) do not cause a detectable suicide inhibition of the BP hydroxylase. The mechanism-based loss of BP hydroxylase activity caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes. In themore » presence of NADPH, /sup 3/H-labeled EP covalently attached to P-450 isozymes with a measured stoichiometry of one mole of EP per mole of the P-450 heme. The results of the effects of these aryl derivatives in the mammalian cell-mediated mutagenesis assay and toxicity assay show that none of the compounds examined nor any of the their metabolites produced in the incubation system are cytotoxic to V79 cells.« less
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Regulation of tyrosine hydroxylase gene expression during differentiation of neuroblastoma cells.
Summerhill, E M; Wood, K; Fishman, M C
1987-07-01
Differentiation of N1E-115 neuroblastoma cells into neuron-like cells, with extension of neurites and acquisition of excitable membranes, can be induced by dimethyl sulfoxide (DMSO). We have found this differentiation to be accompanied by an increase in tyrosine hydroxylase (TH) mRNA, an increase disproportionate to changes in mRNAs for other measured, non-neuron-specific genes. The mRNA increases slowly over several days and falls gradually after removal of DMSO. Nuclear run-on studies suggest that a change in the rate of transcription cannot explain the increase in steady-state mRNA levels. TH mRNA half-life does, however, increase. This suggests that regulation is exerted in this case not at the level of transcription but rather at that of mRNA stability.
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Cherian, Ajeesh Koshy; Briski, Karen P
2011-07-01
Cellular metabolic stasis is monitored in discrete brain sites, including the dorsal vagal complex (DVC), where A2 noradrenergic neurons perform this sensory function. Single-cell qPCR and high-sensitivity immunoblotting were used to determine if A2 neurons adapt to chronic hypoglycemia by increasing substrate fuel transporter expression, and whether such adjustments coincide with decreased cellular energy instability during this systemic metabolic stress. Tyrosine hydroxylase-immunolabeled neurons were laser-microdissected from the caudal DVC 2 hr after single or serial neutral protamine Hagedorn insulin (NPH) dosing. Preceding hypoglycemia suppressed basal A2 MCT2, GLUT3, and GLUT4 profiles and diminished MCT2, GLUT4, and glucokinase responses to recurring hypoglycemia. Acute NPH caused a robust increase in A2 phospho-AMPK protein levels; baseline phospho-AMPK expression was elevated after 3 days of insulin treatment but only slight augmented after a fourth NPH injection. Transcripts encoding the catecholamine biosynthetic enzyme dopamine-β-hydroxylase were unaffected by acute NPH but were diminished by serial insulin dosing. This evidence for diminished basal A2 glucose and lactate uptake and attenuated phospho-AMPK-mediated detection of hypoglycemia-associated energy deficits suggests that these cells acclimate to chronic hypoglycemia by adopting a new metabolic steady state characterized by energy paucity and reduced sensitivity to hypoglycemia. Because dopamine-β-hydroxylase mRNA was reduced after serial, but not single NPH dosing, A2 neurotransmitter biosynthesis may be impervious to acute hypoglycemia but inhibited when posthypoglycemic metabolic deficiency is exacerbated by recurring hypoglycemia. This research suggests that chronic hypoglycemia-associated adjustments in A2-sensory neurotransmission may reflect cellular energetic debilitation rather than adaptive attenuation of cellular metabolic imbalance. Copyright © 2011 Wiley-Liss, Inc.
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Fernández, Cecilia S; Bruque, Carlos D; Taboas, Melisa; Buzzalino, Noemí D; Espeche, Lucia D; Pasqualini, Titania; Charreau, Eduardo H; Alba, Liliana G; Ghiringhelli, Pablo D; Dain, Liliana
2015-09-01
The aim of the current study was to search for the presence of genetic variants in the CYP21A2 Z promoter regulatory region in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Screening of the 10 most frequent pseudogene-derived mutations was followed by direct sequencing of the entire coding sequence, the proximal promoter, and a distal regulatory region in DNA samples from patients with at least one non-determined allele. We report three non-classical patients that presented a novel genetic variant-g.15626A>G-within the Z promoter regulatory region. In all the patients, the novel variant was found in cis with the mild, less frequent, p.P482S mutation located in the exon 10 of the CYP21A2 gene. The putative pathogenic implication of the novel variant was assessed by in silico analyses and in vitro assays. Topological analyses showed differences in the curvature and bendability of the DNA region bearing the novel variant. By performing functional studies, a significantly decreased activity of a reporter gene placed downstream from the regulatory region was found by the G transition. Our results may suggest that the activity of an allele bearing the p.P482S mutation may be influenced by the misregulated CYP21A2 transcriptional activity exerted by the Z promoter A>G variation.
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Gao, Huanyao; Subramanian, Sowmya; Couturier, Jérémy; Naik, Sunil; Kim, Sung-Kun; Leustek, Thomas; Knaff, David B.; Wu, Hui-Chen; Vignols, Florence; Huynh, Boi Hanh; Rouhier, Nicolas; Johnson, Michael K.
2013-01-01
Nfu-type proteins are essential in the biogenesis of iron-sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV-visible absorption and circular dichroism, resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S]2+ and [4Fe-4S]2+ clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for maturation of chloroplastic Fe-S proteins via intact, rapid and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S]2+ cluster donor for glutaredoxin S16, but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4S]2+ cluster donor for adenosine 5′-phosphosulfate reductase (APR1) and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2, but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters. PMID:24032747
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12. PIERS 5S AND 4S, SHOWING TRANSITION AT 4S FROM ...
12. PIERS 5S AND 4S, SHOWING TRANSITION AT 4S FROM GIRDER SPAN TO 'SUSPENDED' TRUSS SPAN AT U0. VIEW LOOKING WEST. - George P. Coleman Memorial Bridge, Spanning York River at U.S. Route 17, Yorktown, York County, VA
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Wood, Ruth I.; Armstrong, Abigail; Fridkin, Vlad; Shah, Vivek; Najafi, Allison; Jakowec, Michael
2013-01-01
In humans and animals, anabolic-androgenic steroids (AAS) increase aggression, but the underlying behavioral mechanisms are unclear. AAS may increase the motivation to fight. Alternatively, AAS may increase impulsive behavior, consistent with the popular image of ‘roid rage. To test this, adolescent male rats were treated chronically with testosterone (7.5 mg/kg) or vehicle and tested for aggressive motivation and impulsivity. Rats were trained to respond on a nose-poke on a 10 min fixed-interval schedule for the opportunity to fight in their home cage with an unfamiliar rat. Although testosterone increased aggression (6.3±1.3 fights/5 min vs 2.4±0.8 for controls, p<0.05), there was no difference in operant responding (28.4±1.6 nose-pokes/ 10 min for testosterone, 32.4±7.0 for vehicle). This suggests that testosterone does not enhance motivation for aggression. To test for impulsivity, rats were trained to respond for food in a delay-discounting procedure. In an operant chamber, one lever delivered one food pellet immediately, the other lever gave 4 pellets after a delay (0, 15, 30 or 45 s). In testosterone- and vehicle-treated rats, body weights and food intake did not differ. However, testosterone-treated rats chose the larger, delayed reward more often (4.5±0.7 times in 10 trials with 45 s delay) than vehicle controls (2.5±0.5 times, p<0.05), consistent with a reduction in impulsive choice. Thus, although chronic high-dose testosterone enhances aggression, this does not include an increase in impulsive behavior or motivation to fight. This is further supported by measurement of tyrosine hydroxylase (TH) by Western immunoblot analysis in brain regions important for motivation (nucleus accumbens, Acb) and executive function (medial prefrontal cortex, PFC). There were no differences in TH between testosterone- and vehicle-treated rats in Acb or PFC. However, testosterone significantly reduced TH (to 76.9±3.1% of controls, p<0.05) in the caudate-putamen, a
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USDA-ARS?s Scientific Manuscript database
Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3’-hydroxylase (C3’H) for CGA biosynthesis has not been isolated or characterized i...
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Genes encoding p-coumarate 3-hydroxylase (C3H) and methods of use
Chapple, Clinton C. S.; Franke, Rochus; Ruegger, Max O.
2006-07-04
The present invention is directed to a method for altering secondary metabolism in plants, specifically phenylpropanoid metabolism. The present invention is further directed to a mutant p-coumarate 3-hydroxylase gene, referred to herein as the ref8 gene, its protein product which can be used to prepare gene constructs and transgenic plants. The gene constructs and transgenic plants are further aspects of the present invention.
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A mutation analysis of the phenylalanine hydroxylase (PAH) gene in the Israeli population.
Bercovich, D; Elimelech, A; Yardeni, T; Korem, S; Zlotogora, J; Gal, N; Goldstein, N; Vilensky, B; Segev, R; Avraham, S; Loewenthal, R; Schwartz, G; Anikster, Y
2008-05-01
Hyperphenylalaninemia (HPA) is a group of diseases characterized by a persistent elevation of phenylalanine levels in tissues and biological fluids. The most frequent form is phenylalanine hydroxylase deficiency, causing phenylketonuria (PKU). Among 159 Israeli patients (Jews, Muslim and Christian Arabs and Druze) with HPA, in whom at least one of the mutations was characterized, a total of 43 different mutations were detected, including seven novel ones. PKU was very rare among Ashkenazi Jews and relatively frequent among Jews from Yemen, the Caucasian Mountains, Bukhara and Tunisia. The mutations responsible for the high frequency were: exon3del (Yemenite Jews), L48S (Tunisian Jews) and E178G, P281L and L48S (Jews from the Caucasian Mountains and Bukhara). Among the non-Jewish Israeli citizens, the disease was relatively frequent in the Negev and in the Nazareth vicinity, and in many localities a unique mutation was detected, often in a single family. While marked genetic heterogeneity was observed in the Arab and Jewish populations, only one mutation A300S, was frequent in all of the communities. Several of the other frequent mutations were shared by the non-Ashkenazi Jews and Arabs; none were mutual to Ashkenazi Jews and Arabs.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lemoine, Pierric, E-mail: pierric.lemoine@univ-rennes1.fr; Bourgès, Cédric; Barbier, Tristan
Ternary copper-containing sulfides Cu{sub 12}Sb{sub 4}S{sub 13} and Cu{sub 4}Sn{sub 7}S{sub 16} have attracted considerable interest since few years due to their high-efficiency conversion as absorbers for solar energy and promising thermoelectric materials. We report therein on the decomposition study of Cu{sub 12}Sb{sub 4}S{sub 13} and Cu{sub 4}Sn{sub 7}S{sub 16} phases using high temperature in situ neutron powder diffraction. Our results obtained at a heating rate of 2.5 K/min indicate that: (i) Cu{sub 12}Sb{sub 4}S{sub 13} decomposes above ≈792 K into Cu{sub 3}SbS{sub 3}, and (ii) Cu{sub 4}Sn{sub 7}S{sub 16} decomposes above ≈891 K into Sn{sub 2}S{sub 3} and amore » copper-rich sulfide phase of sphalerite ZnS-type structure with an assumed Cu{sub 3}SnS{sub 4} stoichiometry. Both phase decompositions are associated to a sulfur volatilization. While the results on Cu{sub 12}Sb{sub 4}S{sub 13} are in fair agreement with recent published data, the decomposition behavior of Cu{sub 4}Sn{sub 7}S{sub 16} differs from other studies in terms of decomposition temperature, thermal stability and products of reaction. Finally, the crystal structure refinements from neutron powder diffraction data are reported and discussed for the Cu{sub 4}Sn{sub 7}S{sub 16} and tetrahedrite Cu{sub 12}Sb{sub 4}S{sub 13} phases at 300 K, and for the high temperature form of skinnerite Cu{sub 3}SbS{sub 3} at 843 K. - Graphical abstract: In situ neutron powder diffraction data (heating rate of 2.5 K/min) indicates that (i) the ternary Cu{sub 12}Sb{sub 4}S{sub 13} phase is stable up to 792 K and decomposes at higher temperature into Cu{sub 3}SbS{sub 3} and Cu{sub 1.5}Sb{sub 0.5}S{sub 2}, and (ii) the Cu{sub 4}Sn{sub 7}S{sub 16} phase is stable up to 891 K and decomposes at higher temperature into Sn{sub 2}S{sub 3} and a cubic phase of sphalerite ZnS-type structure. Sulfur volatilization likely occurs in order to balance the overall stoichiometry.« less
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Ishikawa, Yoshihiro; Bächinger, Hans Peter
2013-11-01
Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.
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Perez, Dale M.; Richards, Mark E.; Parker, Robert; ...
2015-12-13
Turkeys and chickens reared to 5-weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). AcT ingestion per kg body weight was statistically similar between chicken and turkey only when the RRR-AcT containing diet was fed. Metabolites of alpha tocopherol (AT) and gamma tocopherol (GT) were measured in bile. When fed the RRR AcT diet, AT- and GT-metabolites were 8.3 and 5.3-fold elevated in turkey compared to chicken,more » respectively (p<0.001). When fed all-racemic AcT at 58 mg/kg feed, AT- and GT-metabolites were 2.2 and 2.4-fold elevated in turkey compared to chicken, respectively (p<0.01) with AcT ingestion per kg body weight 1.29-fold higher in the chickens (p<0.001). Turkey cytochrome P450 2C29 (CYP2C29) was increased relative to its chicken ortholog based on both RNA-Seq (p<0.001) and activity-based protein profiling (p<0.01) of liver tissue. Turkey CYP3A9 and CYP4F22 were increased compared to their chicken orthologs using as determined by Activity- Based Protein Profiling (p<0.05). The 3A and 4F family are noted as tocopherol hydroxylases in mammals. Alpha tocopherol concentrations in plasma, liver and muscle from turkey were 1.7 to 4.7-fold lower than the respective tissues from chicken (p<0.05). Lipid oxidation occurred more rapidly in turkey thigh compared to chicken thigh (p<0.05). These studies suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylases in turkey liver contribute to the decreased accumulation of tocopherols in turkey tissues compared to that of chicken.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Perez, Dale M.; Richards, Mark E.; Parker, Robert
Turkeys and chickens reared to 5-weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). AcT ingestion per kg body weight was statistically similar between chicken and turkey only when the RRR-AcT containing diet was fed. Metabolites of alpha tocopherol (AT) and gamma tocopherol (GT) were measured in bile. When fed the RRR AcT diet, AT- and GT-metabolites were 8.3 and 5.3-fold elevated in turkey compared to chicken,more » respectively (p<0.001). When fed all-racemic AcT at 58 mg/kg feed, AT- and GT-metabolites were 2.2 and 2.4-fold elevated in turkey compared to chicken, respectively (p<0.01) with AcT ingestion per kg body weight 1.29-fold higher in the chickens (p<0.001). Turkey cytochrome P450 2C29 (CYP2C29) was increased relative to its chicken ortholog based on both RNA-Seq (p<0.001) and activity-based protein profiling (p<0.01) of liver tissue. Turkey CYP3A9 and CYP4F22 were increased compared to their chicken orthologs using as determined by Activity- Based Protein Profiling (p<0.05). The 3A and 4F family are noted as tocopherol hydroxylases in mammals. Alpha tocopherol concentrations in plasma, liver and muscle from turkey were 1.7 to 4.7-fold lower than the respective tissues from chicken (p<0.05). Lipid oxidation occurred more rapidly in turkey thigh compared to chicken thigh (p<0.05). These studies suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylases in turkey liver contribute to the decreased accumulation of tocopherols in turkey tissues compared to that of chicken.« less
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Optimization study for the experimental configuration of CMB-S4
NASA Astrophysics Data System (ADS)
Barron, Darcy; Chinone, Yuji; Kusaka, Akito; Borril, Julian; Errard, Josquin; Feeney, Stephen; Ferraro, Simone; Keskitalo, Reijo; Lee, Adrian T.; Roe, Natalie A.; Sherwin, Blake D.; Suzuki, Aritoki
2018-02-01
The CMB Stage 4 (CMB-S4) experiment is a next-generation, ground-based experiment that will measure the cosmic microwave background (CMB) polarization to unprecedented accuracy, probing the signature of inflation, the nature of cosmic neutrinos, relativistic thermal relics in the early universe, and the evolution of the universe. CMB-S4 will consist of O(500,000) photon-noise-limited detectors that cover a wide range of angular scales in order to probe the cosmological signatures from both the early and late universe. It will measure a wide range of microwave frequencies to cleanly separate the CMB signals from galactic and extra-galactic foregrounds. To advance the progress towards designing the instrument for CMB-S4, we have established a framework to optimize the instrumental configuration to maximize its scientific output. The framework combines cost and instrumental models with a cosmology forecasting tool, and evaluates the scientific sensitivity as a function of various instrumental parameters. The cost model also allows us to perform the analysis under a fixed-cost constraint, optimizing for the scientific output of the experiment given finite resources. In this paper, we report our first results from this framework, using simplified instrumental and cost models. We have primarily studied two classes of instrumental configurations: arrays of large-aperture telescopes with diameters ranging from 2–10 m, and hybrid arrays that combine small-aperture telescopes (0.5-m diameter) with large-aperture telescopes. We explore performance as a function of telescope aperture size, distribution of the detectors into different microwave frequencies, survey strategy and survey area, low-frequency noise performance, and balance between small and large aperture telescopes for hybrid configurations. Both types of configurations must cover both large (~ degree) and small (~ arcmin) angular scales, and the performance depends on assumptions for performance vs. angular scale
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Energies and spin states of FeS(0/-), FeS2(0/-), Fe2S2(0/-), Fe3S4(0/-), and Fe4S4(0/-) clusters.
Li, Yan-Ni; Wang, Shengguang; Wang, Tao; Gao, Rui; Geng, Chun-Yu; Li, Yong-Wang; Wang, Jianguo; Jiao, Haijun
2013-04-15
The structures and energies of the electronic ground states of the FeS(0/-), FeS2(0/-), Fe2S2(0/-), Fe3S4(0/-), and Fe4S4(0/-) neutral and anionic clusters have been computed systematically with nine computational methods in combination with seven basis sets. The computed adiabatic electronic affinities (AEA) have been compared with available experimental data. Most reasonable agreements between theory and experiment have been found for both hybrid B3LYP and B3PW91 functionals in conjugation with 6-311+G* and QZVP basis sets. Detailed comparisons between the available experimental and computed AEA data at the B3LYP/6-311+G* level identified the electronic ground state of (5)Δ for FeS, (4)Δ for FeS(-), (5)B2 for FeS2, (6)A1 for FeS2(-), (1)A1 for Fe2S2, (8)A' for Fe2S2(-), (5)A'' for Fe3S4, (6)A'' for Fe3S4(-), (1)A1 for Fe4S4, and (1)A2 for Fe4S4(-). In addition, Fe2S2, Fe3S4, Fe3S4(-), Fe4S4, and Fe4S4(-) are antiferromagnetic at the B3LYP/6-311+G* level. The magnetic properties are discussed on the basis of natural bond orbital analysis. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Hutabarat, Olly Sanny; Flachowsky, Henryk; Regos, Ionela; Miosic, Silvija; Kaufmann, Christine; Faramarzi, Shadab; Alam, Mohammed Zobayer; Gosch, Christian; Peil, Andreas; Richter, Klaus; Hanke, Magda-Viola; Treutter, Dieter; Stich, Karl; Halbwirth, Heidi
2016-05-01
Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab. The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3'-hydroxylase (F3'H). Using recombinant F3'H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3'H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.
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Repa, J J; Lund, E G; Horton, J D; Leitersdorf, E; Russell, D W; Dietschy, J M; Turley, S D
2000-12-15
Sterol 27-hydroxylase (CYP27) participates in the conversion of cholesterol to bile acids. We examined lipid metabolism in mice lacking the Cyp27 gene. On normal rodent chow, Cyp27(-/-) mice have 40% larger livers, 45% larger adrenals, 2-fold higher hepatic and plasma triacylglycerol concentrations, a 70% higher rate of hepatic fatty acid synthesis, and a 70% increase in the ratio of oleic to stearic acid in the liver versus Cyp27(+/+) controls. In Cyp27(-/-) mice, cholesterol 7alpha-hydroxylase activity is increased 5-fold, but bile acid synthesis and pool size are 47 and 27%, respectively, of those in Cyp27(+/+) mice. Intestinal cholesterol absorption decreases from 54 to 4% in knockout mice, while fecal neutral sterol excretion increases 2.5-fold. A compensatory 2.5-fold increase in whole body cholesterol synthesis occurs in Cyp27(-/-) mice, principally in liver, adrenal, small intestine, lung, and spleen. The mRNA for the cholesterogenic transcription factor sterol regulatory element-binding protein-2 (SREBP-2) and mRNAs for SREBP-2-regulated cholesterol biosynthetic genes are elevated in livers of mutant mice. In addition, the mRNAs encoding the lipogenic transcription factor SREBP-1 and SREBP-1-regulated monounsaturated fatty acid biosynthetic enzymes are also increased. Hepatic synthesis of fatty acids and accumulation of triacylglycerols increases in Cyp27(-/-) mice and is associated with hypertriglyceridemia. Cholic acid feeding reverses hepatomegaly and hypertriglyceridemia but not adrenomegaly in Cyp27(-/-) mice. These studies confirm the importance of CYP27 in bile acid synthesis and they reveal an unexpected function of the enzyme in triacylglycerol metabolism.
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Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene
DOE Office of Scientific and Technical Information (OSTI.GOV)
Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C.
1992-09-01
The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiples of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1,more » 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between theses alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations. 32 refs., 3 figs., 3 tabs.« less
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Li, Jun; Dangott, Lawrence J.; Fitzpatrick, Paul F.
2010-01-01
Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains. PMID:20307070
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Assessment of the chloracnegenic response induced by 3,4,3',4'-tetrachloroazoxybenzene in mice
DOE Office of Scientific and Technical Information (OSTI.GOV)
Horton, V.L.; Yeary, R.A.
Chloracne is a follicular hyperkeratosis produced by exposure to certain halogenated aromatic compounds. The rabbit ear bioassay has been used successfully for testing the acnegenic activity of compounds, but the lack of reference data in this species limits its usefulness in correlating chloracne to other toxic effects such as skin carcinogenesis. In this study, a prototype chloracnegen, 3,4,3',4'-tetrachloroazoxybenzene (TCAOB), was used. Five strains of mice (hairless, rhino, rhino+, DBA/2J, and C57BL/6) were treated topically with 100 ..mu..l of 0.001, 0.01, or 0.1% TCAOB daily for 3-9 wk. Skin and liver histology were performed and hepatic enzyme activities measured. At themore » 0.001% TCAOB level, induction of hepatic aniline hydroxylase and cytochrome P-450 occurred in the C57BL/6 mice and induction of cytochrome c reductase occurred in the rhino mice. Dose-dependent gross and histologic skin lesions, characteristic of follicular hyperkeratosis, were observed in the rhino and hairless strains at the 0.01% and 0.1% levels. These two strains also had induction of hepatic cytochrome c reductase, cytochrome P-450, and aniline hydroxylase at TCAOB concentrations of 0.01 or 0.1%. These results suggest that the rhino and hairless strains of mice may be useful in the study of chloracne.« less
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Bhore, Subhash J; Kassim, Amelia; Loh, Chye Ying; Shah, Farida H
2010-01-01
It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (Chyb) enzyme encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected Chyb amino acid sequences from other plant species and algal members with E. oleifera Chyb using ClustalW and its phylogenetic analysis suggest that Chyb from monocotyledonous plant species, Lilium hubrid, Crocus sativus and Zea mays are the most evolutionary related with E. oleifera Chyb. This study reports the annotation of E. oleifera Chyb. Abbreviations ESTs - expressed sequence tags, EoChyb - Elaeis oleifera beta-carotene hydroxylase, MC - main cluster PMID:21364789
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Synthesis of ( 2S, 4S)-4-phenylamino-5-oxoproline derivatives.
Nizova, I A; Krasnov, V P; Levit, G L; Kodess, M I
2002-01-01
The paper describes the synthesis of ( 2S, 4S)-4-(N-Ts)- and ( 2S, 4S)-4-(N-Boc)-phenylamino-5-oxoprolines (pyroglutamic acid). These derivatives have been shown to be useful for synthesis of their amides and peptides in spite of steric hindrances caused by bulky groups adjacent to the reaction centre. Under the conditions applied no lactam ring opening and no loss of stereochemical integrity of any of the chiral centres were observed, which has been confirmed by NMR techniques.
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Rapid carrier screening using short tandem repeats in the phenylalanine hydroxylase gene.
Shawky, R M; el-Aleem, K A; Rifaat, M M; el-Naggar, R L; Marzouk, G M
2002-01-01
Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by defects in the phenylalanine hydroxylase (PAH) system. Our work aimed to screen the PAH locus for the presence of potentially useful short tandem repeats (STR) as markers for carrier detection in PKU families in Egypt, and to determine the level of PAH heterozygosity within the Egyptian population. The system contains at least eight independent alleles in the Egyptian population, transmitted in a Mendelian fashion. Variations in the number of STR in the 16 families studied gave rise to polymorphisms that proved to be suitable markers for PKU carrier detection and prenatal diagnosis. The most frequent allelic fragment size in PKU patients was 246 bp (35.7%), which together with a fragment of 254 bp accounted for 60.7% of the mutant chromosomes.
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Chronic stress effects on working memory: association with prefrontal cortical tyrosine hydroxylase.
Lee, Young-A; Goto, Yukiori
2015-06-01
Chronic stress causes deficits in cognitive function including working memory, for which transmission of such catecholamines as dopamine and noradrenaline transmission in the prefrontal cortex (PFC) are crucial. Since catecholamine synthesis depends on the rate-limiting enzyme, tyrosine hydroxylase (TH), TH is thought to play an important role in PFC function. In this study, we found that two distinct population existed in Sprague-Dawley rats in terms of working memory capacity, one with higher working memory capacity, and the other with low capacity. This distinction of working memory capacity became apparent after rats were exposed to chronic stress. In addition, such working memory capacity and alterations of working memory function by chronic stress were associated with TH expression in the PFC. Copyright © 2015 Elsevier B.V. All rights reserved.
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SUPPRESSION OF HIF-1α TRANSCRIPTIONAL ACTIVITY BY THE HIF PROLYL HYDROXYLASE EGLN1*
To, Kenneth K. W.; Huang, L. Eric
2005-01-01
The cellular response to hypoxia is, at least in part, mediated by the transcriptional regulation of hypoxia-responsive genes involved in balancing the intracellular ATP production and consumption. Recent evidence suggests that the transcription factor, HIF-1α, functions as a master regulator of oxygen homeostasis by controlling a broad range of cellular events in hypoxia. In normoxia, HIF-1α is targeted for destruction via prolyl hydroxylation, an oxygen-dependent modification that signals for recognition by the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor (VHL). Three HIF prolyl hydroxylases (EGLN1, EGLN2, and EGLN3) have been identified in mammals, among which, EGLN1 and EGLN3, are hypoxia-inducible at their mRNA levels in a HIF-1α-dependent manner. In this study, we demonstrate that apart from promoting HIF-1α proteolysis in normoxia, EGLN1 specifically represses HIF-1α transcriptional activity in hypoxia. Ectopic expression of EGLN1 inhibited HIF-1α transcriptional activity without altering its protein levels in a VHL-deficient cell line, indicating a discrete activity of EGLN1 in transcriptional repression. Conversely, silencing of EGLN1 expression augmented HIF-1α transcriptional activity and its target gene expression in hypoxia. Hence, we propose that the accumulated EGLN1 in hypoxia acts as a negative-feedback mechanism to modulate HIF-1α target gene expression. Our finding also provides new insight into the pharmacological manipulation of the HIF prolyl hydroxylase for ischemic diseases. PMID:16157596
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Pittelkow, Marco; Heider, Johann; Smits, Sander H. J.; Bremer, Erhard
2014-01-01
Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine from the precursor L-aspartate-β-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo
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Rousset, M; Montet, Y; Guigliarelli, B; Forget, N; Asso, M; Bertrand, P; Fontecilla-Camps, J C; Hatchikian, E C
1998-09-29
The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.
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Rousset, Marc; Montet, Yael; Guigliarelli, Bruno; Forget, Nicole; Asso, Marcel; Bertrand, Patrick; Fontecilla-Camps, Juan C.; Hatchikian, E. Claude
1998-01-01
The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed. PMID:9751716
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Wu, Yue; Jiang, Zhensheng; Li, Zhihong; Gu, Jing; You, Qi-Dong; Zhang, Xiaojin
2018-06-01
As a gene associated with anemia, the erythropoiesis gene is physiologically expressed under hypoxia regulated by hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study we applied click chemistry to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time and a series of triazole compounds showed preferable inhibitory activity in fluorescence polarization assay. Of particular note was the orally active HIF-PHD inhibitor 15i (IC50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC50 = 591.4 nM). Furthermore, it can upregulate the hemoglobin of cisplatin induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity observed in vivo. These results confirm that triazole compound 15i is a promising candidate for the treatment of renal anemia.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kwon, Soo-Young; Kang, Beom Sik; Kim, Ghyung-Hwa
2007-11-01
PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collectedmore » to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 94.72, c = 359.68 Å, γ = 120°. The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.« less
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Prolyl hydroxylase regulates axonal rewiring and motor recovery after traumatic brain injury
Miyake, S; Muramatsu, R; Hamaguchi, M; Yamashita, T
2015-01-01
Prolyl 4-hydroxylases (PHDs; PHD1, PHD2, and PHD3) are a component of cellular oxygen sensors that regulate the adaptive response depending on the oxygen concentration stabilized by hypoxia/stress-regulated genes transcription. In normoxic condition, PHD2 is required to stabilize hypoxia inducible factors. Silencing of PHD2 leads to the activation of intracellular signaling including RhoA and Rho-associated protein kinase (ROCK), which are key regulators of neurite growth. In this study, we determined that genetic or pharmacological inhibition of PHD2 in cultured cortical neurons prevents neurite elongation through a ROCK-dependent mechanism. We then explored the role of PHDs in axonal reorganization following a traumatic brain injury in adult mice. Unilateral destruction of motor cortex resulted in behavioral deficits due to disruption of the corticospinal tract (CST), a part of the descending motor pathway. In the spinal cord, sprouting of fibers from the intact side of the CST into the denervated side is thought to contribute to the recovery process following an injury. Intracortical infusion of PHD inhibitors into the intact side of the motor cortex abrogated spontaneous formation of CST collaterals and functional recovery after damage to the sensorimotor cortex. These findings suggest PHDs have an important role in the formation of compensatory axonal networks following an injury and may represent a new molecular target for the central nervous system disorders. PMID:25675298
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Zhang, Kewei; Halitschke, Rayko; Yin, Changxi; Liu, Chang-Jun; Gan, Su-Sheng
2013-01-01
The plant hormone salicylic acid (SA) plays critical roles in plant defense, stress responses, and senescence. Although SA biosynthesis is well understood, the pathways by which SA is catabolized remain elusive. Here we report the identification and characterization of an SA 3-hydroxylase (S3H) involved in SA catabolism during leaf senescence. S3H is associated with senescence and is inducible by SA and is thus a key part of a negative feedback regulation system of SA levels during senescence. The enzyme converts SA (with a Km of 58.29 µM) to both 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA in vitro but only 2,3-DHBA in vivo. The s3h knockout mutants fail to produce 2,3-DHBA sugar conjugates, accumulate very high levels of SA and its sugar conjugates, and exhibit a precocious senescence phenotype. Conversely, the gain-of-function lines contain high levels of 2,3-DHBA sugar conjugates and extremely low levels of SA and its sugar conjugates and display a significantly extended leaf longevity. This research reveals an elegant SA catabolic mechanism by which plants regulate SA levels by converting it to 2,3-DHBA to prevent SA overaccumulation. The research also provides strong molecular genetic evidence for an important role of SA in regulating the onset and rate of leaf senescence. PMID:23959884
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Study of η and dipion transitions in Υ ( 4 S ) decays to lower bottomonia
DOE Office of Scientific and Technical Information (OSTI.GOV)
Guido, E.; Mussa, R.; Tamponi, U.
We study hadronic transitions between bottomonium states using 496 fb -1 data collected at the $Y$(4S) resonance with the Belle detector at the KEKB asymmetric energy e +e - collider. We measure B($Y$(4S)→π +π -$-Y$(1S))=(8.2 ± 0.5(stat) ± 0.4(syst))×10 -5, B($Y$(4S) → π +π -$Y$ð2SÞÞ¼ð7.91.0ðstatÞ 0.4ðsystÞÞ×10-5, and Bð$Y$ð4SÞ → η$Y$ð1SÞÞ ¼ ð1.70 0.23ðstatÞ 0.08ðsystÞÞ × 10-4. We measure the ratio of branching fractions R ¼ Bð$Y$ð4SÞ → η$Y$ð1SÞÞ=Bð$Y$ð4SÞ → πþπ-$Y$ð1SÞÞ ¼ 2.07 0.30ðstatÞ 0.11ðsystÞ. We search for the decay $Y$ð13D1;2 Þ → η$Y$ð1SÞ, but do not find significant evidence for such a transition.We also measure the initial-state radiation production crossmore » sections of the $Y$ð2S; 3SÞ resonances and we find values compatible with the expected ones. Finally, the analysis of the $Y$(4S) → π +π -$Y$(1S) events shows indications for a resonant contribution due to the f 0 (980) meson.« less
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Boronat, M; Carrillo, A; Ojeda, A; Estrada, J; Ezquieta, B; Marín, F; Nóvoa, F J
2004-06-01
The development of Cushing's disease among patients with deficiency of 21-hydroxylase has not been observed to date. The clinical manifestations and the hormonal profile of this exceptional association are herein described through the study of two cases. The first one was a 39-yr-old woman who had undergone non-curative transsphenoidal surgery for a pituitary-dependent Cushing's syndrome 12 yr before. She showed hypertension, central obesity, severe hirsutism, alopecia and hyperpigmentation. Urinary excretion of cortisol was normal, but ACTH levels were very high and hormonal dynamic studies (cortisol circadian rhythm, insulin-induced hypoglycemia and dexamethasone suppression tests) revealed the qualitative disturbances that characterize Cushing's disease. Serum concentrations of androstenedione, free testosterone and 17-hydroxyprogesterone were clearly increased. Reexamination of the tissue samples from previous surgery confirmed the presence of an ACTH-producing pituitary adenoma. CYP21 gene analysis found the splicing 655G mutation at intron 2 and the V281L mutation at exon 7. The second case was a 21-yr-old woman who was diagnosed with pituitary ACTH-dependent Cushing's syndrome according to unequivocal clinical and laboratory findings. However, hirsutism was particularly severe and both serum androgens and 17-hydroxyprogesterone were elevated. The patient was heterozygote for a large conversion of CYP21 gene. In these cases, the clinical and biochemical expression of Cushing's syndrome was determined by the different severity of 21-hydroxylase deficiency and the subsequent residual ability of adrenal cortex to synthesize cortisol.
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Park, Sangkyu; Choi, Min Ji; Lee, Jong Yeol; Kim, Jae Kwang; Ha, Sun-Hwa; Lim, Sun-Hyung
2016-09-13
Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3',4'-dihydroxylated leucocyanidin. 3'-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3'H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3'H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice.
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Hamada, Makoto; Takayama, Tetsuo; Shibata, Tsuyoshi; Hiratate, Akira; Takahashi, Masato; Yashiro, Miyoko; Takayama, Noriko; Okumura-Kitajima, Lisa; Koretsune, Hiroko; Kajiyama, Hiromitsu; Naruse, Takumi; Kato, Sota; Takano, Hiroki; Kakinuma, Hiroyuki
2018-06-01
Prolyl hydroxylase domain-containing protein (PHD) inhibitors are useful as orally administered agents for the treatment of renal anemia. Based on the common structures of known PHD inhibitors, we found novel PHD inhibitor 1 with a 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid motif. The PHD2-inhibitory activity, lipophilicity (CLogP), and PK profiles (hepatocyte metabolism, protein binding, and/or elimination half-life) of this inhibitor were used as the evaluation index to optimize the structure and eventually discovered clinical candidate 42 as the suitable compound. Compound 42 was demonstrated to promote the production of erythropoietin (EPO) following oral administration in mice and rats. The predicted half-life of this compound in humans was 1.3-5.6 h, therefore, this drug may be expected to administer once daily with few adverse effects caused by excessive EPO production. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Monoamine Oxidase and Dopamine β-Hydroxylase Inhibitors from the Fruits of Gardenia jasminoides
Kim, Ji Ho; Kim, Gun Hee; Hwang, Keum Hee
2012-01-01
This research was designed to determine what components of Gardenia jasminoides play a major role in inhibiting the enzymes related antidepressant activity of this plant. In our previous research, the ethyl acetate fraction of G. jasminosides fruits inhibited the activities of both monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B), and oral administration of the ethanolic extract slightly increased serotonin concentrations in the brain tissues of rats and decreased MAO-B activity. In addition, we found through in vitro screening test that the ethyl acetate fraction showed modest inhibitory activity on dopamine-β hydroxylase (DBH). The bioassay-guided fractionation led to the isolation of five bio-active compounds, protocatechuic acid (1), geniposide (2), 6'-O-trans-p-coumaroylgeniposide (3), 3,5-d-ihydroxy-1,7-bis (4-hydroxyphenyl) heptanes (4), and ursolic acid (5), from the ethyl acetate fraction of G. jasminoides fruits. The isolated compounds showed different inhibitory potentials against MAO-A, -B, and DBH. Protocatechuic acid showed potent inhibition against MAO-B (IC50 300 μmol/L) and DBH (334 μmol/L), exhibiting weak MAO-A inhibition (2.41 mmol/L). Two iridoid glycosides, geniposide (223 μmol/L) and 6'-O-trans-p-coumaroylgeniposide (127μmol/L), were selective MAO-B inhibitor. Especially, 6'-O-trans-p-coumaroylgeniposide exhibited more selective MAO-B inhibition than deprenyl, well-known MAO-B inhibitor for the treatment of early-stage Parkinson’s disease. The inhibitory activity of 3,5-di-hydroxy-1,7-bis (4-hydroxyphenyl) heptane was strong for MAO-B (196 μmol/L), modest for MAO-A (400 μmol/L), and weak for DBH (941 μmol/L). Ursolic acid exhibited significant inhibition of DBH (214 μmol/L), weak inhibition of MAO-B (780 μmol/L), and no inhibition against MAO-A. Consequently, G. jasminoides fruits are considerable for development of biofunctional food materials for the combination treatment of depression and neurodegenerative disorders
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Seth, Puneet; Yeowell, Heather N
2010-04-01
Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease
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Tsou, Chih-Chin; Lin, Zong-Sian; Lu, Tsai-Te; Liaw, Wen-Feng
2008-12-17
Transformation of dinitrosyl iron complexes (DNICs) [(NO)(2)Fe(SR)(2)](-) (R = Et, Ph) into [4Fe-4S] clusters [Fe(4)S(4)(SPh)(4)](2-) in the presence of [Fe(SPh)(4)](2-/1-) and S-donor species S(8) via the reassembling process ([(NO)(2)Fe(SR)(2)](-) --> [Fe(4)S(3)(NO)(7)](-) (1)/[Fe(4)S(3)(NO)(7)](2-) (2) --> [Fe(4)S(4)(NO)(4)](2-) (3) --> [Fe(4)S(4)(SPh)(4)](2-) (5)) was demonstrated. Reaction of [(NO)(2)Fe(SR)(2)](-) (R = Et, Ph) with S(8) in THF, followed by the addition of HBF(4) into the mixture solution, yielded complex [Fe(4)S(3)(NO)(7)](-) (1). Complex [Fe(4)S(3)(NO)(7)](2-) (2), obtained from reduction of complex 1 by [Na][biphenyl], was converted into complex [Fe(4)S(4)(NO)(4)](2-) (3) along with byproduct [(NO)(2)Fe(SR)(2)](-) via the proposed [Fe(4)S(3)(SPh)(NO)(4)](2-) intermediate upon treating complex 2 with 1.5 equiv of [Fe(SPh)(4)](2-) and the subsequent addition of 1/8 equiv of S(8) in CH(3)CN at ambient temperature. Complex 3 was characterized by IR, UV-vis, and single-crystal X-ray diffraction. Upon addition of complex 3 to the CH(3)CN solution of [Fe(SPh)(4)](-) in a 1:2 molar ratio at ambient temperature, the rapid NO radical-thiyl radical exchange reaction between complex 3 and the biomimetic oxidized form of rubredoxin [Fe(SPh)(4)](-) occurred, leading to the simultaneous formation of [4Fe-4S] cluster [Fe(4)S(4)(SPh)(4)](2-) (5) and DNIC [(NO)(2)Fe(SPh)(2)](-). This result demonstrates a successful biomimetic reassembly of [4Fe-4S] cluster [Fe(4)S(4)(SPh)(4)](2-) from NO-modified [Fe-S] clusters, relevant to the repair of DNICs derived from nitrosylation of [4Fe-4S] clusters of endonuclease III back to [4Fe-4S] clusters upon addition of ferrous ion, cysteine, and IscS.
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Mechanism-based inactivation of dopamine beta-hydroxylase by p-cresol and related alkylphenols
DOE Office of Scientific and Technical Information (OSTI.GOV)
Goodhart, P.J.; DeWolf, W.E. Jr.; Kruse, L.I.
1987-05-05
The mechanism-based inhibition of dopamine beta-hydroxylase by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate activator addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible,more » is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.« less
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Yang, Seung-Hwan; Choi, Dongsu
2006-11-24
Ethylene and submergence enhance stem elongation of deepwater rice, at least in part, by reducing in the internode the endogenous abscisic acid (ABA) content and increasing the level of gibberellin A1 (GA1). We cloned and characterized the CYP707A5 and CYP707A6 genes, which encode putative ABA 8'-hydroxylase, the enzyme that catalyzes the oxidation of ABA. Expression of CYP707A5 was upregulated significantly by ethylene treatment, whereas that of CYP707A6 was not altered. Recombinant proteins from both genes expressed in yeast cells showed activity of ABA 8'-hydroxylase. This finding indicates that CYP707A5 may play a role in ABA catabolism during submergence- or ethylene-induced stem elongation in deepwater rice. Taken together, these results provide links between the molecular mechanisms and physiological phenomena of submergence- and ethylene-induced stem elongation in deepwater rice.
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Roberts, Kenneth M.; Pavon, Jorge Alex; Fitzpatrick, Paul F.
2013-01-01
Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH4) and O2. A complete kinetic mechanism for PheH was determined by global analysis of single turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH4-phenylalanine complex begins with the rapid binding of BH4 (Kd = 65 µM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (Kd = 130 µM) is approximately ten-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O2 rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, detectable as a decrease in absorbance at 340 nm, with a rate constant of 140 s−1. Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is ten-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines kcat. Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation. PMID:23327364
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Kleppe, Rune; Rosati, Sara; Jorge-Finnigan, Ana; Alvira, Sara; Ghorbani, Sadaf; Haavik, Jan; Valpuesta, José María; Heck, Albert J. R.; Martinez, Aurora
2014-01-01
Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15–0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [32P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18–0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in
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Bratland, Eirik; Magitta, Ng'weina Francis; Bøe Wolff, Anette Susanne; Ekern, Trude; Knappskog, Per Morten; Kämpe, Olle; Haavik, Jan; Husebye, Eystein Sverre
2013-06-01
Patients with autoimmune polyendocrine syndrome type 1 (APS-1) frequently have autoantibodies directed against the aromatic amino acid hydroxylases tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). We aimed to characterize these autoantibodies with regard to their antigenic determinants, their influence on enzymatic activity and their clinical associations. In particular, we wanted to compare autoantibodies against the two different isoforms of TPH, which display different tissue distribution. Using sera from 48 Scandinavian APS-1 patients we identified 36 patients (75%) with antibodies against one or more of these three enzymes. Antibodies against TPH1, but not TPH2, were associated with malabsorption in the whole Scandinavian cohort, while TH antibodies were associated with dental enamel hypoplasia in Norwegian patients. Subsequent experiments with selected patient sera indicated that while the C-terminal domain was the immunodominant part of TPH1, the epitopes of TPH2 and TH were mainly located in the N-terminal regulatory domains. We also identified a TPH1 specific epitope involved in antibody mediated inhibition of enzyme activity, a finding that provides new insight into the enzymatic mechanisms of the aromatic amino acid hydroxylases and knowledge about structural determinants of enzyme autoantigens. In conclusion, TPH1, TPH2 and TH all have unique antigenic properties in spite of their structural similarity. Copyright © 2012 Elsevier GmbH. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nhalil, Hariharan; Han, Dan; Du, Mao-Hua
High temperature synthesis and optical band gaps are reported for three candidate photovoltaic earth-abundant Cu 2PbSiS 4, Ag 2PbGeS 4 and KAg 2SbS 4 semiconductors. The reported synthesis method is found to be more advantageous for KAg 2SbS 4 compared to the literature reported synthesis utilizing supercritical ammonia as a reaction medium, which produces a mixture of chalcogenide products. Based on optical diffuse reflectance data, Cu 2PbSiS 4, Ag 2PbGeS 4 and KAg 2SbS 4 have band gaps in the 1.6–1.8 eV range, and are shown to be stable in ambient air for a period of 6 weeks, making themmore » attractive candidates for solar cell applications. Density functional theory (DFT) calculations indicate indirect band gaps for Cu 2PbSiS 4 and KAg 2SbS 4, and a nearly direct band gap for Ag 2PbGeS 4 with the calculated difference between indirect and direct gaps of only 30 meV. The p-type semiconducting behavior of Cu 2PbSiS 4, Ag 2PbGeS 4 is also verified by the transport measurments. The 3D connectivity of the polyanionic networks in these compounds results in dispersive valence and conduction bands, which is especially noticeable for KAg 2SbS 4. This fact is in part attributed to the presence of formally pentavalent SbV in this compound leading to empty Sb 5s orbitals in the conduction band. Finally, we discuss the potential of Cu 2PbSiS 4, Ag 2PbGeS 4 and KAg 2SbS 4 for photovoltaic applications based on synthesis, stability, band gap and electronic structure considerations.« less
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Nhalil, Hariharan; Han, Dan; Du, Mao-Hua; ...
2018-03-01
High temperature synthesis and optical band gaps are reported for three candidate photovoltaic earth-abundant Cu 2PbSiS 4, Ag 2PbGeS 4 and KAg 2SbS 4 semiconductors. The reported synthesis method is found to be more advantageous for KAg 2SbS 4 compared to the literature reported synthesis utilizing supercritical ammonia as a reaction medium, which produces a mixture of chalcogenide products. Based on optical diffuse reflectance data, Cu 2PbSiS 4, Ag 2PbGeS 4 and KAg 2SbS 4 have band gaps in the 1.6–1.8 eV range, and are shown to be stable in ambient air for a period of 6 weeks, making themmore » attractive candidates for solar cell applications. Density functional theory (DFT) calculations indicate indirect band gaps for Cu 2PbSiS 4 and KAg 2SbS 4, and a nearly direct band gap for Ag 2PbGeS 4 with the calculated difference between indirect and direct gaps of only 30 meV. The p-type semiconducting behavior of Cu 2PbSiS 4, Ag 2PbGeS 4 is also verified by the transport measurments. The 3D connectivity of the polyanionic networks in these compounds results in dispersive valence and conduction bands, which is especially noticeable for KAg 2SbS 4. This fact is in part attributed to the presence of formally pentavalent SbV in this compound leading to empty Sb 5s orbitals in the conduction band. Finally, we discuss the potential of Cu 2PbSiS 4, Ag 2PbGeS 4 and KAg 2SbS 4 for photovoltaic applications based on synthesis, stability, band gap and electronic structure considerations.« less
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AsS-Tl/sub 3/AsS/sub 4/ and AsS-Tl/sub 2/S systems
DOE Office of Scientific and Technical Information (OSTI.GOV)
Vorob'ev, Yu.I.; Velikova, N.G.; Kirilenko, V.V.
1987-11-01
Using DTA and XPA as well as microstructural analysis and microhardness measurements, the AsS-Tl/sub 3/AsS/sub 4/ and AsS-Tl/sub 2/S phase diagrams are studied. Two new ternary compounds, Tl/sub 2/As/sub 3/S/sub 4/ and Tl/sub 2/As/sub 2/S/sub 3/, are revealed and their interplanar distances are calculated.
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Reduction and Oxidation of the Active Site Iron in Tyrosine Hydroxylase: Kinetics and Specificity†
Frantom, Patrick A.; Seravalli, Javier; Ragsdale, Stephen W.; Fitzpatrick, Paul F.
2006-01-01
Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze–quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 ± 0.1 mM−1 s−1. 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 ± 0.1 mM−1 s−1 and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated Km for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH4 does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM−1 s−1. S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms. PMID:16475826
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Improved GaSb surfaces using a (NH4)2S/(NH4)2S04 solution
NASA Astrophysics Data System (ADS)
Murape, D. M.; Eassa, N.; Nyamhere, C.; Neethling, J. H.; Betz, R.; Coetsee, E.; Swart, H. C.; Botha, J. R.; Venter, A.
2012-05-01
Bulk (1 0 0) n-GaSb surfaces have been treated with a sulphur based solution ((NH4)2S/(NH4)2SO4) to which sulphur has been added, not previously reported for the passivation of GaSb surfaces. Au/n-GaSb Schottky barrier diodes (SBDs) fabricated on the treated material show significant improvement compared to that of the similar SBDs on the as-received material as evidenced by the lower ideality factor (n), higher barrier height (ϕb) and lower contact resistance obtained. Additionally, the reverse leakage current, although not saturating, has been reduced by almost an order of magnitude at -0.2 V. The sample surfaces were studied by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The native oxide, Sb-O, present on the as-received material is effectively removed on treating with ([(NH4)2S/(NH4)2SO4]+S) and (NH4)2S. Analysis of the as-received surface by XPS, prior to and after argon sputtering, suggests that the native oxide layer is ≤8.5 nm.
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Protonation and Proton-Coupled Electron Transfer at S-Ligated [4Fe-4S] Clusters
Morris, Wesley D.; Darcy, Julia W.; Mayer, James M.
2015-01-01
Biological [Fe-S] clusters are increasingly recognized to undergo proton-coupled electron transfer (PCET), but the site of protonation, mechanism, and role for PCET remains largely unknown. Here we explore this reactivity with synthetic model clusters. Protonation of the arylthiolate-ligated [4Fe-4S] cluster [Fe4S4(SAr)4]2- (1, SAr = S-2,4-6-(iPr)3C6H2) leads to thiol dissociation, reversibly forming [Fe4S4(SAr)3L]1- (2) + ArSH (L = solvent, and/or conjugate base). Solutions of 2 + ArSH react with the nitroxyl radical TEMPO to give [Fe4S4(SAr)4]1- (1ox) and TEMPOH. This reaction involves PCET coupled to thiolate association and may proceed via the unobserved protonated cluster [Fe4S4(SAr)3(HSAr)]1-(1-H). Similar reactions with this and related clusters proceed comparably. An understanding of the PCET thermochemistry of this cluster system has been developed, encompassing three different redox levels and two protonation states. PMID:25965413
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Brown, Philip M.; Drossman, Douglas A.; Wood, Alastair J. J.; Cline, Gary A.; Frazier, Kenny S.; Jackson, Jessica I.; Bronner, Johanna; Freiman, Joel; Zambrowicz, Brian; Sands, Arthur; Gershon, Michael D.
2016-01-01
BACKGROUND & AIMS Serotonin (5-hydroxytryptamine [5-HT]) has an important role in gastrointestinal function. LX1031 is an oral, locally acting, small molecule inhibitor of tryptophan hydroxylase (TPH). Local inhibition of TPH in the gastrointestinal tract might reduce mucosal production of serotonin (5-HT) and be used to treat patients with nonconstipating irritable bowel syndrome (IBS). METHODS We evaluated 2 dose levels of LX1031 (250 mg or 1000 mg, given 4 times/day) in a 28-day, multicenter, randomized, double-blind, placebo-controlled study of 155 patients with nonconstipating IBS. 5-hydroxyindoleacetic acid (5-HIAA), a biomarker of pharmacodynamic activity, was measured in urine samples at baseline (24 hours after LX1031 administration), and at weeks 4 and 6 (n = 76). RESULTS Each dose of LX1031 was safe and well-tolerated. The primary efficacy end point, relief of IBS pain and discomfort, improved significantly in patients given 1000 mg LX1031 (25.5%), compared with those given placebo, at week 1 (P = .018); with nonsignificant improvements at weeks 2, 3, and 4 (17.9%, 16.3%, and 11.6%, respectively). Symptom improvement correlated with a dose-dependent reduction in 5-HIAA, a marker for TPH inhibition, from baseline until week 4. This suggests the efficacy of LX1031 is related to the extent of inhibition of 5-HT biosynthesis. Stool consistency significantly improved, compared with the group given placebo, at weeks 1 and 4 (P < .01) and at week 2 (P < .001). CONCLUSIONS In a phase 2 study, LX1031 was well tolerated, relieving symptoms and increasing stool consistency in patients with nonconstipating IBS. Symptom relief was associated with reduced levels of 5-HIAA in urine samples. This marker might be used to identify patients with nonconstipating IBS who respond to inhibitors of 5-HT synthesis. PMID:21684281
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Sham, Adeline; Martinez, Eliana C; Beyer, Sebastian; Trau, Dieter W; Raghunath, Michael
2015-03-01
Clinical applications of tissue engineering are constrained by the ability of the implanted construct to invoke vascularization in adequate extent and velocity. To overcome the current limitations presented by local delivery of single angiogenic factors, we explored the incorporation of prolyl hydroxylase inhibitors (PHIs) into scaffolds as an alternative vascularization strategy. PHIs are small molecule drugs that can stabilize the alpha subunit of hypoxia-inducible factor-1 (HIF-1), a key transcription factor that regulates a variety of angiogenic mechanisms. In this study, we conjugated the PHI pyridine-2,4-dicarboxylic acid (PDCA) through amide bonds to a gelatin sponge (Gelfoam(®)). Fibroblasts cultured on PDCA-Gelfoam were able to infiltrate and proliferate in these scaffolds while secreting significantly more vascular endothelial growth factor than cells grown on Gelfoam without PDCA. Reporter cells expressing green fluorescent protein-tagged HIF-1α exhibited dose-dependent stabilization of this angiogenic transcription factor when growing within PDCA-Gelfoam constructs. Subsequently, we implanted PDCA-Gelfoam scaffolds into the perirenal fat tissue of Sprague Dawley rats for 8 days. Immunostaining of explants revealed that the PDCA-Gelfoam scaffolds were amply infiltrated by cells and promoted vascular ingrowth in a dose-dependent manner. Thus, the incorporation of PHIs into scaffolds appears to be a feasible strategy for improving vascularization in regenerative medicine applications.
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Sim, Jingwei; Cowburn, Andrew S; Palazon, Asis; Madhu, Basetti; Tyrakis, Petros A; Macías, David; Bargiela, David M; Pietsch, Sandra; Gralla, Michael; Evans, Colin E; Kittipassorn, Thaksaon; Chey, Yu C J; Branco, Cristina M; Rundqvist, Helene; Peet, Daniel J; Johnson, Randall S
2018-04-03
Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
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A Tyrosine-Hydroxylase Characterization of Dopaminergic Neurons in the Honey Bee Brain
Tedjakumala, Stevanus R.; Rouquette, Jacques; Boizeau, Marie-Laure; Mesce, Karen A.; Hotier, Lucie; Massou, Isabelle; Giurfa, Martin
2017-01-01
Dopamine (DA) plays a fundamental role in insect behavior as it acts both as a general modulator of behavior and as a value system in associative learning where it mediates the reinforcing properties of unconditioned stimuli (US). Here we aimed at characterizing the dopaminergic neurons in the central nervous system of the honey bee, an insect that serves as an established model for the study of learning and memory. We used tyrosine hydroxylase (TH) immunoreactivity (ir) to ensure that the neurons detected synthesize DA endogenously. We found three main dopaminergic clusters, C1–C3, which had been previously described; the C1 cluster is located in a small region adjacent to the esophagus (ES) and the antennal lobe (AL); the C2 cluster is situated above the C1 cluster, between the AL and the vertical lobe (VL) of the mushroom body (MB); the C3 cluster is located below the calyces (CA) of the MB. In addition, we found a novel dopaminergic cluster, C4, located above the dorsomedial border of the lobula, which innervates the visual neuropils of the bee brain. Additional smaller processes and clusters were found and are described. The profuse dopaminergic innervation of the entire bee brain and the specific connectivity of DA neurons, with visual, olfactory and gustatory circuits, provide a foundation for a deeper understanding of how these sensory modules are modulated by DA, and the DA-dependent value-based associations that occur during associative learning. PMID:28740466
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Bogus-Nowakowska, Krystyna; Równiak, Maciej; Hermanowicz-Sobieraj, Beata; Wasilewska, Barbara; Najdzion, Janusz; Robak, Anna
2016-12-01
The present study examines the distribution of tyrosine hydroxylase (TH) immunoreactivity and its morphological relationships with neuropeptide Y (NPY)- and gonadoliberin (GnRH)-immunoreactive (IR) structures in the preoptic area (POA) of the male guinea pig. Tyrosine hydroxylase was expressed in relatively small population of perikarya and they were mostly observed in the periventricular preoptic nucleus and medial preoptic area. The tyrosine hydroxylase-immunoreactive (TH-IR) fibers were dispersed troughout the whole POA. The highest density of these fibers was observed in the median preoptic nucleus, however, in the periventricular preoptic nucleus and medial preoptic area they were only slightly less numerous. In the lateral preoptic area, the density of TH-IR fibers was moderate. Two morphological types of TH-IR fibers were distinguished: smooth and varicose. Double immunofluorescence staining showed that TH and GnRH overlapped in the guinea pig POA but they never coexisted in the same structures. TH-IR fibers often intersected with GnRH-IR structures and many of them touched the GnRH-IR perikarya or dendrites. NPY wchich was abundantly present in the POA only in fibers showed topographical proximity with TH-IR structures. Althoug TH-IR perikarya and fibers were often touched by NPY-IR fibers, colocalization of TH and NPY in the same structures was very rare. There was only a small population of fibers which contained both NPY and TH. In conclusion, the morphological evidence of contacts between TH- and GnRH-IR nerve structures may be the basis of catecholaminergic control of GnRH release in the preoptic area of the male guinea pig. Moreover, TH-IR neurons were conatcted by NPY-IR fibers and TH and NPY colocalized in some fibers, thus NPY may regulate catecholaminergic neurons in the POA. Copyright © 2016 Elsevier B.V. All rights reserved.
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Bujňáková, Z; Baláž, M; Zdurienčíková, M; Sedlák, J; Čaplovičová, M; Čaplovič, Ľ; Dutková, E; Zorkovská, A; Turianicová, E; Baláž, P; Shpotyuk, O; Andrejko, S
2017-02-01
Arsenic sulfide compounds have a long history of application in a traditional medicine. In recent years, realgar has been studied as a promising drug in cancer treatment. In this study, the arsenic sulfide (As 4 S 4 ) nanoparticles combined with zinc sulfide (ZnS) ones in different molar ratio have been prepared by a simple mechanochemical route in a planetary mill. The successful synthesis and structural properties were confirmed and followed via X-ray diffraction and high-resolution transmission electron microscopy measurements. The morphology of the particles was studied via scanning electron microscopy and transmission electron microscopy methods and the presence of nanocrystallites was verified. For biological tests, the prepared As 4 S 4 /ZnS nanoparticles were further milled in a circulation mill in a water solution of Poloxamer 407 (0.5wt%), in order to cover the particles with this biocompatible copolymer and to obtain stable nanosuspensions with unimodal distribution. The average size of the particles in the nanosuspensions (~120nm) was determined by photon cross-correlation spectroscopy method. Stability of the nanosuspensions was determined via particle size distribution and zeta potential measurements, confirming no physico-chemical changes for several months. Interestingly, with the increasing amount of ZnS in the sample, the stability was improved. The anti-cancer effects were tested on two melanoma cell lines, A375 and Bowes, with promising results, confirming increased efficiency of the samples containing both As 4 S 4 and ZnS nanocrystals. Copyright © 2016 Elsevier B.V. All rights reserved.
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Estrogen-2-hydroxylase in the brain of the male African catfish, Clarias gariepinus
DOE Office of Scientific and Technical Information (OSTI.GOV)
Timmers, R.J.; Granneman, J.C.; Lambert, J.G.
1988-11-01
Estrogen-2-hydroxylase activity, involved in the biosynthesis of catecholestrogens, was localized in the brain of the male African catfish, Clarias gariepinus, by means of a radiometric assay using (2-TH)estradiol as substrate. Fore- and midbrain were divided in 18, 500-microns thick, transverse sections from which small defined areas were punched out and assayed. The estrogen-2-hydroxylase activity was calculated from the release of tritium during hydroxylation, and expressed in femtomole catecholestradiol.milligram-1 tissue.hour-1. The enzyme could be demonstrated throughout the brain. A high activity (greater than 350 fmol) was observed in the telencephalon, in particularly the rostral part and the area ventralis pars dorsalis;more » in the diencephalon in the preoptic region, including the magnocellular part of the preoptic nucleus and the rostral part of the anterior periventricular nucleus; and in the area tuberalis, including the nucleus lateralis tuberis, the rostral part of the nucleus anterior tuberis, the caudal part of the nucleus posterior periventricularis, and in the nucleus recessus posterioris. Also a high activity was detected in the mesencephalic tectum opticum and the dorsolateral part of the torus semicircularis. The ventral mesencephalon showed a moderate (200-350 fmol) to low (less than 200 fmol) activity, whereas the lowest activity was found in the hindbrain (118 fmol). The significance of the biosynthesis of catecholestrogens in the brain is discussed in light of the negative feedback mechanism of gonadal steroids on gonadotropin release.« less
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Punchaichira, Toyanji Joseph; Dey, Sanjay Kumar; Mukhopadhyay, Anirban; Kundu, Suman; Thelma, B K
2017-07-01
Dopamine-β-hydroxylase (DBH, EC 1.14.17.1), an oxido-reductase that catalyses the conversion of dopamine to norepinephrine, is largely expressed in sympathetic neurons and adrenal medulla. Several regulatory and structural variants in DBH associated with various neuropsychiatric, cardiovascular diseases and a few that may determine enzyme activity have also been identified. Due to paucity of studies on functional characterization of DBH variants, its structure-function relationship is poorly understood. The purpose of the study was to characterize five non-synonymous (ns) variants that were prioritized either based on previous association studies or Sorting Tolerant From Intolerant (SIFT) algorithm. The DBH ORF with wild type (WT) and site-directed mutagenized variants were transfected into HEK293 cells to generate transient and stable lines expressing these variant enzymes. Activity was determined by UPLC-PDA and corresponding quantity by MRM HR on a TripleTOF 5600 MS respectively of spent media from stable cell lines. Homospecific activity computed for the WT and variant proteins showed a marginal decrease in A318S, W544S and R549C variants. In transient cell lines, differential secretion was observed in the case of L317P, W544S and R549C. Secretory defect in L317P was confirmed by localization in ER. R549C exhibited both decreased homospecific activity and differential secretion. Of note, all the variants were seen to be destabilizing based on in silico folding analysis and molecular dynamics (MD) simulation, lending support to our experimental observations. These novel genotype-phenotype correlations in this gene of considerable pharmacological relevance have implications for dopamine-related disorders.
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Zhang, Bo; Bandyopadhyay, Sibali; Shakamuri, Priyanka; Naik, Sunil G.; Huynh, Boi Hanh; Couturier, Jérémy; Rouhier, Nicolas; Johnson, Michael K.
2013-01-01
Saccharomyces cerevisiae mitochondrial glutaredoxin 5 (Grx5) is the archetypical member of a ubiquitous class of monothiol glutaredoxins with a strictly conserved CGFS active-site sequence that has been shown to function in biological [Fe2S2]2+ cluster trafficking. In this work, we show that recombinant S. cerevisiae Grx5 purified aerobically after prolonged exposure of the cell-free extract to air or after anaerobic reconstitution in the presence of glutathione, predominantly contains a linear [Fe3S4]+ cluster. The excited state electronic properties and ground state electronic and vibrational properties of the linear [Fe3S4]+ cluster have been characterized using UV-visible absorption/CD/MCD, EPR, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic S = 5/2 linear [Fe3S4]+ cluster with properties similar to those reported for synthetic linear [Fe3S4]+ clusters and the linear [Fe3S4]+ clusters in purple aconitase. Moreover, the results indicate that the Fe-S cluster content previously reported for many monothiol Grxs has been misinterpreted exclusively in terms of [Fe2S2]2+ clusters, rather than linear [Fe3S4]+ clusters or mixtures of linear [Fe3S4]+ and [Fe2S2]2+ clusters. In the absence of GSH, anaerobic reconstitution of Grx5 yields a dimeric form containing one [Fe4S4]2+ cluster that competent for in vitro activation of apo-aconitase, via intact cluster transfer. The ligation of the linear [Fe3S4]+ and [Fe4S4]2+ clusters in Grx5 has been assessed by spectroscopic, mutational and analytical studies. Potential roles for monothiol Grx5 in scavenging and recycling linear [Fe3S4]+ clusters released during protein unfolding under oxidative stress conditions and in maturation of [Fe4S4]2+ cluster-containing proteins are discussed in light of these results. PMID:24032439
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NASA Astrophysics Data System (ADS)
Fu, You-Jun; Laskin, Julia; Wang, Lai-Sheng
2006-09-01
Collision-induced dissociation (CID) experiments on a series of [4Fe-4S] cluster ions, [Fe4S4Cl4 - x(SC2H5)x]2-/1- (x = 0-4), revealed that their fragmentation channels change with the coordination environment. Among the three Coulomb repulsion related channels for the doubly charged species, the collision induced electron detachment channel was found to become more significant from x = 0 to 4 due to the decreasing electron binding energies and the magnitude of repulsion Coulomb barrier, while both the ligand detachment of Cl- and the fission of the [Fe4S4]2+ core became more and more significant with the increase of the Cl- coordination, and eventually became the dominant channel at x = 0. From the parents containing the SC2H5 ligand, neutral losses of HSC2H5 (62 u) and/or HSCHCH2 (60 u) were observed. It was proposed that inter- and intra-ligand proton transfer could happen during the CID process, resulting in hydrogen coordination to the [4Fe-4S] cluster. In the presence of O2, [Fe4S4Cl3(SC2H5)]2- and [Fe4S4Cl4]2- can form the O2-substituted products [Fe4S4Cl2(SC2H5)O2]- and [Fe4S4Cl3O2]-, respectively. It was shown that the O2 complexation occurs by coordination to the empty iron site of the [4Fe-4S] cubane core after dissociation of one Cl- ligand.
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Lan, Xiaozhong; Zeng, Junlan; Liu, Ke; Zhang, Fangyuan; Bai, Ge; Chen, Min; Liao, Zhihua; Huang, Luqi
2018-02-26
Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6β-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production. Copyright © 2018 Elsevier Inc. All rights reserved.
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Effect of gamma-oryzanol on cytochrome P450 activities in human liver microsomes.
Umehara, Ken; Shimokawa, Yoshihiko; Miyamoto, Gohachiro
2004-07-01
The effects of gamma-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, on several cytochrome P450 (CYP) specific reactions in human liver microsomes were investigated to predict drug interactions with gamma-oryzanol in vivo from in vitro data. The following eight CYP catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6beta-hydroxylation. gamma-Oryzanol had little inhibitory effects on CYP activities, indicating that this compound would not be expected to cause clinically significant interactions with other CYP-metabolized drugs at expected therapeutic concentrations.
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Ohnishi, Mutsuko; Nakatani, Teruyo; Lanske, Beate; Razzaque, M. Shawkat
2011-01-01
Changes in the expression of klotho, a β-glucuronidase, contribute to the development of features that resemble those of premature aging, as well as chronic renal failure. Klotho knockout mice have increased expression of the sodium/phosphate cotransporter (NaPi2a) and 1α-hydroxylase in their kidneys, along with increased serum levels of phosphate and 1,25-dihydroxyvitamin D. These changes are associated with widespread soft-tissue calcifications, generalized tissue atrophy, and a shorter lifespan in the knockout mice. To determine the role of the increased vitamin D activities in klotho knockout animals, we generated klotho and 1α-hydroxylase double-knockout mice. These double mutants regained body weight and developed hypophosphatemia with a complete elimination of the soft-tissue and vascular calcifications that were routinely found in klotho knockout mice. The markedly increased serum fibroblast growth factor 23 and the abnormally low serum parathyroid hormone levels, typical of klotho knockout mice, were significantly reversed in the double-knockout animals. These in vivo studies suggest that vitamin D has a pathologic role in regulating abnormal mineral ion metabolism and soft-tissue anomalies of klotho-deficient mice. PMID:19225558
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USDA-ARS?s Scientific Manuscript database
This experiment was designed to test the effect of polymorphism in the cholesterol 7-alpha hydroxylase (CYP7) gene locus, and dietary cholesterol (C) on cerebrum C in neonatal pigs fed sow's milk formulas. Thirty-six pigs (18 male and 18 female) genetically selected for high (HG), or low (LG) plasma...
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Investigation of magnetic properties on spin-ordering effects of FeGa2S4 and FeIn2S4
NASA Astrophysics Data System (ADS)
Myoung, Bo Ra; Lim, Jung Tae; Kim, Chul Sung
2017-09-01
We have studied crystal and magnetic properties of chalcogenides FeGa2S4 and FeIn2S4 with X-ray diffractometer (XRD), magnetic property measurement system (MPMS), magnetometer, physical property measurement system (PPMS), and Mössbauer spectrometer. The crystal structure has 2-dimension triangular lattice structure with P-3m1 of FeGa2S4, while FeIn2S4 has inverse spinel with space group Fd3m. The AC magnetic susceptibility measurements show that FeGa2S4 is an insulating spin glass material, exhibiting geometrical frustration, unlike in the antiferromagnetic [AFM] metallic spin glass FeIn2S4. From hysteresis (M-H) curves at 4.2 K, FeGa2S4 has spin-flop behavior with an angle of 120° of triangle, as against linear slope of FeIn2S4 due to anti-parallel spin. The gap energy by splitting of 5T2g, Δ1 and electric quadrupole splitting ΔEQ of FeIn2S4 are much higher than that of FeGa2S4 at 4.2 K because FeGa2S4 is geometrically frustrated magnet having degenerate ground state at low temperature.
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Witsch, Thilo J; Turowski, Pawel; Sakkas, Elpidoforos; Niess, Gero; Becker, Simone; Herold, Susanne; Mayer, Konstantin; Vadász, István; Roberts, Jesse D; Seeger, Werner; Morty, Rory E
2014-02-01
Bronchopulmonary dysplasia (BPD) is a common and serious complication of premature birth, characterized by a pronounced arrest of alveolar development. The underlying pathophysiological mechanisms are poorly understood although perturbations to the maturation and remodeling of the extracellular matrix (ECM) are emerging as candidate disease pathomechanisms. In this study, the expression and regulation of three members of the lysyl hydroxylase family of ECM remodeling enzymes (Plod1, Plod2, and Plod3) in clinical BPD, as well as in an experimental animal model of BPD, were addressed. All three enzymes were localized to the septal walls in developing mouse lungs, with Plod1 also expressed in the vessel walls of the developing lung and Plod3 expressed uniquely at the base of developing septa. The expression of plod1, plod2, and plod3 was upregulated in the lungs of mouse pups exposed to 85% O2, an experimental animal model of BPD. Transforming growth factor (TGF)-β increased plod2 mRNA levels and activated the plod2 promoter in vitro in lung epithelial cells and in lung fibroblasts. Using in vivo neutralization of TGF-β signaling in the experimental animal model of BPD, TGF-β was identified as the regulator of aberrant plod2 expression. PLOD2 mRNA expression was also elevated in human neonates who died with BPD or at risk for BPD, compared with neonates matched for gestational age at birth or chronological age at death. These data point to potential roles for lysyl hydroxylases in normal lung development, as well as in perturbed late lung development associated with BPD.
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The upregulation of immune responses in tyrosine hydroxylase (TH) silenced Litopenaeus vannamei.
Mapanao, Ratchaneegorn; Chang, Chin-Chyuan; Cheng, Winton
2017-02-01
Catecholamines (CAs) play a crucial role in maintaining physiological and immune homeostasis in invertebrates and vertebrates under stressful conditions. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in CA synthesis. To develop an effective CA-related immunological defense system against stress and pathogen infection, various criteria, were evaluated in TH double-stranded (ds) RNA-injected white shrimp, Litopenaeus vannamei. Specifically, the relative transcript quantification of TH, dopamine β-hydroxylase (DBH), crustacean hyperglycemic hormone (CHH), and other immune-related genes; TH activity in the haemolymph; and the estimation of l-dihydroxyphenylalanine (l-DOPA), glucose, and lactate levels in the haemolymph were examined. TH depletion revealed a significant increase in the total haemocyte count; granular cells; semigranular cells; respiratory bursts (RBs, release of superoxide anion); superoxide dismutase (SOD) activity; phagocytic activity and clearance efficiency; and the expression of lipopolysaccharide and β-1,3-glucan-binding protein and peroxinectin, SOD, crustin, and lysozyme genes. In addition, the reduction of TH gene expression and activity was accompanied by a decline of phenoloxidase (PO) activity per granulocyte, lower glucose and lactate levels, and significantly low expression of DBH and CHH genes. However, the number of hyaline cells, activity of PO, RBs per haemocyte, and expression of POI and POII genes were not significantly different in the LvTH-silenced shrimp. Notably, the survival ratio of LvTH-silenced shrimp was significantly higher than that of shrimp injected with diethyl pyrocarbonate-water and nontargeting dsRNA when challenged with Vibrio alginolyticus. Therefore, the depletion of TH can enhance disease resistance in shrimp by upregulating specific immune parameters but downregulating the levels of carbohydrate metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Huang, Mei; Nguyen, Patricia; Jia, Fangjun; Hu, Shijun; Gong, Yongquan; de Almeida, Patricia E.; Wang, Li; Nag, Divya; Kay, Mark A.; Giaccia, Amato J; Robbins, Robert C.; Wu, Joseph C.
2011-01-01
Background Under normoxic conditions, hypoxia inducible factor-1 alpha (HIF-1α) is rapidly degraded by two hydroxylases, prolyl hydroxylase (PHD) and factor inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its up-regulation may be an effective therapeutic option for ischemic heart failure. Methods and Results PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin sequences for inhibiting PHD isoenzyme 2 (shPHD2) and FIH (shFIH) were inserted into novel non-viral minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit+ cardiac progenitor cells (CPCs) demonstrated higher expression of angiogenesis factors in the double knockdown group compared to the single knockdown and shScramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially following LAD ligation in adult FVB mice (n=60). Functional studies using magnetic resonance imaging (MRI), echocardiography, and pressure-volume (PV) loops showed greater improvement in cardiac function in the double knockdown group. To assess mechanism(s) of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double knockdown group. Fluorescence activated cell sorting (FACS) showed significantly higher activation of endogenous c-kit+ cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser capture microdissection (LCM) analysis confirmed up-regulation of HIF-1α protein and angiogenesis genes, respectively. Conclusions We demonstrated that HIF-1α up-regulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function. PMID
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Rajathei, David Mary; Preethi, Jayakumar; Singh, Hemant K; Rajan, Koilmani Emmanuvel
2014-08-01
Tryptophan hydroxylase (TPH) catalyses l-tryptophan into 5-hydroxy-l-tryptophan, which is the first and rate-limiting step of serotonin (5-HT) biosynthesis. Earlier, we found that TPH2 up-regulated in the hippocampus of postnatal rats after the oral treatment of Bacopa monniera leaf extract containing the active compound bacosides. However, the knowledge about the interactions between bacosides with TPH is limited. In this study, we take advantage of in silico approach to understand the interaction of bacoside-TPH complex using three different docking algorithms such as HexDock, PatchDock and AutoDock. All these three algorithms showed that bacoside A and A3 well fit into the cavity consists of active sites. Further, our analysis revealed that major active compounds bacoside A3 and A interact with different residues of TPH through hydrogen bond. Interestingly, Tyr235, Thr265 and Glu317 are the key residues among them, but none of them are either at tryptophan or BH4 binding region. However, its note worthy to mention that Tyr 235 is a catalytic sensitive residue, Thr265 is present in the flexible loop region and Glu317 is known to interacts with Fe. Interactions with these residues may critically regulate TPH function and thus serotonin synthesis. Our study suggested that the interaction of bacosides (A3/A) with TPH might up-regulate its activity to elevate the biosynthesis of 5-HT, thereby enhances learning and memory formation.
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Bachelot, Anne; Grouthier, Virginie; Courtillot, Carine; Dulon, Jérôme; Touraine, Philippe
2017-04-01
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is characterized by cortisol and in some cases aldosterone deficiency associated with androgen excess. Goals of treatment are to replace deficient hormones and control androgen excess, while avoiding the adverse effects of exogenous glucocorticoid. Over the last 5 years, cohorts of adults with CAH due to 21-hydroxylase deficiency from Europe and the United States have been described, allowing us to have a better knowledge of long-term complications of the disease and its treatment. Patients with CAH have increased mortality, morbidity and risk for infertility and metabolic disorders. These comorbidities are due in part to the drawbacks of the currently available glucocorticoid therapy. Consequently, novel therapies are being developed and studied in an attempt to improve patient outcomes. New management strategies in the care of pregnancies at risk for congenital adrenal hyperplasia using fetal sex determination and dexamethasone have also been described, but remain a subject of debate. We focused the present overview on the data published in the last 5 years, concentrating on studies dealing with cardiovascular risk, fertility, treatment and prenatal management in adults with classic CAH to provide the reader with an updated review on this rapidly evolving field of knowledge. © 2017 European Society of Endocrinology.
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Nguyen, Xuan-Khanh Thi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F.; Kanthasamy, Anumantha G.; Cadet, Jean L.; Nabeshima, Toshitaka; Kim, Hyoung-Chun
2014-01-01
The present study was designed to evaluate the specific role of protein kinase C (PKC) δ in methamphetamine (MA)-induced dopaminergic toxicity. A multiple-dose administration regimen of MA significantly increases PKCδ expression, while rottlerin, a PKCδ inhibitor, significantly attenuates MA-induced hyperthermia and behavioural deficits. These behavioural effects were not significantly observed in PKCδ antisense oligonucleotide (ASO)-treated- or PKCδ knockout (−/−)-mice. There were no MA-induced significant decreases of dopamine (DA) content or tyrosine hydroxylase (TH) expression in the striatum in rottlerin-treated-, ASO-treated- or PKCδ (−/−)-mice. The administration of MA also results in a significant decrease of TH phosphorylation at ser 40, but not ser 31, while the inhibition of PKCδ consistently and significantly attenuates MA-induced reduction in the phosphorylation of TH at ser 40. Therefore, these results suggest that the MA-induced enhancement of PKCδ expression is a critical factor in the impairment of TH phosphorylation at ser 40 and that pharmacological or genetic inhibition of PKCδ may be protective against MA-induced dopaminergic neurotoxicity in vivo. PMID:21672585
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Sulfur K-Edge XAS Studies of the Effect of DNA Binding on the [Fe 4 S 4 ] Site in EndoIII and MutY
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ha, Yang; Arnold, Anna R.; Nuñez, Nicole N.
S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe 4S 4] clusters in the DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation on Fe–S bond covalencies (i.e., the amount of S 3p character mixed into the Fe 3d valence orbitals). Increased covalencies in both iron–thiolate and iron–sulfide bonds would stabilize the oxidized state of the [Fe 4S 4] clusters. Our results are compared to those on previously studied [Fe 4S 4] model complexes, ferredoxin (Fd), and to new data on high-potential iron–sulfur protein (HiPIP). A limited decrease in covalency ismore » observed upon removal of solvent water from EndoIII and MutY, opposite to the significant increase observed for Fd, where the [Fe 4S 4] cluster is solvent exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA binding, which is due to the effect of its negative charge on the iron–sulfur bonds. Furthermore, in EndoIII, this change in covalency can be quantified and makes a significant contribution to the observed decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP-like redox behavior.« less
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Sulfur K-Edge XAS Studies of the Effect of DNA Binding on the [Fe 4 S 4 ] Site in EndoIII and MutY
Ha, Yang; Arnold, Anna R.; Nuñez, Nicole N.; ...
2017-07-18
S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe 4S 4] clusters in the DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation on Fe–S bond covalencies (i.e., the amount of S 3p character mixed into the Fe 3d valence orbitals). Increased covalencies in both iron–thiolate and iron–sulfide bonds would stabilize the oxidized state of the [Fe 4S 4] clusters. Our results are compared to those on previously studied [Fe 4S 4] model complexes, ferredoxin (Fd), and to new data on high-potential iron–sulfur protein (HiPIP). A limited decrease in covalency ismore » observed upon removal of solvent water from EndoIII and MutY, opposite to the significant increase observed for Fd, where the [Fe 4S 4] cluster is solvent exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA binding, which is due to the effect of its negative charge on the iron–sulfur bonds. Furthermore, in EndoIII, this change in covalency can be quantified and makes a significant contribution to the observed decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP-like redox behavior.« less
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Sato, M; Yoshida, S; Nagao, K; Imaizumi, K
2000-06-01
The exogenously hypercholesterolemic (ExHC) rat is a strain segregated from SD rats with a high response to dietary cholesterol. To understand the underlying mechanism(s) for this hypercholesterolemia, the interactive effects of dietary fatty acid and the susceptibility of rats to dietary cholesterol on the serum cholesterol concentration and hepatic mRNA abundance of the low-density lipoprotein (LDL) receptor, cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) and 3-hydroxyl-3methylglutaryl (HMG) CoA reductase were examined. Both strains were fed on a diet supplemented with 10% each of olive, safflower or coconut oil with or without the addition of 1% cholesterol for one week. The ExHC rats fed on olive, safflower and coconut oil in combination with cholesterol respectively resulted in a 3.5-, 2.0- and 2.1-fold higher serum cholesterol concentration than that in the animals fed on the corresponding dietary fats without any supplementation of cholesterol (p < 0.01 by dietary cholesterol or type of fat). The dietary cholesterol dependent-elevation of serum cholesterol in the SD rats was less than 1.5-fold (p<0.01) and there was no dietary fat effect. The ExHC rats fed on the safflower oil-containing diet supplemented with cholesterol resulted in a higher mRNA abundance of the LDL receptor and 7alpha-hydroxylase than in the corresponding fat-fed rats without cholesterol (p<0.05). There was no dietary cholesterol-dependent change of mRNA abundance in either strain fed on olive or coconut oil, except for a decreased abundance of HMG CoA reductase mRNA in the olive oil-fed ExHC rats and coconut oil-fed Sprague-Dawley (SD) rats (p<0.05). These results indicate that the hepatic mRNA abundance of the LDL receptor and of 7alpha-hydroxylase depended on the dietary combination of cholesterol and a fatty acid and suggest that a linoleic acid-rich diet may alleviate exogenous hypercholesterolemia by activating the process involved in the hepatic uptake and biliary excretion of
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Ishii, Tomohiro; Adachi, Masanori; Takasawa, Kei; Okada, Satoshi; Kamasaki, Hotaka; Kubota, Takuo; Kobayashi, Hironori; Sawada, Hirotake; Nagasaki, Keisuke; Numakura, Chikahiko; Harada, Shohei; Minamitani, Kanshi; Sugihara, Shigetaka; Tajima, Toshihiro
2018-01-01
We aimed to evaluate the incidence and characteristics of adrenal crisis in Japanese children with 21-hydroxylase deficiency (21-OHD). We conducted a retrospective nationwide survey for the councilors of the Japanese Society for Pediatric Endocrinology (JSPE) regarding adrenal crisis in children under 7 years with 21-OHD, admitted to hospitals from 2011 through 2016. We defined adrenal crisis as the acute impairment of general health due to glucocorticoid deficiency with at least two of symptoms, signs, or biochemical abnormalities. The councilors of the JSPE in 83 institutions responded to this survey (response rate, 60.1%). Data analyses of 378 patients with 1,101.4 person-years (PYs) revealed that 67 patients (17.7%) experienced at least 1 episode of hospital admission for adrenal crisis at the median age of 2 years. The incidence of adrenal crisis was calculated as 10.9 per 100 PYs (95% confidence interval [CI] 9.6-12.2). Infections were the most common precipitating factors, while no factor was observed in 12.5%. Hypoglycemia occurred concomitantly in 27.4%. One patient died from severe hypoglycemia, resulting in a mortality rate of 0.09 per 100 PYs (95% CI 0.0-0.2). Adrenal crisis is not rare and can be accompanied by disastrous hypoglycemia in children with 21-OHD. © 2018 S. Karger AG, Basel.
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Marumoto, Shinsuke; Okuno, Yoshiharu; Hagiwara, Yuki; Miyazawa, Mitsuo
2017-08-01
Using biotransformation as a biocatalytic process has the advantage of being able to proceed under mild conditions and with high regio- and enantioselectivity. This study investigated the biotransformation of (-)-(1R,4S)-menthone (1) and (+)-(1S,4R)-menthone (2) by Spodoptera litura larvae. Compound 1 was converted to (-)-(1R,4S)-7-hydroxymenthone (1-1), (+)-(1R,3S,4S)-7-hydroxyneomenthol (1-2) and (-)-(1R,4S,8R)-p-menth-3-one-9-oic acid (1-3). The metabolism of substrate 2 generated three enantiomers of the above metabolites, designated as 2-1 to 2-3, respectively. The C-9 position of (-)-menthone and (+)-menthone was oxidized to carboxylic acid by S. litura, which is a metabolic pathway not observed in any other example of biocatalysis.
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Böttcher, M; Lentini, S; Arens, E R; Kaiser, A; van der Mey, D; Thuss, U; Kubitza, D; Wensing, G
2018-07-01
Insufficient erythropoietin (EPO) synthesis is a relevant cause of renal anaemia in patients with chronic kidney disease. Molidustat, a selective hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor, increases endogenous EPO levels dose dependently in preclinical models. We examined the pharmacokinetics, safety, tolerability and effect on EPO levels of single oral doses of molidustat in healthy male volunteers. This was a single-centre, randomized, single-blind, placebo-controlled, group-comparison, dose-escalation study. Molidustat was administered at doses of 5, 12.5, 25, 37.5 or 50 mg as a polyethylene glycol-based solution. In total, 45 volunteers received molidustat and 14 received placebo. Molidustat was absorbed rapidly, and the mean maximum plasma concentration and area under the concentration-time curve increased dose dependently. The mean terminal half-life was 4.64-10.40 h. A significant increase in endogenous EPO was observed following single oral doses of molidustat of 12.5 mg and above. Geometric mean peak EPO levels were 14.8 IU l -1 (90% confidence interval 13.0, 16.9) for volunteers who received placebo and 39.8 IU l -1 (90% confidence interval: 29.4, 53.8) for those who received molidustat 50 mg. The time course of EPO levels resembled the normal diurnal variation in EPO. Maximum EPO levels were observed approximately 12 h postdose and returned to baseline after approximately 24-48 h. All doses of molidustat were well tolerated and there were no significant changes in vital signs or laboratory safety parameters. Oral administration of molidustat to healthy volunteers elicited a dose-dependent increase in endogenous EPO. These results support the ongoing development of molidustat as a potential new treatment for patients with renal anaemia. © 2018 The British Pharmacological Society.
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Structural interactions between lipids, water and S1-S4 voltage-sensing domains.
Krepkiy, Dmitriy; Gawrisch, Klaus; Swartz, Kenton J
2012-11-02
Membrane proteins serve crucial signaling and transport functions, yet relatively little is known about their structures in membrane environments or how lipids interact with these proteins. For voltage-activated ion channels, X-ray structures suggest that the mobile voltage-sensing S4 helix would be exposed to the membrane, and functional studies reveal that lipid modification can profoundly alter channel activity. Here, we use solid-state NMR to investigate structural interactions of lipids and water with S1-S4 voltage-sensing domains and to explore whether lipids influence the structure of the protein. Our results demonstrate that S1-S4 domains exhibit extensive interactions with lipids and that these domains are heavily hydrated when embedded in a membrane. We also find evidence for preferential interactions of anionic lipids with S1-S4 domains and that these interactions have lifetimes on the timescale of ≤ 10(-3)s. Arg residues within S1-S4 domains are well hydrated and are positioned in close proximity to lipids, exhibiting local interactions with both lipid headgroups and acyl chains. Comparative studies with a positively charged lipid lacking a phosphodiester group reveal that this lipid modification has only modest effects on the structure and hydration of S1-S4 domains. Taken together, our results demonstrate that Arg residues in S1-S4 voltage-sensing domains reside in close proximity to the hydrophobic interior of the membrane yet are well hydrated, a requirement for carrying charge and driving protein motions in response to changes in membrane voltage. Published by Elsevier Ltd.
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Structural interactions between lipids, water and S1-S4 voltage-sensing domains
Krepkiy, Dmitriy; Gawrisch, Klaus; Swartz, Kenton J.
2012-01-01
Membrane proteins serve crucial signaling and transport functions, yet relatively little is known about their structures in membrane environments or how lipids interact with these proteins. For voltage-activated ion channels, X-ray structures suggest that the mobile voltage-sensing S4 helix would be exposed to the membrane, and functional studies reveal that lipid modification can profoundly alter channel activity. Here we use solid-state NMR to investigate structural interactions of lipids and water with S1-S4 voltage-sensing domains, and to explore whether lipids influence the structure of the protein. Our results demonstrate that S1-S4 domains exhibit extensive interactions with lipids, and that these domains are heavily hydrated when embedded in a membrane. We also find evidence for preferential interactions of anionic lipids with S1-S4 domains, and that these interactions have lifetimes on the timescale of 10−3s. Arg residues within S1-S4 domains are well-hydrated and are positioned in close proximity to lipids, exhibiting local interactions with both lipid head groups and acyl chains. Comparative studies with a positively charged lipid lacking a phosphodiester group reveal that this lipid modification has only modest effects on the structure and hydration of S1-S4 domains. Taken together, our results demonstrate that Arg residues in S1-S4 voltage-sensing domains reside in close proximity to the hydrophobic interior of the membrane, yet are well-hydrated, a requirement for carrying charge and driving protein motions in response to changes in membrane voltage. PMID:22858867
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Autoantibodies to human tryptophan hydroxylase and aromatic L-amino acid decarboxylase.
Dal Pra, Chiara; Chen, Shu; Betterle, Corrado; Zanchetta, Renato; McGrath, Vivienne; Furmaniak, Jadwiga; Rees Smith, Bernard
2004-03-01
To assess the prevalence of autoantibodies (Abs) to tryptophan hydroxylase (TPH) and aromatic l-amino acid decarboxylase (AADC) in patients with different autoimmune diseases and to analyse their respective epitopes. TPH and AADC Abs were measured in an immunoprecipitation assay using (35)S-labelled full-length and fragments of TPH and AADC. Patients with different autoimmune adrenal diseases (n=84), non-adrenal autoimmune diseases (n=37), idiopathic vitiligo (n=8) and 56 healthy blood donors were studied. Fourteen of twenty-three (61%) of patients with autoimmune polyglandular syndrome (APS) type I and 1/34 (3%) of patients with isolated Addison's disease (AD) were positive for TPH Abs. None of the patients with APS type II (n=27), coeliac disease (n=10), autoimmune thyroid disease (AITD) (n=11), type 1 diabetes mellitus (DM) (n=16) or idiopathic vitiligo (n=8) was positive for TPH Abs. AADC Abs were detected in 12/23 (52%) patients with APS type I, in 1/29 (3%) patients with APS type II and 1/34 (3%) patients with isolated AD. None of the patients with coeliac disease, type 1 DM, AITD or idiopathic vitiligo was positive for AADC Abs. TPH Abs were found to interact with the C-terminal amino acids (aa) 308-423, central aa 164-205 and N-terminal aa 1-105 of the TPH molecule. AADC Ab binding epitopes were within the C-terminal aa 382-483, the central aa 243-381 and the N-terminal aa 1-167. Our study suggests that TPH Abs and AADC Abs react with several different epitopes and that different epitopes are recognized by different sera. The prevalence of TPH Abs and AADC Abs in patients with APS type I in our study is in agreement with previous reports. TPH Abs and AADC Abs were found very rarely in patients with other forms of autoimmune adrenal disease and were not detected in patients with non-adrenal autoimmune diseases.
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Nguyen, Thi Phuong Mai; Nguyen, Thu Hien; Ngo, Diem Ngoc; Vu, Chi Dung; Nguyen, Thi Kim Lien; Nong, Van Hai; Nguyen, Huy Hoang
2015-07-10
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is characterized by a deficiency of one of the enzymes involved in the synthesis of cortisol from cholesterol by the adrenal cortex. CAH cases arising from impaired 11β-hydroxylase are the second most common form. Mutations in the CYP11B1 gene are the cause of 11β-hydroxylase deficiency. This study was performed on a patient with congenital adrenal hyperplasia and with premature development such as enlarged penis, muscle development, high blood pressure, and bone age equivalent of 5 years old at 2 years of chronological age. Biochemical tests for steroids confirmed the diagnosis of CAH. We used PCR and sequencing to screen for mutations in CYP11B1 gene. Results showed that the patient has a novel homozygous mutation of guanine (G) to thymine (T) in intron 6 (IVS6+5G>T). The analysis of this mutation by MaxEntScan boundary software indicated that this mutant could affect the gene splicing during transcription. Copyright © 2015 Elsevier B.V. All rights reserved.
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Zhu, Kai; Lai, Hao; Guo, Changfa; Li, Jun; Wang, Yulin; Wang, Lingyan; Wang, Chunsheng
2014-01-01
Mesenchymal stem cell (MSC) transplantation has attracted much attention in myocardial infarction therapy. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. Small interfering RNA-mediated prolyl hydroxylase domain protein 2 (PHD2) silencing in MSCs holds tremendous potential to enhance their survival and paracrine effect after transplantation. However, an efficient and biocompatible PHD2 silencing system for clinical application is lacking. Herein, we developed a novel PHD2 silencing system based on arginine-terminated generation 4 poly(amidoamine) (Arg-G4) nanoparticles. The system exhibited effective and biocompatible small interfering RNA delivery and PHD2 silencing in MSCs in vitro. After genetically modified MSC transplantation in myocardial infarction models, MSC survival and paracrine function of IGF-1 were enhanced significantly in vivo. As a result, we observed decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angiogenesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This work demonstrated that an Arg-G4 nanovector-based PHD2 silencing system could enhance the efficiency of MSC transplantation for infarcted myocardium repair.
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Lin, Zhiwei
2014-01-01
The infrared spectra of (1'S, 6'S)-1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0] non-8-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride salt (CLF-HCl) were studied and compared with free base. Their fragmentation pathways were investigated using tandem mass spectrometric (MS/MS) techniques on Fourier-transform ion cyclotron resonance spectrum, and many characteristic fragment ions were found. Copyright © 2013 Elsevier B.V. All rights reserved.
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Harnoss, Jonathan Michael; Strowitzki, Moritz Johannes; Radhakrishnan, Praveen; Platzer, Lisa Katharina; Harnoss, Julian Camill; Hank, Thomas; Cai, Jun; Ulrich, Alexis; Schneider, Martin
2015-01-01
Oxygen is essential for metazoans to generate energy. Upon oxygen deprivation adaptive and protective pathways are induced, mediated by hypoxia-inducible factors (HIFs) and prolyl hydroxylase domain-containing enzymes (PHDs). Both play a pivotal role in various conditions associated with prolonged ischemia and inflammation, and are promising targets for therapeutic intervention. This review focuses on aspects of therapeutic PHD modulation in surgically relevant disease conditions such as hepatic and intestinal disorders, wound healing, innate immune responses, and tumorigenesis, and discusses the therapeutic potential and challenges of PHD inhibition in surgical patients. PMID:27774478
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USDA-ARS?s Scientific Manuscript database
The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2, which encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rod...
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Gjetting, Torben; Petersen, Marie; Guldberg, Per; Güttler, Flemming
2001-01-01
Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46–48 (GAL) and 65–69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2–120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency. PMID:11326337
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Estradiol or fluoxetine alters depressive behavior and tryptophan hydroxylase in rat raphe.
Yang, Fu-Zhong; Wu, Yan; Zhang, Wei-Guo; Cai, Yi-Yun; Shi, Shen-Xun
2010-03-10
The effects of 17beta-estradiol and fluoxetine on behavior of ovariectomized rats subjected to the forced swimming test and the expression of tryptophan hydroxylase (TPH) in dorsal and median raphe were investigated, respectively through time sampling technique of behavior scoring and immunohistochemistry. Both estradiol and fluoxetine increased swimming and decreased immobility in the forced swimming test. The forced swimming stress decreased integrated optical density of TPH-positive regions in dorsal and median raphe. Both estradiol and fluoxetine administration prevented integrated optical density of TPH-positive regions from being decreased by forced swimming stress. These observations suggest that both estradiol and fluoxetine have protective bearing on ovariectomized rats enduring forced swimming stress.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Datta, Soumendu, E-mail: soumendu@bose.res.in; Rahaman, Badiur
2015-11-15
Spin-polarized DFT has been used to perform a comparative study of the geometric structures and electronic properties for isolated M{sub 4}X{sub 4} nano clusters between their two stable isomers - a planar rhombus-like 2D structure and a cubane-like 3D structure with M = Mn, Fe, Co, Ni, Cu ; X = O, S. These two structural patterns of the M{sub 4}X{sub 4} clusters are commonly found as building blocks in several poly-nuclear transition metal complexes in inorganic chemistry. The effects of the van der Waals corrections to the physical properties have been considered in the electronic structure calculations employing themore » empirical Grimme’s correction (DFT+D2). We report here an interesting trend in their relative structural stability - the isolated M{sub 4}O{sub 4} clusters prefer to stabilize more in the planar structure, while the cubane-like 3D structure is more favorable for most of the isolated M{sub 4}S{sub 4} clusters than their planar 2D counterparts. Our study reveals that this contrasting trend in the relative structural stability is expected to be driven by an interesting interplay between the s-d and p-d hybridization effects of the constituents’ valence electrons.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Skraastad, M.I.; Van de Vosse, E.; Belfroid, R.
1992-10-01
Significant linkage disequilibrium has been found between the Huntington disease (HD) gene and DNA markers located around D4S95 and D4S98. The linkage-disequilibrium studies favor the proximal location of the HD gene, in contrast to the conflicting results of recombination analyses. The authors have analyzed 45 Dutch HD families with 19 DNA markers and have calculated the strength of linkage disequilibrium. Highly significant linkage disequilibrium has been detected with D4S95, consistent with the studies in other populations. In contrast with most other studies, however, the area of linkage disequilibrium extends from D4S10 proximally to D4S95, covering 1,100 kb. These results confirmmore » that the HD gene most likely maps near D4S95. 28 refs., 1 fig., 2 tabs.« less
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Skrzypecki, Janusz; Gawlak, Maciej; Huc, Tomasz; Szulczyk, Paweł; Ufnal, Marcin
2017-01-01
The effect of renal denervation on the efficacy of antihypertensive drugs has not yet been elucidated. Twenty-week-old spontaneously hypertensive rats were treated with metoprolol, losartan, indapamide, or saline (controls) and assigned to renal denervation or a sham procedure. Acute hemodynamic measurements were performed ten days later. Series showing a significant interaction between renal denervation and the drugs were repeated with chronic telemetry measurements. In the saline series, denervated rats showed a significantly lower mean arterial blood pressure (blood pressure) than the sham-operated rats. In contrast, in the metoprolol series denervated rats showed a significantly higher blood pressure than sham rats. There were no differences in blood pressure between denervated and sham rats in the losartan and indapamide series. In chronic studies, a 4-week treatment with metoprolol caused a decrease in blood pressure. Renal denervation and sham denervation performed 10 days after the onset of metoprolol treatment did not affect blood pressure. Denervated rats showed markedly reduced renal nerve tyrosine hydroxylase levels. In conclusion, renal denervation decreases blood pressure in hypertensive rats. The hypotensive action of metoprolol, indapamide, and losartan is not augmented by renal denervation, suggesting the absence of synergy between renal denervation and the drugs investigated in this study.
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Metabolism of 2,4-dichlorophenol in tobacco engineered with bacterial degradative genes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Perkins, E.J.; Sekine, M.; Gordon, M.P.
1990-05-01
The potential use of plants in toxic waste remediation has been overlooked. While chlorophenols are relatively slowly metabolized in Nicotiana tabacum var. Xanthi leaf extracts, chlorocatechols are rapidly metabolized, presumably by polyphenol oxidases. Our initial focus has been the fate of 2,4-dichlorophenol (2,4DCP) in var. Xanthi plants which express a bacterial 2,4DCP hydroxylase, which converts 2,4DCP to 3,5-dichlorocatechol. The roots of wild type and 2,4DCP hydroxylase transgenic plants growing in hydroponics were exposed to {sup 14}C-2,4DCP. Approximately 95% of {sup 14}C-2,4DCP metabolites remained in the roots when exposed to 2,4DCP. Upon extraction of root tissue, three major metabolites were foundmore » in untransformed plants and four major metabolites in transformed plants. Upon digestion with beta-D-glucosidase, these metabolites disappeared concomitant with the appearance of free 2,4DCP in wild type plants and 2,4DCP and 3,5-dichlorocatechol in transgenic plants. It is apparent that the chlorophenols are not readily available substrates for polyphenol oxidases in whole plants.« less
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Post-transcriptional Regulation of Tyrosine Hydroxylase Expression in Adrenal Medulla and Brain
Tank, A. William; Xu, Lu; Chen, Xiqun; Radcliffe, Pheona; Sterling, Carol R.
2009-01-01
It is well-established that long-term stress leads to induction of tyrosine hydroxylase (TH) mRNA and TH protein in adrenal medulla and brain. This induction is usually associated with stimulation of TH gene transcription rate. However, a number of studies have reported major discrepancies between the stress-induced changes in TH gene transcription, TH mRNA and TH protein. These discrepancies suggest that post-transcriptional mechanisms also play an important role in regulating TH expression in response to stress and other stimuli. In this report we summarize some of our findings and literature reports that demonstrate these discrepancies in adrenal medulla, locus coeruleus and midbrain dopamine neurons. We then describe our recent work investigating the molecular mechanisms that mediate this post-transcriptional regulation in adrenal medulla and midbrain. Our results suggest that trans-acting factors binding to the polypyrimidine-rich region of the 3′UTR of TH mRNA play a role in these post-transcriptional mechanisms. A hypothetical cellular model describing this post-transcriptional regulation is proposed. PMID:19120116
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NASA Astrophysics Data System (ADS)
Wang, Yue; Zou, Xiaochuan; Feng, Xia; Shi, Yongfang; Wu, Liming
2017-01-01
A new rare-earth metal gallium thioantimonate, Y4GaSbS9, has been synthesized successfully via high-temperature solid-state method. Single-crystal X-ray diffraction analyses revealed it adopted a known RE4GaSbS9-structure type in the orthorhombic space group Aba2 (no.41) with a=13.480(4) Å, b=13.790(4) Å, c=13.990(4) Å, V=2600.6(2) Å3 and Z=8. The structure is composed of bimetallic polar (Sb2S5) units and dimeric (GaS4)2 tetrahedra that share vertexes to form a 1D infinite chains 2 ∞ 1Ga 10-, inside which the isolated Y3+ cations and S2- anions. Polycrystalline Y4GaSbS9 shows the weak powder second harmonic generation (SHG) responses of this family, which is about 7.5 times that of the benchmark α-SiO2 in the particle size of 74-106 μm at the laser radiation wavelength of 2050 nm with a non-phase-matchable behavior. In addition, the synthesis, structural characterization, and optical properties as well as theoretical studies are also discussed.
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Polymorphism of the Tryptophan Hydroxylase 2 (TPH2) Gene Is Associated with Chimpanzee Neuroticism
Morimura, Naruki; Udono, Toshifumi; Hayasaka, Ikuo; Humle, Tatyana; Murayama, Yuichi; Ito, Shin'ichi; Inoue-Murayama, Miho
2011-01-01
In the brain, serotonin production is controlled by tryptophan hydroxylase 2 (TPH2), a genotype. Previous studies found that mutations on the TPH2 locus in humans were associated with depression and studies of mice and studies of rhesus macaques have shown that the TPH2 locus was involved with aggressive behavior. We previously reported a functional single nucleotide polymorphism (SNP) in the form of an amino acid substitution, Q468R, in the chimpanzee TPH2 gene coding region. In the present study we tested whether this SNP was associated with neuroticism in captive and wild-born chimpanzees living in Japan and Guinea, respectively. Even after correcting for multiple tests (Bonferroni p = 0.05/6 = 0.008), Q468R was significantly related to higher neuroticism (β = 0.372, p = 0.005). This study is the first to identify a genotype linked to a personality trait in chimpanzees. In light of the prior studies on humans, mice, and rhesus macaques, these findings suggest that the relationship between neuroticism and TPH2 has deep phylogenetic roots. PMID:21765945
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Palafox-Carlos, H; Contreras-Vergara, C A; Muhlia-Almazán, A; Islas-Osuna, M A; González-Aguilar, G A
2014-05-16
Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.
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Chen, Zhenya; Shen, Xiaolin; Wang, Jian; Wang, Jia; Yuan, Qipeng; Yan, Yajun
2017-11-01
Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
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NASA Technical Reports Server (NTRS)
Zuo, M.; Smith, Steven J.; Chutjian, A.; Williams, I. D.; Tayal, S. S.; Mclaughlin, Brendan M.
1995-01-01
Experimental and theoretical excitation cross sections are reported for the first forbidden transition 4S(O) -- 2S(2)2p(3) 2D(O) (lambda-lambda 3726, 3729) and the first allowed (resonance) transition 4S(O) -- 2s2p(4) 4P(lambda-833) in O II. Use is made of electron energy loss and merged-beams methods. The electron energy range covered is 3.33 (threshold) to 15 eV for the S -- D transition, and 14.9 (threshold) to 40 eV for the S -- P transition. Care was taken to assess and minimize the metastable fraction of the O II beam. An electron mirror was designed and tested to reflect inelastically backscattered electrons into the forward direction to account for the full range of polar scattering angles. Comparisons are made between present experiments and 11-state R-matrix calculations. Calculations are also presented for the 4S(O) -- 2s(2)2p(3)2P(O) (lambda-2470) transition.
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Are Striatal Tyrosine Hydroxylase Interneurons Dopaminergic?
Xenias, Harry S.; Ibáñez-Sandoval, Osvaldo; Koós, Tibor
2015-01-01
Striatal GABAergic interneurons that express the gene for tyrosine hydroxylase (TH) have been identified previously by several methods. Although generally assumed to be dopaminergic, possibly serving as a compensatory source of dopamine (DA) in Parkinson's disease, this assumption has never been tested directly. In TH–Cre mice whose nigrostriatal pathway had been eliminated unilaterally with 6-hydroxydopamine, we injected a Cre-dependent virus coding for channelrhodopsin-2 and enhanced yellow fluorescent protein unilaterally into the unlesioned midbrain or bilaterally into the striatum. Fast-scan cyclic voltammetry in striatal slices revealed that both optical and electrical stimulation readily elicited DA release in control striata but not from contralateral striata when nigrostriatal neurons were transduced. In contrast, neither optical nor electrical stimulation could elicit striatal DA release in either the control or lesioned striata when the virus was injected directly into the striatum transducing only striatal TH interneurons. This demonstrates that striatal TH interneurons do not release DA. Fluorescence immunocytochemistry in enhanced green fluorescent protein (EGFP)–TH mice revealed colocalization of DA, l-amino acid decarboxylase, the DA transporter, and vesicular monoamine transporter-2 with EGFP in midbrain dopaminergic neurons but not in any of the striatal EGFP–TH interneurons. Optogenetic activation of striatal EGFP–TH interneurons produced strong GABAergic inhibition in all spiny neurons tested. These results indicate that striatal TH interneurons are not dopaminergic but rather are a type of GABAergic interneuron that expresses TH but none of the other enzymes or transporters necessary to operate as dopaminergic neurons and exert widespread GABAergic inhibition onto direct and indirect spiny neurons. PMID:25904808
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Far-infrared study of the mechanochemically synthesized Cu2FeSnS4 (stannite) nanocrystals
NASA Astrophysics Data System (ADS)
Trajic, J.; Romcevic, M.; Paunovic, N.; Curcic, M.; Balaz, P.; Romcevic, N.
2018-05-01
The analysis of the optical properties of mechanochemically synthesized stannite Cu2FeSnS4 nanocrystals has been performed using far-infrared spectroscopy. The Cu2FeSnS4 stannite nanocrystals were synthesized mechanochemically from elemental precursors Cu, Fe, Sn, and S. Milling time was 45, 60, 90 and 120 min. Reflectivity spectra were analyzed using the classical form of the dielectric function, which includes the phonon and the free carrier contribution. The influence of milling time on synthesis of stannite Cu2FeSnS4 is observed. Among the modes that are characteristic for the stannite Cu2FeSnS4, we registered the modes of binary phases of FeS and SnS. The total disappearance of the binary phases of FeS and SnS and forming pure Cu2FeSnS4 is observed when the milling time is 120 min. Effective permittivity of Cu2FeSnS4 and binary phases of FeS and SnS were modeled by Maxwell - Garnet approximation.
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Tristán-Noguero, Alba; Díez, Héctor; Jou, Cristina; Pineda, Mercè; Ormazábal, Aida; Sánchez, Aurora; Artuch, Rafael; Garcia-Cazorla, Àngels
2016-06-01
Tyrosine hydroxylase (TH) deficiency is an inborn error of dopamine synthesis. Two clinical phenotypes have been described. The THD "B" phenotype produces a severe encephalopathy of early-onset with sub-optimal L-Dopa response, whereas the "A" phenotype has a better L-Dopa response and outcome. The objective of the study is to describe the expression of key synaptic proteins and neurodevelopmental markers in a fetal brain of THD "B" phenotype. The brain of a 16-week-old miscarried human fetus was dissected in different brain areas and frozen until the analysis. TH gene study revealed the p.R328W/p.T399M mutations, the same mutations that produced a B phenotype in her sister. After protein extraction, western blot analyses were performed to assess protein expression. The results were compared to an age-matched control. We observed a decreased expression in TH and in other dopaminergic proteins, such as VMAT 1 and 2 and dopamine receptors, especially D2DR. GABAergic and glutamatergic proteins such as GABA VT, NMDAR1 and calbindin were also altered. Developmental markers for synapses, axons and dendrites were decreased whereas markers of neuronal volume were preserved. Although this is an isolated case, this brain sample is unique and corresponds to the first reported study of a THD brain. It provides interesting information about the influence of dopamine as a regulator of other neurotransmitter systems, brain development and movement disorders with origin at the embryological state. This study could also contribute to a better understanding of the pathophysiology of THD at early fetal stages.
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In-crystal reaction cycle of a toluene-bound diiron hydroxylase
NASA Astrophysics Data System (ADS)
Acheson, Justin F.; Bailey, Lucas J.; Brunold, Thomas C.; Fox, Brian G.
2017-03-01
Electrophilic aromatic substitution is one of the most important and recognizable classes of organic chemical transformation. Enzymes create the strong electrophiles that are needed for these highly energetic reactions by using O2, electrons, and metals or other cofactors. Although the nature of the oxidants that carry out electrophilic aromatic substitution has been deduced from many approaches, it has been difficult to determine their structures. Here we show the structure of a diiron hydroxylase intermediate formed during a reaction with toluene. Density functional theory geometry optimizations of an active site model reveal that the intermediate is an arylperoxo Fe2+/Fe3+ species with delocalized aryl radical character. The structure suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the O-O bond by transferring a proton from a metal-bound water. Our work provides the spatial and electronic constraints needed to propose a comprehensive mechanism for diiron enzyme arene hydroxylation that accounts for many prior experimental results.
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NASA Technical Reports Server (NTRS)
Simsic, P. L.
1974-01-01
Excitation of neutral atoms by inelastic scattering of incident electrons in gaseous nebulae were investigated using Slater Wave functions to describe the initial and final states of the atom. Total cross sections using the Born Approximation are calculated for: Li(2s yields 2p), Na(3s yields 4p), k(4s yields 4p). The intensity of emitted radiation from gaseous nebulae is also calculated, and Maxwell distribution is employed to average the kinetic energy of electrons.
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Alonso, Hernan; Roujeinikova, Anna
2012-11-01
The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C(12)E(8)]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-D-maltopyranoside (DM), n-dodecyl-β-D-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism.
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Alonso, Hernan
2012-01-01
The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C12E8]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-d-maltopyranoside (DM), n-dodecyl-β-d-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism. PMID:22941083
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2010-01-01
Background Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism. Results The cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins. Conclusions CYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism. PMID:20128892
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Taboas, Melisa; Fernández, Cecilia; Belli, Susana; Buzzalino, Noemi; Alba, Liliana; Dain, Liliana
2013-01-01
Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90%-95% of cases. This autosomal recessive disorder has a broad spectrum of clinical forms, ranging from severe or classical, which includes the salt-wasting and simple virilizing forms, to the mild late onset or nonclassical form. Most of the disease-causing mutations described are likely to be the consequence of nonhomologous recombination or gene conversion events between the active CYP21A2 gene and its homologous CYP21A1P pseudogene. Nevertheless, an increasing number of naturally occurring mutations have been found. The change p.H62L is one of the most frequent rare mutations of the CYP21A2 gene. It was suggested that the p.H62L represents a mild mutation that may be responsible for a more severe enzymatic impairment when presented with another mild mutation on the same allele. In this report, a 20-year-old woman carrying an isolated p.H62L mutation in compound heterozygosity with c.283-13A/C>G mutation is described. Although a mildly nonclassical phenotype was expected, clinical signs and hormonal profile of the patient are consistent with a more severe simple virilizing form of 21-hydroxylase deficiency. The study of genotype-phenotype correlation in additional patients would help in defining the role of p.H62L in disease manifestation.
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S4AC Case Study: Enhancing Underserved Seniors' Access to Health Promotion Programs.
Koehn, Sharon; Habib, Sanzida; Bukhari, Syeda
2016-03-01
The Seniors Support Services for South Asian Community (S4AC) project was developed in response to the underutilization of available recreation and seniors' facilities by South Asian seniors who were especially numerous in a suburban neighbourhood in British Columbia. Addressing the problem required the collaboration of the municipality and a registered non-profit agency offering a wide range of services and programs to immigrant and refugee communities. Through creative outreach and accommodation, the project has engaged more than 100 Punjabi-speaking seniors annually in diverse exercise activities. Case study research methods with staff and current and former senior participants of S4AC include participant observation, individual interviews, and focus groups. Viewed through the critical interpretive lens of the "candidacy framework", findings reveal the myriad ways in which access to health promotion and physical activity for immigrant older adults is a complex iterative process of negotiation at multiple levels.
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Kolet, Swati P; Haldar, Saikat; Niloferjahan, Siddiqui; Thulasiram, Hirekodathakallu V
2014-07-01
Transformation of testosterone and progesterone into synthetically challenging 14α-hydroxy derivatives was achieved by using fungal strain Mucor hiemalis. Prolonged incubation led to the formation of corresponding 6β/7α,14α-dihydroxy metabolites. The position and stereochemistry of newly introduced hydroxyl group was determined by detailed spectroscopic analyses. The time course experiment indicated that fungal strain initiated transformation by hydroxylation at 14α-position followed by at 6β- or 7α-positions. Studies using cell-free extracts suggest that the 14α-hydroxylase activity is NADPH dependent and belongs to the cytochrome P450 family. Copyright © 2014 Elsevier Inc. All rights reserved.
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Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase.
Selak, Mary A; Armour, Sean M; MacKenzie, Elaine D; Boulahbel, Houda; Watson, David G; Mansfield, Kyle D; Pan, Yi; Simon, M Celeste; Thompson, Craig B; Gottlieb, Eyal
2005-01-01
Several mitochondrial proteins are tumor suppressors. These include succinate dehydrogenase (SDH) and fumarate hydratase, both enzymes of the tricarboxylic acid (TCA) cycle. However, to date, the mechanisms by which defects in the TCA cycle contribute to tumor formation have not been elucidated. Here we describe a mitochondrion-to-cytosol signaling pathway that links mitochondrial dysfunction to oncogenic events: succinate, which accumulates as a result of SDH inhibition, inhibits HIF-alpha prolyl hydroxylases in the cytosol, leading to stabilization and activation of HIF-1alpha. These results suggest a mechanistic link between SDH mutations and HIF-1alpha induction, providing an explanation for the highly vascular tumors that develop in the absence of VHL mutations.
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Super reduced Fe4S4 cluster of Balch's dithiolene series.
Begum, Ameerunisha; Moula, Golam; Bose, Moumita; Sarkar, Sabyasachi
2012-03-28
A super reduced Fe(4)S(4) cluster with a sulfur based radical, [NBu(4)](4)[Fe(3)(III)Fe(II)(μ(3)-S)(4)(mnt)(3)(6-)(mnt)(1-)˙](4-)˙, (1) (mnt, maleonitrile dithiolate) which evolves H(2)S gas on treatment with acid under ambient conditions has been synthesized and structurally characterized. The Fe-S distances in 1 are in the range 2.246-2.383 Å, in stark contrast to that of the known n = -2 member of the series based on the [Fe(4)(μ(3)-S)(4)(S(2)C(2)R(2))(4)](n) unit (R = CF(3), Ph) with Fe-S bond lengths of 2.149-2.186 Å. The EPR of 1 displays very weak signals at g, 4.03 and 2.38 along with a strong S-based radical EPR signal at g, 2.003 associated with five structured components tentatively assigned to hyperfine interaction arising out of the naturally abundant (57)Fe with = 88 G. The EPR profile resembles the reduced Fe-S cluster of CO inhibited Clostridium pasteurianum W5 hydrogenase or the Fe(4)S(4) centers of wild-type enzyme, IspH treated with HMBPP or IPP.
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N4H9Cu7S4: a hydrazinium-based salt with a layered Cu7S4- framework.
Mitzi, David B
2007-02-05
Crystals of a hydrazinium-based copper(I) sulfide salt, N4H9Cu7S4 (1), have been isolated by an ambient temperature solution-based process. In contrast to previously reported hydrazinium salts of main-group metal chalcogenides, which consist of isolated metal chalcogenide anions, and ACu7S4 (A = NH4+, Rb+, Tl+, K+), which contains a more three-dimensional Cu7S4- framework with partial Cu-site occupancy, the structure of 1 [P21, a = 6.8621(4) A, b = 7.9851(4) A, c = 10.0983(5) A, beta = 99.360(1) degrees , Z = 2] is composed of extended two-dimensional Cu7S4- slabs with full Cu-site occupancy. The Cu7S4- slabs are separated by a mixture of hydrazinium and hydrazine moieties. Thermal decomposition of 1 into copper(I) sulfide proceeds at a significantly lower temperature than that observed for analogous hydrazinium salts of previously considered metal chalcogenides, completing the transition at temperatures as low as 120 degrees C. Solutions of 1 may be used in the solution deposition of a range of Cu-containing chalcogenide films.
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Treatment for GaSb surfaces using a sulphur blended (NH4)2S/(NH4)2SO4 solution
NASA Astrophysics Data System (ADS)
Murape, D. M.; Eassa, N.; Neethling, J. H.; Betz, R.; Coetsee, E.; Swart, H. C.; Botha, J. R.; Venter, A.
2012-07-01
A sulphur based chemical, [(NH4)2S/(NH4)2SO4] to which S has been added, not previously reported for the treatment of (1 0 0) n-GaSb surfaces, is introduced and benchmarked against the commonly used passivants Na2S·9H2O and (NH4)2S. The surfaces of the treated material were studied by scanning electron microscopy (SEM), Auger electron spectroscopy (AES) and X-ray photoelectron spectroscopy (XPS). It has been found that the native oxides present on the GaSb surface are more effectively removed when treated with ([(NH4)2S/(NH4)2SO4] + S) than with (NH4)2S or Na2S·9H2O, as evidenced by the ratio of the O506 eV to Sb457 eV AES peaks. XPS results reveal that Sb2S3/Sb2S5 "replaces" Sb2O3/Sb2O5, suggesting that sulphur atoms substitute oxygen atoms in Sb2O3/Sb2O5 to form Sbsbnd S. It seems sulphurization only partially removes Ga2O3. Treatment with ([(NH4)2S/(NH4)2SO4] + S) also results in a noteworthy improvement in the current-voltage (I-V) characteristics of Au/n-GaSb Schottky contacts compared to those fabricated on as-received material.
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Hildebrand, Alexandra; Lönnecke, Peter; Silaghi-Dumitrescu, Luminita; Hey-Hawkins, Evamarie
2008-09-14
PhP(2-SHC6H4)2 (PS2H2) reacts with WCl6 with reduction of tungsten to give the air-sensitive tungsten(IV) complex [W{PhP(2-SC6H4)2-kappa(3)S,S',P}2] (1). 1 is oxidised in air to [WO{PhPO(2-SC6H4)2-kappa(3)S,S',O}{PhP(2-SC6H4)2-kappa(3)S,S',P}] (2). The attempted synthesis of 2 by reaction of 1 with iodosobenzene as oxidising agent was unsuccessful. [W{P(2-SC6H4)3-kappa(4)S,S',S",P}2] (3) was formed in the reaction of P(2-SHC6H4)3 (PS3H3) with WCl6. The W(VI) complex 3 contains two PS3(3-) ligands, each coordinated in a tetradentate fashion resulting in a tungsten coordination number of eight. The reaction of 3 with AgBF4 yields the dinuclear tungsten complex [W2{P(2-SC6H4)3-kappa(4)S,S',S",P}3]BF4 (4). Complexes 1-4 were characterised by spectral methods and X-ray structure determination.
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[15]aneN4S: synthesis, thermodynamic studies and potential applications in chelation therapy.
Torres, Nuno; Gonçalves, Sandrina; Fernandes, Ana S; Machado, J Franco; de Brito, Maria J Villa; Oliveira, Nuno G; Castro, Matilde; Costa, Judite; Cabral, Maria F
2014-01-03
The purpose of this work was to synthesize and characterize the thiatetraaza macrocycle 1-thia-4,7,10,13-tetraazacyclopentadecane ([15]aneN4S). Its acid-base behaviour was studied by potentiometry at 25 °C and ionic strength 0.10 M in KNO3. The protonation sequence of this ligand was investigated by 1H-NMR titration that also allowed the determination of protonation constants in D2O. Binding studies of [15]aneN4S with Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+ metal ions were further performed under the same experimental conditions. The results demonstrated that this compound has a higher selectivity and thermodynamic stability for Hg2+ and Cu2+, followed by Ni2+. The UV-visible-near IR spectroscopies and magnetic moment data for the Co(II) and Ni(II) complexes indicated a tetragonal distorted coordination geometry for both metal centres. The value of magnetic moment and the X-band EPR spectra of the Cu(II) complex are consistent with a distorted square pyramidal geometry.
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Castellarin, Simone D; Di Gaspero, Gabriele; Marconi, Raffaella; Nonis, Alberto; Peterlunger, Enrico; Paillard, Sophie; Adam-Blondon, Anne-Francoise; Testolin, Raffaele
2006-01-01
Background Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines. Results Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals. Conclusion We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly, in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between
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Astuti, Dewi; Ricketts, Christopher J; Chowdhury, Rasheduzzaman; McDonough, Michael A; Gentle, Dean; Kirby, Gail; Schlisio, Susanne; Kenchappa, Rajappa S; Carter, Bruce D; Kaelin, William G; Ratcliffe, Peter J; Schofield, Christopher J; Latif, Farida; Maher, Eamonn R
2011-02-01
Germline mutations in the von Hippel-Lindau disease (VHL) and succinate dehydrogenase subunit B (SDHB) genes can cause inherited phaeochromocytoma and/or renal cell carcinoma (RCC). Dysregulation of the hypoxia-inducible factor (HIF) transcription factors has been linked to VHL and SDHB-related RCC; both HIF dysregulation and disordered function of a prolyl hydroxylase domain isoform 3 (PHD3/EGLN3)-related pathway of neuronal apoptosis have been linked to the development of phaeochromocytoma. The 2-oxoglutarate-dependent prolyl hydroxylase enzymes PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) have a key role in regulating the stability of HIF-α subunits (and hence expression of the HIF-α transcription factors). A germline PHD2 mutation has been reported in association with congenital erythrocytosis and recurrent extra-adrenal phaeochromocytoma. We undertook mutation analysis of PHD1, PHD2 and PHD3 in two cohorts of patients with features of inherited phaeochromocytoma (n=82) and inherited RCC (n=64) and no evidence of germline mutations in known susceptibility genes. No confirmed pathogenic mutations were detected suggesting that mutations in these genes are not a frequent cause of inherited phaeochromocytoma or RCC.
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Michiel, Magalie; Perchat, Nadia; Perret, Alain; Tricot, Sabine; Papeil, Aude; Besnard, Marielle; de Berardinis, Véronique; Salanoubat, Marcel; Fischer, Cécile
2012-12-01
In aerobic cells, urate is oxidized to 5-hydroxyisourate by two distinct enzymes: a coenzyme-independent urate oxidase (EC 1.7.3.3) found in eukaryotes and bacteria like Bacillus subtilis and a prokaryotic flavoprotein urate hydroxylase (HpxO) originally found in some Klebsiella species. More cases of analogous or non-homologous isofunctional enzymes (NISE) for urate catabolism have been hypothesized by inspecting bacterial genomes. Here, we used a functional complementation approach in which a candidate gene for urate oxidation is integrated by homologous recombination in the Acinetobacter baylyi ADP1 genome at the locus of its original hpxO gene. Catabolism of urate was restored in A. baylyi ADP1 expressing a FAD-dependent protein from Xanthomonas campestris, representing a new urate hydroxylase family that we called HpyO. This enzyme was kinetically characterized and compared with other HpxO enzymes. In contrast to the latter, HpyO is a typical Michaelian enzyme. This work provides the first experimental evidences for the function of HpyO in bacterial urate catabolism and establishes it as a NISE of HpxO. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.
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Chen, Hsi-Chuan; Li, Quanzi; Shuford, Christopher M.; Liu, Jie; Muddiman, David C.; Sederoff, Ronald R.; Chiang, Vincent L.
2011-01-01
The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (Vmax/km) for any of the complexes is 70–6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex–mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis. PMID:22160716
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Bai, Chao; Berman, Judit; Farre, Gemma; Capell, Teresa; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu
2017-02-01
Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.
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Predicting the onset of Addison's disease: ACTH, renin, cortisol and 21-hydroxylase autoantibodies.
Baker, Peter R; Nanduri, Priyaanka; Gottlieb, Peter A; Yu, Liping; Klingensmith, Georgeanna J; Eisenbarth, George S; Barker, Jennifer M
2012-05-01
Autoantibodies to 21-hydroxylase (21OH-AA) precede onset of autoimmune Addison's disease (AD). Progression to AD can take months to years, and early detection of metabolic decompensation may prevent morbidity and mortality. To define optimal methods of predicting progression to overt AD (defined by subnormal peak cortisol response to Cosyntropin) in 21OH-AA+ individuals. Individuals were screened for 21OH-AA at the Barbara Davis Center from 1993 to 2011. Subjects positive for 21OH-AA (n = 87) were tested, and the majority prospectively followed for the development of Addison's disease, including seven diagnosed with AD upon 21OH-AA discovery (discovered), seven who progressed to AD (progressors) and 73 nonprogressors. Plasma renin activity (PRA), ACTH, baseline cortisol, peak cortisol and 21OH-AA were measured at various time points relative to diagnosis of AD or last AD-free follow-up. Compared with nonprogressors, in the time period 2 months-2 years prior to the onset of AD, progressors were significantly more likely to have elevated ACTH (11-22 pM, P < 1E-4), with no significant differences in mean PRA (P = 0·07) or baseline cortisol (P = 0·08), and significant but less distinct differences seen with 21OH-AA levels (P < 1E-4) and poststimulation cortisol levels (P = 6E-3). Moderately elevated ACTH is a more useful early indicator of impending AD than 21OH-AA, PRA or peak cortisol, in the 2 months-2 years preceding the onset of AD. © 2012 Blackwell Publishing Ltd.
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The [4Fe-4S](2+) cluster in reconstituted biotin synthase binds S-adenosyl-L-methionine.
Cosper, Michele Mader; Jameson, Guy N L; Davydov, Roman; Eidsness, Marly K; Hoffman, Brian M; Huynh, Boi Hanh; Johnson, Michael K
2002-11-27
The combination of resonance Raman, electron paramagnetic resonance and Mössbauer spectroscopies has been used to investigate the effect of S-adenosyl-l-methionine (SAM) on the spectroscopic properties of the [4Fe-4S]2+ cluster in biotin synthase. The results indicate that SAM interacts directly at a unique iron site of the [4Fe-4S]2+ cluster in BioB and support the hypothesis of a common inner-sphere mechanism for the reductive cleavage of SAM in the radical SAM family of Fe-S enzymes.
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Carrera, P; Barbieri, A M; Ferrari, M; Righetti, P G; Perego, M; Gelfi, C
1997-11-01
A quick diagnosis of the classic form of 21-hydroxylase deficiency (simple virilizing and salt wasting) is of great importance, especially for prenatal diagnosis and treatment in pregnancies at risk. A method for simultaneous detection of common point mutations in the P450c21 B gene is here proposed by combining a nested PCR amplification refractory mutation system (ARMS) with capillary zone electrophoresis (CZE) in sieving liquid polymers. In the first PCR, B genes are selectively amplified. In the nested reaction, ARMS-detected wild-type and mutated alleles are separately pooled and resolved by CZE. CZE is performed in coated capillaries in the presence of 30 g/L hydroxyethyl cellulose in the background electrolyte for size separation of the DNA analytes. For high-sensitivity detection the electrophoresis buffer contains the fluorescent dye SYBR Green I. Laser-induced fluorescence detection is obtained by excitation at 488 nm and signal collection at 520 nm. Specificity and reproducibility of the protocols were established by using samples from 75 Italian families with 21-hydroxylase deficiency already genotyped by allele-specific oligonucleotide hybridization or direct sequencing. Whereas dot-blot is time consuming because of the high number of hybridizations with radioactive probes, this present protocol is more rapid, giving sufficient separation on CZE after PCR reactions without preconcentration or desalting of samples.
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Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain
Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.
2016-01-01
The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649
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Bouzid, Assil; Le Roux, Sébastien; Ori, Guido; Boero, Mauro; Massobrio, Carlo
2015-07-21
First-principles molecular dynamics simulations based on density functional theory are employed for a comparative study of structural and bonding properties of two stoichiometrically identical chalcogenide glasses, GeSe4 and GeS4. Two periodic cells of 120 and 480 atoms are adopted. Both glasses feature a coexistence of Ge-centered tetrahedra and Se(S) homopolar connections. Results obtained for N = 480 indicate substantial differences at the level of the Se(S) environment, since Ge-Se-Se connections are more frequent than the corresponding Ge-S-S ones. The presence of a more prominent first sharp diffraction peak in the total neutron structure factor of glassy GeS4 is rationalized in terms of a higher number of large size rings, accounting for extended Ge-Se correlations. Both the electronic density of states and appropriate electronic localization tools provide evidence of a higher ionic character of Ge-S bonds when compared to Ge-Se bonds. An interesting byproduct of these investigations is the occurrence of discernible size effects that affect structural motifs involving next nearest neighbor distances, when 120 or 480 atoms are used.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Li, Chao; Feng, Kai; Tu, Heng
Four new chalcohalides, namely NaBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Br, and CsBa{sub 2}SnS{sub 4}Cl, have been synthesized by the conventional high temperature solid-state reactions. They crystallize in three different space groups: space group I4/mcm for NaBa{sub 2}SnS{sub 4}Cl and KBa{sub 2}SnS{sub 4}Cl, Pnma for KBa{sub 2}SnS{sub 4}Br, and P2{sub 1}/c for CsBa{sub 2}SnS{sub 4}Cl. In all four compounds, the X{sup −} halide anions are only connected to six alkali metal or Ba cations, and the Sn atoms are only tetrahedrally enjoined to four S atoms. However, the M–X–Ba pseudo layers and the SnS{sub 4} tetrahedra are arrangedmore » in different ways in the three structural types, which demonstrates the interesting effect of ionic radii on the crystal structures. UV–vis–NIR spectroscopy measurements indicate that NaBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Br, and CsBa{sub 2}SnS{sub 4}Cl have band gaps of 2.28, 2.30, 1.95, and 2.06 eV, respectively. - Graphical abstract: A new series of chalcohalides, NaBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Br and CsBa{sub 2}SnS{sub 4}Cl have been obtained. They present three different space groups: NaBa{sub 2}SnS{sub 4}Cl and KBa{sub 2}SnS{sub 4}Cl in space group I4/mcm, KBa{sub 2}SnS{sub 4}Br in Pnma and CsBa{sub 2}SnS{sub 4}Cl in space group P2{sub 1}/c. UV–vis–NIR spectroscopy measurements indicate that NaBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Br and CsBa{sub 2}SnS{sub 4}Cl have band gaps of 2.28, 2.30 1.95, and 2.06 eV, respectively. - Highlights: • Four new chalcohalides, NaBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Br and CsBa{sub 2}SnS{sub 4}Cl were obtained. • They adopt three different structures owing to different ionic radii and elemental electronegativity. • NaBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Cl, KBa{sub 2}SnS{sub 4}Br and CsBa{sub 2}SnS{sub 4}Cl have
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Cloning and Characterization of a Flavonoid 3′-Hydroxylase Gene from Tea Plant (Camellia sinensis)
Zhou, Tian-Shan; Zhou, Rui; Yu, You-Ben; Xiao, Yao; Li, Dong-Hua; Xiao, Bin; Yu, Oliver; Yang, Ya-Jun
2016-01-01
Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3′H, designated as CsF3′H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3′H was highly homologous with the characterized F3′Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3′H-specific conserved motifs were discovered in the protein sequence of CsF3′H. Enzymatic analysis of the heterologously expressed CsF3′H in yeast demonstrated that tea F3′H catalyzed the 3′-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min−1, respectively. Transcription level of CsF3′H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3′,4′-flavan-3-ols, 3′,4′,5′-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3′H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3′H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3′H in the biosynthesis of 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols in tea leaves. PMID:26907264
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Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sun, Zhichao; Yu, Xuemei; Wu, Weibin
2012-07-13
Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated thatmore » the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.« less
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A new quaternary semiconductor compound (Ba2Sb4GeS10): Ab initio study
NASA Astrophysics Data System (ADS)
Ozisik, Havva Bogaz; Ozisik, Haci; Deligoz, Engin
2017-03-01
The newly synthesised Ba2Sb4GeS10 compound is notable because of the interesting features of the quaternary Sb-containing materials. The first principle method has been used to determine the physical properties of this compound. In particular, the electronic structure has been analysed using both conventional GGA-PBE and HSE06 functional. The values of the band gap for PBE and HSE06 calculations were 1.324 and 1.84 eV, respectively. The calculated elastic constants were used to predict polycrystalline mechanical properties. The estimated Vickers hardness (2.7 GPa) values show that Ba2Sb4GeS10 is soft matter. Moreover, the vibrational properties of the compound have been studied. The calculation of the elastic constants and phonon dispersion curves indicates that the Ba2Sb4GeS10 compound is stable both mechanically and dynamically. Furthermore, the minimum thermal conductivity and optical properties, such as dielectric functions and energy loss function, have also been discussed in detail in this paper.
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Electronic structure and fragmentation properties of [Fe4S4(SEt)4-x(SSEt)x]2-
NASA Astrophysics Data System (ADS)
Fu, You-Jun; Laskin, Julia; Wang, Lai-Sheng
2007-06-01
A limited exposure of (n-Bu4N)2[Fe4S4(SEt)4] solutions in acetonitrile to air was found to produce a new series of [4Fe-4S] cluster complexes, [Fe4S4(SEt)4-x(SSEt)x]2- (x = 1-4), with the original -SEt ligands substituted by -SSEt di-sulfide ligands, which were formed due to partial decomposition of the [4Fe-4S] core in parent [Fe4S4(SEt)4]2-. The products were first observed in the experiments with an ESI-ion Trap-TOF mass spectrometer and were further identified using high resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Photoelectron spectra of the [Fe4S4(SEt)4-x(SSEt)x]2- dianions revealed that the -SSEt coordination induced little change in the electronic structure of the [4Fe-4S] cluster, but the electron binding energies of [Fe4S4(SEt)4-x(SSEt)x]2- increased from 0.52 to 0.73 eV with increase in x from 0 to 4, suggesting a greater electron withdrawing ability of -SSEt than -SEt. In high resolution MS/MS experiments on [Fe4S4(SEt)3(SSEt)]2-/1-, clusters with both charge states yielded fragment [Fe4S4(SEt)3]-, suggesting that -SSEt could be lost either as a negatively charged ion SSEt- from the doubly charged precursor, or as a radical SSEt from the singly charged species. The biological implication of the interaction between [Fe4S4(SEt)4]2- and O2 is discussed in comparison to the air exposure of [4Fe-4S] proteins to the air.
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Yoo, Su Jeong; Kim, Hea Ok; Lim, Yoongho; Kim, Jeongmin; Jeong, Lak Shin
2002-01-01
Novel (2R,4R)- and (2S,4S)-iso dideoxynucleosides with exocyclic methylene have been designed and synthesized, based on the lead BMS-200475 (3) which exhibited potent anti-HBV activity. For the synthesis of D types of (2R,4R)-nucleosides, L-xylose was converted to the key intermediate 14. The intermediate 14 was converted to the uracil derivative 4a and the cytosine derivative 4b. Compound 14 was also converted to the purine derivatives such as adenine derivative 4c, hypoxanthine derivative 4d, and guanine derivative 4e. The corresponding L types of (2S,4S)-enantiomers were more efficiently synthesized from the commercially available 1,2-isopropylidene-D-xylose (20) than the synthetic method used in the synthesis of (2R,4R)-nucleosides. The key intermediate 25 was converted to the pyrimidine analogues 5a and 5b and the purine derivatives 5c, 5d, and 5e using the similar method used in the preparation of 4c, 4d, and 4e. The synthesized final (2R,4R)- and (2S,4S)-nucleosides were tested against several viruses such as HIV-1, HSV-1, HSV-2, HCMV and HBV. (2R,4R)-Adenine analogue 4c exhibited potent anti-HBV activity (EC(50)=1.5 microM in 2.2.15 cells) among compounds tested, while (2R,4R)-uracil derivative 4a was the most active against HCMV among compounds tested and (2R,4R)-adenine derivative 4c was found to be moderately active against the same virus. However, the corresponding (2S,4S)-isomers were found to be totally inactive against all tested viruses. Both (2R,4R)-adenine derivative 4c and (2S,4S)-adenine analogue 5c were totally resistant to the adenosine deaminase like iso-ddA (1). From the molecular modeling study the hydroxymethyl side chains of BMS-200475 (3) and 4c were almost overlapped, indicating that 4c may be suitable for phosphorylation by cellular kinases like the lead 3, but some discrepancy between two bases was observed, indicating why 4c is less potent against HBV than 3. It is concluded that discovery of (2R,4R)-adenine analogue 4c as potent
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8 CFR 236.4 - Removal of S-5, S-6, and S-7 nonimmigrants.
Code of Federal Regulations, 2010 CFR
2010-01-01
... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Removal of S-5, S-6, and S-7 nonimmigrants. 236.4 Section 236.4 Aliens and Nationality DEPARTMENT OF HOMELAND SECURITY IMMIGRATION REGULATIONS... of Aliens Prior to Order of Removal § 236.4 Removal of S-5, S-6, and S-7 nonimmigrants. (a) Condition...
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Isolated p.H62L Mutation in the CYP21A2 Gene in a Simple Virilizing 21-Hydroxylase Deficient Patient
Fernández, Cecilia; Belli, Susana; Buzzalino, Noemi; Dain, Liliana
2013-01-01
Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90%–95% of cases. This autosomal recessive disorder has a broad spectrum of clinical forms, ranging from severe or classical, which includes the salt-wasting and simple virilizing forms, to the mild late onset or nonclassical form. Most of the disease-causing mutations described are likely to be the consequence of nonhomologous recombination or gene conversion events between the active CYP21A2 gene and its homologous CYP21A1P pseudogene. Nevertheless, an increasing number of naturally occurring mutations have been found. The change p.H62L is one of the most frequent rare mutations of the CYP21A2 gene. It was suggested that the p.H62L represents a mild mutation that may be responsible for a more severe enzymatic impairment when presented with another mild mutation on the same allele. In this report, a 20-year-old woman carrying an isolated p.H62L mutation in compound heterozygosity with c.283-13A/C>G mutation is described. Although a mildly nonclassical phenotype was expected, clinical signs and hormonal profile of the patient are consistent with a more severe simple virilizing form of 21-hydroxylase deficiency. The study of genotype-phenotype correlation in additional patients would help in defining the role of p.H62L in disease manifestation. PMID:23936690
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Fex, J; Wenthold, R J
1976-06-18
Activities of choline acetyltransferase (ChAC), glutamate decarboxylase (GAD) and tyrosine hydroxylase (TH), enzymes catalyzing the synthesis of acetylcholine (ACh), gamma-aminobutyric acid (GABA) and catecholamines, respectively, were measured in the cochlea and cochlear nucleus of the guinea pig. ChAc activity in the organ of Corti, third turn, was 1270 pmole ACh formed/min/mg protein (ChAc, 1270) and was higher than in turn 4 (ChAc, 543). ChAc activity was higher when the preparation included the inner hair cell region than when not. GAD activity in samples of turn 3 and 4 combined was low, 0.17 nmole GABA formed/min/mg protein (GAD, 0.17). All 3 enzymes were low in auditory nerve: ChAc, 1.7, GAD, 0.10 and TH, 1.0 pmole DOPA formed/min/mg protein. In the cochlear nucleus, the values were: ChAc, 129, GAD, 1.70 and TH, 2.7. The findings on the distribution of ChAc activity in the organ of Corti fit the hypothesis that the olivocochlear nerve fibers are cholinergic. Because of low GAD in the cochlea, GABA is unlikely to be transmitter in the organ of Corti. Similarly, it is unlikely that ACh, GABA or a catecholamine is a transmitter between the auditory nerve and the cochlear nucleus.
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NASA Astrophysics Data System (ADS)
Syverson, D. D.; Ono, S.; Seyfried, W. E., Jr.
2017-12-01
conditions. Geochimica et Cosmochimica Acta 122, 170-183. [4] Syverson, D.D., Ono, S., Shanks, W.C. & Seyfried Jr., W.E. 2015 Multiple sulfur isotope fractionation and mass transfer processes during pyrite precipitation and recrystallization: An experimental study at 300 and 350°C. Geochimica et Cosmochimica Acta 165, 418-434.
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Distribution of neurons expressing tyrosine hydroxylase in the human cerebral cortex
Benavides-Piccione, Ruth; DeFelipe, Javier
2007-01-01
Since the very first detailed description of the different types of cortical interneurons by Cajal, the tremendous variation in the morphology, physiology and neurochemical properties of these cells has become apparent. However, it still remains unclear whether all types of interneurons are present in all cortical areas and species. Here we have focused on tyrosine hydroxylase (TH)-immunoreactive cortical interneurons, which although only present in certain species, are particularly abundant in the human neocortex. We argue that this type of interneuron is more widespread in the human neocortex than in any other species examined so far and that, therefore, it is probably involved in a larger variety of cortical circuits. In addition, notable regional variation can be seen in relation to these interneurons. These differences further emphasize the variability in the design of microcircuits between cortical areas and species, and they probably reflect an evolutionary adaptation of cortical circuits to particular functions. PMID:17593221
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Absolute structure and structure-function relationships of 4R,2‧R and 4S,2‧S Pidotimod®
NASA Astrophysics Data System (ADS)
Sarno, Simone; Manzo, Angelo M.; Ferraris, Davide M.; Miggiano, Riccardo; Rizzi, Menico; Palin, Luca; Boccaleri, Enrico; Milanesio, Marco
2017-11-01
Pidotimod® is a dipeptide with widely recognized immunomodulatory properties and with particularly beneficial effects for the treatment of acute respiratory and urinary tract infections. Pidotimod® presents two chiral centres which originate four stereoisomers. (4R,2‧S, 4S,2‧R, 4R,2‧R and 4S,2‧S). To date, only the 4R,2‧S and 4S,2‧R stereoisomers are reported in the literature. We report here the absolute crystal structure of the 4R,2‧R and 4S,2‧S diastereoisomers of Pidotimod®, obtained by crystals grown by slow evaporation of a mixture of water and ethanol. The analysis of the crystal structures revealed the key role of a solvent water molecule in the crystal packing engaged in an extended hydrogen bonds network. This water-assisted H-bond network explained the recalcitrance of 4R,2‧R and 4S,2‧S Pidotimod® to crystallize in pure ethanol, despite their high solubility, and the growth of well-diffracting crystals only in presence of water. Hence, Pidotimod®4R,2‧R and 4S,2‧S stereoisomers markedly differ from the 4R,2‧S and 4S,2‧R ones, which crystallize in absence of water. The molecular and crystal structures of the 4R,2‧R and 4S,2‧S Pidotimod® stereoisomers here presented gave some hints on the differences in bioactivity with respect to the 4R,2‧S stereoisomer. In fact, beyond an expected different dispositions of hydrophilic ligands, 4R,2‧R and 4S,2‧S showed an incremented tendency to intermolecular H-bonds with water.
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ERIC Educational Resources Information Center
Leppanen, Jukka M.; Peltola, Mikko J.; Puura, Kaija; Mantymaa, Mirjami; Mononen, Nina; Lehtimaki, Terho
2011-01-01
Background: Allelic variation in the promoter region of a gene that encodes tryptophan hydroxylase isoform 2 (TPH2), a rate-limiting enzyme of serotonin synthesis in the central nervous system, has been associated with variations in cognitive function and vulnerability to affective spectrum disorders. Little is known about the effects of this gene…
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Houben, C H; Tsui, S Y; Mou, J W; Chan, K W; Tam, Y H; Lee, K H
2014-12-01
To present the results of feminising genitoplasty done in female patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Case series. A tertiary referral centre in Hong Kong. Female patients with congenital adrenal hyperplasia undergoing corrective surgery for virilisation between 1993 and 2012. The operative result was judged with a scoring system (1-3) for four areas: appearance of clitoris, labia and vagina, plus requirement for revision surgery. A total of 23 female patients with congenital adrenal hyperplasia with a median age of 17.5 (range, 1.5-33.8) years were identified. Of these individuals, 17 presented in the neonatal period and early infancy, of which four had an additional salt-losing crisis. Six patients-including four migrants from mainland China-were late presenters at a median age of 2 (range, 0.5-14) years. Twenty-two patients had corrective surgery at a median age of 2 (range, 1-14) years. Clitoral reduction was performed in all, and further surgery in 21 patients. The additional surgery was flap vaginoplasty in 10 patients, a modified Passerini procedure in six, and a labial reconstruction in five; one patient with prominent clitoris was for observation only. Minor revision surgery (eg mucosal trimming) was required in three patients; a revision vaginoplasty was done in one individual. Of the 23 patients, 18 (78%) with a median age of 20 (range, 9.3-33.8) years participated in the outcome evaluation: a 'good' outcome (4 points) was seen in 12 patients and a 'satisfactory' (5-9 points) result in five patients. Nearly three quarters of our cohort (n=17) presented with classic virilising form of 21-hydroxylase deficiency. Only four (25%) patients experienced a salt-losing crisis. Female gender assignment at birth was maintained for all individuals in this group. 'Good' and 'satisfactory' outcomes of surgery were reported in nearly all participants.
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8 CFR 1236.4 - Removal of S-5, S-6, and S-7 nonimmigrants.
Code of Federal Regulations, 2010 CFR
2010-01-01
... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Removal of S-5, S-6, and S-7 nonimmigrants. 1236.4 Section 1236.4 Aliens and Nationality EXECUTIVE OFFICE FOR IMMIGRATION REVIEW, DEPARTMENT OF... OF ALIENS ORDERED REMOVED Detention of Aliens Prior to Order of Removal § 1236.4 Removal of S-5, S-6...
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Alanine and glycine conjugates of (2S,4R)-4-[18F]fluoroglutamine for tumor imaging.
Zha, Zhihao; Ploessl, Karl; Lieberman, Brian P; Wang, Limin; Kung, Hank F
2018-05-01
Glutamine is an essential source of energy, metabolic substrates, and building block for supporting tumor proliferation. Previously, (2S,4R)-4-[ 18 F]fluoroglutamine (4F-Gln) was reported as a glutamine-related metabolic imaging agent. To improve the in vivo kinetics of this radiotracer, two new dipeptides, [ 18 F]Gly-(2S,4R)4-fluoroglutamine (Gly-4F-Gln) and [ 18 F]Ala-(2S,4R)4-fluoroglutamine (Ala-4F-Gln) were investigated. Radiolabeling was performed via 2-steps 18 F-fluorination. Cell uptake studies of Gly-4F-Gln and Ala-4F-Gln were investigated in 9 L cell lines. In vitro and in vivo metabolism studies were carried out in Fisher 344 rats. Biodistribution and microPET imaging studies were performed in 9 L tumor-bearing rats. In vitro incubation of these [ 18 F]dipeptides in rat and human blood showed a rapid conversion to (2S,4R)-4-[ 18 F]fluoroglutamine (t 1/2 = 2.3 and 0.2 min for [ 18 F]Gly-4F-Gln and [ 18 F]Ala-4F-Gln, respectively for human blood). Biodistribution and PET imaging in Fisher 344 rats bearing 9 L tumor xenografts showed that these dipeptides rapidly localized in the tumors, comparable to that of (2S,4R)-4-[ 18 F]fluoroglutamine (4F-Gln). The results support that these dipeptides, [ 18 F]Gly-4F-Gln and [ 18 F]Ala-4F-Gln, are prodrugs, which hydrolyze in the blood after an iv injection. They appear to be selectively taken up and trapped by tumor tissue in vivo. The dipeptide, [ 18 F]Ala-4F-Gln, may be suitable as a PET tracer for imaging glutaminolysis in tumors. Copyright © 2018 Elsevier Inc. All rights reserved.
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Zanello, Piero
2018-06-01
In the context of the plethora of proteins harboring iron-sulfur clusters we have already reviewed structure/electrochemistry of metalloproteins expressing single types of iron-sulfur clusters (namely: {Fe(Cys) 4 }, {[Fe 2 S 2 ](Cys) 4 }, {[Fe 2 S 2 ](Cys) 3 (X)} (X = Asp, Arg, His), {[Fe 2 S 2 ](Cys) 2 (His) 2 }, {[Fe 3 S 4 ](Cys) 3 }, {[Fe 4 S 4 ](Cys) 4 } and {[Fe 4 S 4 ](S γ Cys ) 3 (nonthiolate ligand)} cores) and their synthetic analogs. More recently we are focussing on structure/electrochemistry of metalloproteins harboring iron-sulfur centres of different nuclearities. Having started such a subject with proteins harboring [4Fe-4S] and [2Fe-2S] clusters, we now depict the state of art of proteins containing [4Fe-4S] and [3Fe-4S] clusters. Copyright © 2018 Elsevier Inc. All rights reserved.
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Kalani, Amir; Thomas, Nithin; Sacerdote, Alan; Bahtiyar, Gül
2013-03-18
Non-classic adrenal hyperplasia (NCAH) has been associated with insulin resistance (IR). Therapies such as metformin, thiazolidinediones and lifestyle alterations improve IR and also ameliorate the biochemical and clinical abnormalities of NCAH, much as they do in polycystic ovarian syndrome (PCOS). More recently, bariatric surgery, such as Roux-en-Y gastric bypass (RYGBP), has also been associated with improvement in IR and amelioration of PCOS and may, therefore, be beneficial in NCAH. We report a case of a 39-year-old, deaf-mute, obese woman with NCAH due to 11-hydroxylase deficiency who underwent RYGBP followed by improvement of NCAH manifestations. She was initially treated with metformin and pioglitazone, which lowered serum 11-deoxycortisol from 198 ng/dl (<51) to 26 ng/dl. Five weeks after undergoing RYGBP her body mass index fell from 44.18 kg/m(2) to 39.54 kg/m(2) and, despite not taking metformin or pioglitazone, serum 11-deoxycortisol remained normal at <40 ng/dl. Concurrently and subsequently, her NCAH symptoms, for example, alopecia, hirsutism and irregular menses normalised as well. We conclude that RYGBP, like other interventions that reduce IR, may be another way of treating non-classic 11-hydroxylase deficiency in selected patients.
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Liu, Shi; Zainuddin, Ima M; Vanderschuren, Herve; Doughty, James; Beeching, John R
2017-05-01
Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Vorob'ev, Yu.I.; Velikova, N.G.; Kirilenko, V.V.
1987-12-01
Using DTA and XPA methods, microstructural investigations, and microhardness measurements, phase diagrams of the quasibinary sections As/sub 2/S/sub 3/-Tl/sub 3/AsS/sub 4/, Tl/sub 3/AsS/sub 4/-S, and Tl/sub 3/AsS/sub 4/-Tl/sub 2/S, are characterized by five ternary compounds Tl/sub 3/As/sub 5/S/sub 10/, Tl/sub 9/As/sub 5/S/sub 15/, Tl/sub 9/As/sub 3/S/sub 13/, Tl/sub 3/AsS/sub 6/, and Tl/sub 8/As/sub 2/S/sub 9/, which decompose by peritectic reactions at 198, 307, 408, 362, and 318/degree/C, respectively. Interplanar spacings and line intensities are given for the detected compounds. Glass formation is considered in the Tl-As-S system.
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Space shuttle/food system study. Package feasibility study, modifications 3S, 4C and 5S
NASA Technical Reports Server (NTRS)
1974-01-01
An optimum feeding system for the space shuttle was presented. This system consisted of all rehydratable type foods which were enclosed in a 4 in. x 4 in. x 1 in. flexible package. A feasibility follow-on study was conducted, and two acceptable, feasible prototypes for this package are described.
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Hannemann, Frank; Virus, Cornelia; Bernhardt, Rita
2006-06-25
The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.
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Predicting the onset of Addison's disease: ACTH, renin, cortisol and 21-hydroxylase autoantibodies
Baker, Peter R.; Nanduri, Priyaanka; Gottlieb, Peter A.; Yu, Liping; Klingensmith, Georgeanna J.; Eisenbarth, George S.; Barker, Jennifer M.
2016-01-01
Summary Context Autoantibodies to 21-hydroxylase (21OH-AA) precede onset of autoimmune Addison's disease (AD). Progression to AD can take months to years, and early detection of metabolic decompensation may prevent morbidity and mortality. Objective To define optimal methods of predicting progression to overt AD (defined by subnormal peak cortisol response to Cosyntropin) in 21OH-AA+ individuals. Design, Setting and Participants Individuals were screened for 21OH-AA at the Barbara Davis Center from 1993 to 2011. Subjects positive for 21OH-AA (n = 87) were tested, and the majority prospectively followed for the development of Addison's disease, including seven diagnosed with AD upon 21OH-AA discovery (discovered), seven who progressed to AD (progressors) and 73 nonprogressors. Main Outcome Measured Plasma renin activity (PRA), ACTH, baseline cortisol, peak cortisol and 21OH-AA were measured at various time points relative to diagnosis of AD or last AD-free follow-up. Results Compared with nonprogressors, in the time period 2 months–2 years prior to the onset of AD, progressors were significantly more likely to have elevated ACTH (11–22 pm, P < 1E-4), with no significant differences in mean PRA (P = 0·07) or baseline cortisol (P = 0·08), and significant but less distinct differences seen with 21OH-AA levels (P < 1E-4) and poststimulation cortisol levels (P = 6E-3). Conclusion Moderately elevated ACTH is a more useful early indicator of impending AD than 21OH-AA, PRA or peak cortisol, in the 2 months–2 years preceding the onset of AD. PMID:22066755
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Thórólfsson, Matthías; Ibarra-Molero, Beatriz; Fojan, Peter; Petersen, Steffen B; Sanchez-Ruiz, Jose M; Martínez, Aurora
2002-06-18
Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii
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Functional Implication of β-Carotene Hydroxylases in Soybean Nodulation1[C][W][OA
Kim, Yun-Kyoung; Kim, Sunghan; Um, Ji-Hyun; Kim, Kyunga; Choi, Sun-Kang; Um, Byung-Hun; Kang, Suk-Woo; Kim, Jee-Woong; Takaichi, Shinichi; Song, Seok-Bo; Lee, Choon-Hwan; Kim, Ho-Seung; Kim, Ki Woo; Nam, Kyoung Hee; Lee, Suk-Ha; Kim, Yul-Ho; Park, Hyang-Mi; Ha, Sun-Hwa; Verma, Desh Pal S.; Cheon, Choong-Ill
2013-01-01
Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative β-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation. PMID:23700351
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Xiao, Yuming; Koutmos, Markos; Case, David A; Coucouvanis, Dimitri; Wang, Hongxin; Cramer, Stephen P
2006-05-14
We have used four vibrational spectroscopies--FT-IR, FT-Raman, resonance Raman, and 57Fe nuclear resonance vibrational spectroscopy (NRVS)--to study the normal modes of the Fe-S cluster in [(n-Bu)4N]2[Fe4S4(SPh)4]. This [Fe4S4(SR)4]2- complex serves as a model for the clusters in 4Fe ferredoxins and high-potential iron proteins (HiPIPs). The IR spectra exhibited differences above and below the 243 K phase transition. Significant shifts with 36S substitution into the bridging S positions were also observed. The NRVS results were in good agreement with the low temperature data from the conventional spectroscopies. The NRVS spectra were interpreted by normal mode analysis using optimized Urey-Bradley force fields (UBFF) as well as from DFT theory. For the UBFF calculations, the parameters were refined by comparing calculated and observed NRVS frequencies and intensities. The frequency shifts after 36S substitution were used as an additional constraint. A D 2d symmetry Fe4S4S'4 model could explain most of the observed frequencies, but a better match to the observed intensities was obtained when the ligand aromatic rings were included for a D 2d Fe4S4(SPh)4 model. The best results were obtained using the low temperature structure without symmetry constraints. In addition to stretching and bending vibrations, low frequency modes between approximately 50 and 100 cm(-1) were observed. These modes, which have not been seen before, are interpreted as twisting motions with opposing sides of the cube rotating in opposite directions. In contrast with a recent paper on a related Fe4S4 cluster, we find no need to assign a large fraction of the low frequency NRVS intensity to 'rotational lattice modes'. We also reassign the 430 cm(-1) band as primarily an elongation of the thiophenolate ring, with approximately 10% terminal Fe-S stretch character. This study illustrates the benefits of combining NRVS with conventional Raman and IR analysis for characterization of Fe-S centers. DFT
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Bulk superconductivity in bismuth oxysulfide Bi4O4S3.
Singh, Shiva Kumar; Kumar, Anuj; Gahtori, Bhasker; Shruti; Sharma, Gyaneshwar; Patnaik, Satyabrata; Awana, Veer P S
2012-10-10
A very recent report on the observation of superconductivity in Bi(4)O(4)S(3) [Mizuguchi, Y.; http://arxiv.org/abs/1207.3145] could potentially reignite the search for superconductivity in a broad range of layered sulfides. We report here the synthesis of Bi(4)O(4)S(3) at 500 °C by a vacuum encapsulation technique and its basic characterizations. The as-synthesized Bi(4)O(4)S(3) was contaminated with small amounts of Bi(2)S(3) and Bi impurities. The majority phase was found to be tetragonal (space group I4/mmm) with lattice parameters a = 3.9697(2) Å and c = 41.3520(1) Å. Both AC and DC magnetization measurements confirmed that Bi(4)O(4)S(3) is a bulk superconductor with a superconducting transition temperature (T(c)) of 4.4 K. Isothermal magnetization (M-H) measurements indicated closed loops with clear signatures of flux pinning and irreversible behavior. The lower critical field (H(c1)) at 2 K for the new superconductor was found to be ~15 Oe. Magnetotransport measurements showed a broadening of the resistivity (ρ) and a decrease in T(c) (ρ = 0) with increasing magnetic field. The extrapolated upper critical field H(c2)(0) was ~31 kOe with a corresponding Ginzburg-Landau coherence length of ~100 Å . In the normal state, the ρ ~ T(2) dependence was not indicated. Hall resistivity data showed a nonlinear magnetic field dependence. Our magnetization and electrical transport measurements substantiate the appearance of bulk superconductivity in as-synthesized Bi(4)O(4)S(3). On the other hand, Bi heat-treated at the same temperature is not superconducting, thus excluding the possibility of impurity-driven superconductivity in the newly discovered superconductor Bi(4)O(4)S(3).
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Sakai, R; Esaki, Y; Hasuwa, H; Ikawa, M; Lo, P; Matsuura, R; Nakahata, K; Zenitani, M; Asada, M; Maeda, A; Eguchi, H; Okuyama, H; Miyagawa, S
2016-05-01
We attempted to knock out the expression of Hanganutziu-Deicher (H-D) antigens through the use of a CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9 system for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Plasmids expressing hCas9 and sgRNA for pCMAH were prepared by ligating oligos into the BbsI site of pX330. The N-terminal and C-terminal EGFP coding regions overlapping 482 bp were PCR-amplified and placed under a ubiquitous CAG promoter. The approximately 400-bp genomic fragments containing the sgRNA target sequence of pCMAH were placed into the multi-cloning sites flanked by the EGFP fragments. The pCAG-EGxxFP-target was mixed with pX330 with/without the sgRNA sequences and then introduced into HEK293T cells. Four oligos and primers, gSO1, gSO3, gSO4, and gSO8, were nominated from 8 candidates. Among them, gSO1 showed the best efficiency. Pig endothelial cells (PECs) from an α-Gal knockout pig were then used to examine the changes in the expression of the H-D antigen by the knockout of the CMAH genome by the pX330-gS01. Changes in the expression of the H-D antigen in the PECs with the CRISPR (gS01) were clear in comparison with those in the parental cells, on the basis of FACS analysis data. The expression of the H-D antigen can be knocked out by use of the CRISPR system for pCMAH, thus confirming that this system is a very convenient system for producing knockout pigs. Copyright © 2016 Elsevier Inc. All rights reserved.
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Israel, A S; Barbella, Y R; Cubeddu, L X
1982-06-01
The effect of acute stresses on plasma norepinephrine, epinephrine and dopamine-beta-hydroxylase (DBH) were evaluated in control and 6-hydroxydopamine-treated, awake cannulated guinea pigs. Forced immobolization for 1 hr caused a 3- and 5-fold increase in plasma DBH and norepinephrine, respectively. Pretreatment with 6-hydroxydopamine (23 mg/kg b.wt.i.a., 72 and 48 hr before stress) reduced by 70% the increase in plasma DBH and totally prevented the rise in plasma catecholamines evoked by the restraining stress. Injection of insulin (5 U/kg b.wt.i.a.) induced a 60% decrease in blood glucose, a 1-fold increase in plasma DBH and a selective 4-fold increase in plasma epinephrine; these effects were not modified by chemical sympathectomy. Our results indicate that forced immobilization and hypoglycemia produce a preferential activation of the sympathetic postganglionic nerves and of the adrenal medulla, respectively, and that in guinea pigs both stresses increase plasma DBH. The kinetics of disappearance of plasma DBH were studied after subjecting the guinea pigs for 1 hr to forced immobilization. Although 7 of 12 animals showed a biphasic rate of fall of plasma DBH, in each case there was a rapid initial fall possibly due to the "distribution" of the enzyme with a T1/2 of 1.65 hr. Similar findings were observed in 6-hydroxydopamine-treated guinea pigs. These results suggest that the distribution of DBH is the most important process in reducing the augmented plasma DBH levels elicited by a short-term stress and that this process is not dependent on the integrity of the sympathetic nerves nor on the adrenal or sympathetic origin of the enzyme. This study supports the view that the ratio, content of releasable DBH present in sympathetic nerves and adrenal glands/total circulating pool of DBH, is the factor that determines whether an increase in plasma DBH would occur in animals exposed to an acute stress.
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Synthesis, Structure, and Characterization of Cu4S10(4-methylpyridine)4
NASA Technical Reports Server (NTRS)
Hepp, Aloysius F.; Richman, Robert M.; Duraj, Stan A.; Andras, Maria T.; Moore, Hall L.; Sabat, Michal; Eckles, William E.; Martuch, Robert A.
1996-01-01
The title compound, Cu4S10(4-methylpyridine)(sub 4) (dot) 4-methylpyridine was prepared by three different reactions: the oxidation of copper powder by sulfur and the reaction of copper (I) sulfide (or CuBr (dot) SMe2) with excess sulfur, both in the coordinating solvent, 4-methylpyridine. Red crystals of the compound obtained by layering with hexanes were subjected to single crystal X-ray diffraction. The structure was refined to R = 0.026 and R(sub w) = 0.036 in a space group P1bar (No. 2), with Z = 2, a = 13.983 (2) A, b = 15.384 (2) A, c = 9.660 (1) A, alpha = 93.87 (1)deg., beta = 93.38 (1)deg., gamma = 99.78 (1)deg., V = 2037.9 (9) A(exp 3). The compound has approximate S(sub 4) symmetry and consists of two pentasulfide chains linking four Cu(I) ions, each with a corrdinating 2-methylpyridine. The infrared spectrum was dominated by absorption due to coordinated 4-methylpyridine with several low-energy peaks attributable to S-S stretches, which were also observed by Raman spectroscopy. A featureless electronic absorption spectrum yielded a single peak in the near ultraviolet upon computer enhancement (lambda = 334 nm, epsilon = 10,000), most likely an intraligand transition. Cyclic voltammetry indicates that the polysulfide complex undergoes irrversible oxidation and reduction at +0.04 and -0.34 V vs. SCE, respectively, at 298 K in 4-methylpyridine when swept at 20 mV/sec. The electrochemical behavior was unvaried even at sweep rates as high as 100 V/sec.
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Szymanski, Pawel T; Ahmed, Safwat A; Radwan, Mohamed M; Khalifa, Sherief I; Fahmy, Hesham
2014-08-01
Three natural cembranoids from the Red Sea soft coral Sarcophyton glaucum namely (1S,2E,4R,6E,8R,11S,12R)-8,12-epoxy-2,6-cembradiene-4,11-diol, (1S,2E,4R,6E,8S,11R,12S)-8,11-epoxy-4,12-epoxy-2,6-cembradiene and (1S,4R,13S)-cembra-2E,7E,11E-trien-4,13-diol were evaluated for their inhibitory effects on mouse melanoma B16F10 cell growth. Results show that all the cembranoids strongly inhibit viability of melanoma cells particularly during 48 -72 hrs treatment and also inhibit de novo DNA synthesis and PARP activity and stimulate fragmentation of DNA. (1S,2E,4R,6E,8R,11S,12R)-8,12-epoxy-2,6-cembradiene-4,11-diol was not cytotoxic to monkey kidney CV-1 cells at the concentration that produces the anti-melanoma effects which indicates that this compound may be a good candidate for further development. (1S,2E,4R,6E,8S,11R,12S)-8,11-epoxy-4,12-epoxy-2,6-cembradiene and (1S,4R,13S)-cembra-2E,7E,11E-trien-4,13-diol were found to be cytotoxic to healthy cells.
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Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma
Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S.; Watanabe, Takeshi; Kim, Min P.; Blackmon, Shanda H.; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I.; Minelli, Rosalba; Scott, Kenneth L.; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R.; Creighton, Chad J.; Martinez, Elisabeth D.; Zal, Tomasz; Grande-Allen, K. Jane; Yamauchi, Mitsuo; Kurie, Jonathan M.
2015-01-01
Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. PMID:25664850
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Al Suwaidi, Hana; Attia, Salima; Al Ameri, Ahlam
2015-01-01
Summary Combined17α-hydroxylase/17,20-lyase deficiency is a rare cause of congenital adrenal hyperplasia and hypogonadism. Hypertension and hypokalemia are essential presenting features. We report an Arab family with four affected XX siblings. The eldest presented with abdominal pain and was diagnosed with a retroperitoneal malignant mixed germ cell tumour. She was hypertensive and hypogonadal. One sibling presented with headache due to hypertension while the other two siblings were diagnosed with hypertension on a routine school check. A homozygous R96Q missense mutation in P450c17 was detected in the index case who had primary amenorrhea and lack of secondary sexual characters at 17 years. The middle two siblings were identical twins and had no secondary sexual characters at the age of 14. All siblings had hypokalemia, very low level of adrenal androgens, high ACTH and high levels of aldosterone substrates. Treatment was commenced with steroid replacement and puberty induction with estradiol. The index case had surgical tumor resection and chemotherapy. All siblings required antihypertensive treatment and the oldest remained on two antihypertensive medications 12 years after diagnosis. Her breast development remained poor despite adequate hormonal replacement. Combined 17α-hydroxylase/17,20-lyase deficiency is a rare condition but might be underdiagnosed. It should be considered in young patients presenting with hypertension, particularly if there is a family history of consanguinity and with more than one affected sibling. Antihypertensive medication might continue to be required despite adequate steroid replacement. Breast development may remain poor in mutations causing complete form of the disease. Learning points Endocrine hypertension due to rarer forms of CAH should be considered in children and adolescents, particularly if more than one sibling is affected and in the presence of consanguinity. 17α-hydroxylase/17,20-lyase deficiency is a rare form of
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Yasutake, Yoshiaki; Fujii, Yoshikazu; Cheon, Woo-Kwang; Arisawa, Akira; Tamura, Tomohiro
2009-01-01
Vitamin D3 hydroxylase (Vdh) is a novel cytochrome P450 monooxygenase isolated from the actinomycete Pseudonocardia autotrophica and consisting of 403 amino-acid residues. Vdh catalyzes the activation of vitamin D3 via sequential hydroxylation reactions: these reactions involve the conversion of vitamin D3 (cholecalciferol or VD3) to 25-hydroxyvitamin D3 [25(OH)VD3] and the subsequent conversion of 25(OH)VD3 to 1α,25-dihydroxyvitamin D3 [calciferol or 1α,25(OH)2VD3]. Overexpression of recombinant Vdh was carried out using a Rhodococcus erythropolis expression system and the protein was subsequently purified and crystallized. Two different crystal forms were obtained by the hanging-drop vapour-diffusion method at 293 K using polyethylene glycol as a precipitant. The form I crystal belonged to the trigonal space group P31, with unit-cell parameters a = b = 61.7, c = 98.8 Å. There is one Vdh molecule in the asymmetric unit, with a solvent content of 47.6%. The form II crystal was grown in the presence of 25(OH)VD3 and belonged to the orthorhombic system P212121, with unit-cell parameters a = 63.4, b = 65.6 c = 102.2 Å. There is one Vdh molecule in the asymmetric unit, with a solvent content of 46.7%. Native data sets were collected to resolutions of 1.75 and 3.05 Å for form I and form II crystals, respectively, using synchrotron radiation. The structure solution was obtained by the molecular-replacement method and model refinement is in progress for the form I crystal. PMID:19342783
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Korenev, Vladimir S; Floquet, Sébastien; Marrot, Jérôme; Haouas, Mohamed; Mbomekallé, Israël-Martyr; Taulelle, Francis; Sokolov, Maxim N; Fedin, Vladimir P; Cadot, Emmanuel
2012-02-20
Reaction of the cyclic lacunary [H(7)P(8)W(48)O(184)](33-) anion (noted P(8)W(48)) with the [Mo(2)S(2)O(2)(H(2)O)(6)](2+) oxothiocation led to two compounds, namely, [K(4){Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(2)(WO(2))(P(8)W(48)O(184))](30-) (denoted 1) and [{Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(2)(P(8)W(48)O(184))](36-) (denoted 2), which were characterized in the solid state and solution. In the solid state, the structure of [K(4){Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(2)(WO(2))(P(8)W(48)O(184))](30-) reveals the presence of two disordered {Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(2+) "handles" connected on both sides of the P(8)W(48) ring. Such a disorder is consistent with the presence of two geometrical isomers where the relative disposition of the two {Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(2+) handles are arranged in a perpendicular or parallel mode. Such an interpretation is fully supported by (31)P and (183)W NMR solution studies. The relative stability of both geometrical isomers appears to be dependent upon the nature of the internal alkali cations, i.e., Na(+) vs K(+), and increased lability of the two {Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(2+) handles, compared to the oxo analogous, was clearly identified by significant broadening of the (31)P and (183)W NMR lines. Solution studies carried out by UV-vis spectroscopy showed that formation of the adduct [{Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(2)(P(8)W(48)O(184))](36-) occurs in the 1.5-4.7 pH range and corresponds to a fast and quantitative condensation process. Furthermore, (31)P NMR titrations in solution reveal formation of the "monohandle" derivative [{Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(P(8)W(48)O(184))](38-) as an intermediate prior to formation of the "bishandle" derivatives. Furthermore, the electrochemical behavior of [{Mo(4)O(4)S(4)(H(2)O)(3)(OH)(2)}(2)(P(8)W(48)O(184))](36-) was studied in aqueous medium and compared with the parent anion P(8)W(48).
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mei Luo, E-mail: Lmhh5523385@yahoo.cn; Hai Zhangjia; Hao Yin
2010-12-15
Two oxazolines compound 1a and 1b, 2-[4(S)-4,5-dihydro-4-phenyl-2-ozazolinyl-benzenamine, and (C{sub 15}H{sub 14}N{sub 2}O), 2-[4(S)-4,5-dihydro-4-benzyl-2-ozazolinyl-benzenamine (C{sub 16}H{sub 16}N{sub 2}O) were obtained in moderate yield from the reactions of 2-aminobenzonitrile with optically active L-(+)-Phenylglycinol and L-(+)-Phenylalaninol, respectively, in chlorobenzene under dry, anaerobic conditions. ZnCl{sub 2} was dried under vacuum and acted as a Lewis acid catalyst in this reaction. The structures of 1a and 1b were determined by X-ray diffraction and NMR. There exist intra- and intermolecular N-H-N hydrogen bonds in the crystal structure.
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EPA’s SPECIATE 4.4 Database:Development and Uses
SPECIATE is the U.S. Environmental Protection Agency's (EPA)repository of volatile organic gas and particulate matter (PM) speciation profiles for air pollution sources. EPA released SPECIATE 4.4 in early 2014 and, in total, the SPECIATE 4.4 database includes 5,728 PM, VOC, total...
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EPA’s SPECIATE 4.4 Database: Development and Uses
SPECIATE is the U.S. Environmental Protection Agency's (EPA)repository of volatile organic gas and particulate matter (PM) speciation profiles for air pollution sources. EPA released SPECIATE 4.4 in early 2014 and, in total, the SPECIATE 4.4 database includes 5,728 PM, VOC, total...
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Nakano, Masashi; Kino, Kuniki
2015-01-01
We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA. PMID:25795668
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Assessment of deoxyhypusine hydroxylase as a putative, novel drug target.
Kerscher, B; Nzukou, E; Kaiser, A
2010-02-01
Antimalarial drug resistance has nowadays reached each drug class on the market for longer than 10 years. The focus on validated, classical targets has severe drawbacks. If resistance is arising or already present in the field, a target-based High-Throughput-Screening (HTS) with the respective target involves the risk of identifying compounds to which field populations are also resistant. Thus, it appears that a rewarding albeit demanding challenge for target-based drug discovery is to identify novel drug targets. In the search for new targets for antimalarials, we have investigated the biosynthesis of hypusine, present in eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine hydroxylase (DOHH), which has recently been cloned and expressed from P. falciparum, completes the modification of eIF5A through hydroxylation. Here, we assess the present druggable data on Plasmodium DOHH and its human counterpart. Plasmodium DOHH arose from a cyanobacterial phycobilin lyase by loss of function. It has a low FASTA score of 27 to its human counterpart. The HEAT-like repeats present in the parasite DOHH differ in number and amino acid identity from its human ortholog and might be of considerable interest for inhibitor design.
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Selvaraju, Vaithinathan; Parinandi, Narasimham L.; Adluri, Ram Sudheer; Goldman, Joshua W.; Hussain, Naveed; Sanchez, Juan A.
2014-01-01
Abstract Significance: In this review, we have discussed the efficacy and effect of small molecules that act as prolyl hydroxylase domain inhibitors (PHDIs). The use of these compounds causes upregulation of the pro-angiogenic factors and hypoxia inducible factor-1α and -2α (HIF-1α and HIF-2α) to enhance angiogenic, glycolytic, erythropoietic, and anti-apoptotic pathways in the treatment of various ischemic diseases responsible for significant morbidity and mortality in humans. Recent Advances: Sprouting of new blood vessels from the existing vasculature and surgical intervention, such as coronary bypass and stent insertion, have been shown to be effective in attenuating ischemia. However, the initial reentry of oxygen leads to the formation of reactive oxygen species that cause oxidative stress and result in ischemia/reperfusion (IR) injury. This apparent “oxygen paradox” must be resolved to combat IR injury. During hypoxia, decreased activity of PHDs initiates the accumulation and activation of HIF-1α, wherein the modulation of both PHD and HIF-1α appears as promising therapeutic targets for the pharmacological treatment of ischemic diseases. Critical Issues: Research on PHDs and HIFs has shown that these molecules can serve as therapeutic targets for ischemic diseases by modulating glycolysis, erythropoiesis, apoptosis, and angiogenesis. Efforts are underway to identify and synthesize safer small-molecule inhibitors of PHDs that can be administered in vivo as therapy against ischemic diseases. Future Directions: This review presents a comprehensive and current account of the existing small-molecule PHDIs and their use in the treatment of ischemic diseases with a focus on the molecular mechanisms of therapeutic action in animal models. Antioxid. Redox Signal. 20, 2631–2665. PMID:23992027
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Jensen, Kasper P; Ooi, Bee-Lean; Christensen, Hans E M
2008-12-18
The aim of this work is to understand the molecular evolution of iron-sulfur clusters in terms of electronic structure and function. Metal-substituted models of biological [Fe(4)S(4)] clusters in oxidation states [M(x)Fe(4-x)S(4)](3+/2+/1+) have been studied by density functional theory (M = Cr, Mn, Fe, Co, Ni, Cu, Zn, and Pd, with x = 1 or 2). Most of these clusters have not been characterized before. For those that have been characterized experimentally, very good agreement is obtained, implying that also the predicted structures and properties of new clusters are accurate. Mean absolute errors are 0.024 A for bond lengths ([Fe(4)S(4)], [NiFe(3)S(4)], [CoFe(3)S(4)]) and 0.09 V for shifts in reduction potentials relative to the [Fe(4)S(4)] cluster. All structures form cuboidal geometries similar to the all-iron clusters, except the Pd-substituted clusters, which instead form highly distorted trigonal and tetragonal local sites in compromised, pseudocuboidal geometries. In contrast to other electron-transfer sites, cytochromes, blue copper proteins, and smaller iron-sulfur clusters, we find that the [Fe(4)S(4)] clusters are very insensitive to metal substitution, displaying quite small changes in reorganization energies and reduction potentials upon substitution. Thus, the [Fe(4)S(4)] clusters have an evolutionary advantage in being robust to pollution from other metals, still retaining function. We analyze in detail the electronic structure of individual clusters and rationalize spin couplings and redox activity. Often, several configurations are very close in energy, implying possible use as spin-crossover systems, and spin states are predicted accurately in all but one case ([CuFe(3)S(4)]). The results are anticipated to be helpful in defining new molecular systems with catalytic and magnetic properties.
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de Sousa, B G; Oliveira, J I N; Albuquerque, E L; Fulco, U L; Amaro, V E; Blaha, C A G
2017-10-01
Many species of bacteria involved in degradation of n-alkanes have an important constitutional metabolic enzyme, the alkane hydroxylase called AlkB, specialized in the conversion of hydrocarbons molecules that can be used as carbon and/or energy source. This enzyme plays an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. A number of these enzymes has been biochemically characterized in detail because the potential of alkane hydroxylases to catalyse high added-value reactions is widely recognized. Nevertheless, the industrial and process bioremediation application of them is restricted, owing to their complex biochemistry, challenging process requirements, and the limited number of their three-dimensional structures. Furthermore, AlkB has great potential as biocatalysts for selective transformation of a wide range of chemically inert unreactive alkanes into reactive chemical precursors that can be used as tools for bioremediation and bioprocesses. Aiming to understand the possible ways the AlkB enzyme Pseudomonas putida P1 interacts with octane, octanol and 1-octyne, we consider its suitable biochemical structure taking into account a 3-D homology modelling. Besides, by using a quantum chemistry computational model based on the density functional theory (DFT), we determine possible protein-substrate interaction regions measured by means of its binding energy simulated throughout the Molecular Fractionation with Conjugated Caps (MFCC) approach. Copyright © 2017 Elsevier Inc. All rights reserved.
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Tarfarosh, Shah Faisal Ahmad; Dar, Mohammad Maqbool; Hussain, Arshad; Shoib, Sheikh; Shah, Tabindah; Shah, Sahil; Manzoor, Mushbiq
2016-01-01
Background The progress that man has made in all domains of life, during all these years of reign over the earth, is utterly remarkable. However, it always came at a price. Each epoch of progress has seen human beings inflicted with trauma and cynical consequences. During the last two decades, Kashmiri (Indian) people have experienced continuous violence, a reign of terror, and political turmoil. Each of these disastrous events has contributed to the increase in psychiatric disorders in this part of the world, especially major depressive disorders. We can observe that besides the environmental influences, gene polymorphism also plays a crucial role in the development of depressive disorders. The role of Tryptophan Hydroxylase 1 (TPH1) gene is implicated in various psychiatric disorders, including depression. However, no study has investigated TPH1 A779C gene polymorphism in depressive disorders in a distressed society like Kashmir (India). Aims To study TPH1 A779C single nucleotide polymorphism in depressive disorders in Kashmiri (Indian) population. Materials and Methods Two hundred and forty patients diagnosed with depressive disorder, and 160 unrelated healthy volunteers (control), were studied in a case-control study design. Polymorphism was determined using polymerase chain reaction (PCR) and agarose gel electrophoresis, after digestion with HAP II enzyme. Genotypes and allele frequencies were compared using Chi-square tests, Fisher’s exact test, odds ratio, 95% confidence interval (C.I.) and a p-value of <0.05 was considered to be statistically significant. Results The mean age ± standard deviation (SD) of depression and control group was 32.02±10.99 and 31.75±9.93, respectively (p= 0.512). It was found that the patients from depression group had AA genotype (51.7%) in comparison to control group (17.5%) and these results were statistically significant (p≤0.0001). Calculation of allelic frequency revealed a stronger association of A allele with
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Mahajan, Monika; Yadav, Sudesh Kumar
2014-08-01
Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Jokilehto, Terhi; Turku Graduate School of Biomedical Sciences, Turku; Hoegel, Heidi
2010-04-15
Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1{alpha} and -2{alpha}). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells.more » The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1{alpha} or -2{alpha} expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.« less
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Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.
Nagatsu, T; Oka, K; Kato, T
1979-07-21
A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.
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Capyk, Jenna K.; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C.; Eltis, Lindsay D.
2011-01-01
Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (kcat/Km) of KshAB for the CoA thioester substrates was 20–30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent KmO2 was 90 ± 10 μm in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ1 ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism. PMID:21987574
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Capyk, Jenna K; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C; Eltis, Lindsay D
2011-11-25
Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (k(cat)/K(m)) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent K(m)(O(2)) was 90 ± 10 μM in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ(1) ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.
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Yeung, John H K; Or, Penelope M Y
2011-10-15
Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited CYP2C11-mediated tolbutamide 4-hydroxylation in the rat both in vitro and in vivo. In this study, the effects of water extractable fraction of PSP on tolbutamide 4-hydroxylation was investigated in pooled human liver microsomes and in specific human CYP2C9 isoform. PSP (2.5-20μM) dose-dependently decreased the biotransformation of tolbutamide to 4-hydroxy-tolbutamide. Enzyme kinetics studies showed inhibition of tolbutamide 4-hydroxylase activity was competitive and concentration-dependent. In pooled human liver microsomes, PSP had a K(i) value of 14.2μM compared to sulfaphenazole, a human CYP2C9 inhibitor, showed a K(i) value of 0.32μM. In human CYP2C9 isoform, the K(i) value of PSP was 29.5μM and the K(i) value of sulfaphenazole was 0.04μM. This study demonstrated that PSP can competitively inhibit tolbutamide 4-hydroxylation in both pooled human liver microsomes and specific human CYP2C9 in vitro. This study compliments previous findings in the rat that PSP can inhibit human tolbutamide 4-hydroxylase, but the relatively high K(i) values in human CYP2C9 would suggest a low potential for PSP to cause herb-drug interaction. Copyright © 2011 Elsevier GmbH. All rights reserved.
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López-Gutiérrez, A U; Riba, L; Ordoñez-Sánchez, M L; Ramírez-Jiménez, S; Cerrillo-Hinojosa, M; Tusié-Luna, M T
1998-01-01
Congenital adrenal hyperplasia (CAH) is an inherited recessive disorder of adrenal steroidogenesis caused by mutations in the steroid 21-hydroxylase gene (CYP21) in more than 90% of affected patients. The CYP21 gene is located within the HLA complex locus on chromosome 6 (6p21.3). During a molecular characterisation study of a group of 47 Mexican families with 21-hydroxylase deficiency, we identified nine in which the mutation or mutations found in the patient did not appear to originate from one of the parents. Through DNA fingerprinting, paternity was established in all nine families with a probability of non-paternity in the range of 10(-19) to 10(-23). Among these families, we identified one patient with exclusive paternal inheritance of all eight markers tested on chromosome 6p, despite normal maternal and paternal contributions for eight additional markers on three different chromosomes. We did not identify duplication of paternal information for markers in the 6q region, consistent with lack of expression of transient neonatal diabetes owing to genomic imprinting in this patient. Our results substantiate evidence for the existence of different genetic mechanisms involved in the expression of this recessive condition in a substantial portion (approximately 19%) of affected Mexican families. In addition to the identification of a patient with paternal uniparental disomy, the occurrence of germline mutations may explain the unusual pattern of segregation in the majority of the remaining eight families. PMID:9863599
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Eslavath, Ravi Kumar; Sharma, Deepshikha; Bin Omar, Nabil A M; Chikati, Rajasekhar; Teli, Mahesh Kumar; Rajanikant, G K; Singh, Surya S
2016-11-15
Hypoxia inducible factor (HIF)-1α, a subunit of HIF transcription factor, regulates cellular response to hypoxia. In normoxic conditions, it is hydroxylated by prolyl hydroxylase (PHD)-2 and targeted for proteosomal degradation. Drugs which inhibit PHD-2 have implications in conditions arising from insufficient blood supply. β-ODAP (β-N- oxalyl-L-α, β- diaminopropionic acid), a non-protein excitatory amino acid present in Lathyrus sativus, is an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor agonist known to activate conventional protein kinase C and stabilize HIF-1α under normoxic conditions. However, the mechanism of HIF-1α stabilization by this compound is unknown. In silico approach was used to understand the mechanism of stabilization of HIF-1α which revealed β-ODAP interacts with key amino acid residues and Fe 2+ at the catalytic site of PHD-2. These results were further corroborated with luciferase HRE (hypoxia response element) reporter system in HeLa cells. Different chemical modulators of PHD-2 activity and HIF-1α levels were included in the study for comparison. Results obtained indicate that β-ODAP inhibits PHD-2 and facilitates HIF dependent HRE expression and hence, might be helpful in conditions arising from hypoxia. Copyright © 2016 Elsevier B.V. All rights reserved.
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Park, Sang Won; He, Zhi; Shen, Xine; Roman, Richard J; Ma, Tangeng
2012-06-01
Extensive anatomical and functional interactions exist between central dopaminergic and opioidergic systems and both systems are proposed to be targets for amphetamine-like drugs. We have previously reported that μ-opioid receptor (μ-OR) knockout mice are resistant to the loss of dopamine in the striatum and the development of behavioral sensitization induced by repeated methamphetamine (METH) treatment. The present study assessed whether METH-treated μ-OR knockout mice exhibit a differential response of the expression of dopamine transporter and tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis and maintaining dopamine levels. Mice daily received intraperitoneal injection of METH (0, 0.6, 2.5, or 10 mg/kg) for 7 days and sacrificed on day 11 (4 days after the last injection). The expression of TH protein in the striatum and the levels of TH mRNA and number of TH positive neurons in the substantia nigra were reduced in wild-type mice treated with METH (2.5 and 10 mg/kg), but not in the μ-OR knockout mice. In contrast, METH exposure at the highest dose (10 mg/kg) reduced dopamine transporter levels in both strains of mice. These results suggest that the μ-OR contributes to METH-induced loss of dopamine and behavioral sensitization by decreasing the expression of TH.
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Insight into Environmental Effects on Bonding and Redox Properties of [4Fe-4S] Clusters in Proteins
DOE Office of Scientific and Technical Information (OSTI.GOV)
Niu, Shuqiang; Ichiye, Toshiko
The large differences in redox potentials between the HiPIPs and ferredoxins are generally attributed to hydrogen bonds and electrostatic effects from the protein and solvent. Recent ligand K-edge X-ray absorption studies by Solomon and co-workers show that the Fe-S covalencies of [4Fe-4S] clusters in the two proteins differ considerably apparently because of hydrogen bonds from water, indicating electronic effects may be important. However, combined density function theory (DFT) and photoelectron spectroscopy studies by our group and Wang and co-workers indicate that hydrogen bonds tune the potential of [4Fe-4S] clusters by mainly electrostatics. The DFT studies here rationalize both results, namelymore » that the observed change in the Fe-S covalency is due to differences in ligand conformation between the two proteins rather than hydrogen bonds. Moreover, the ligand conformation affects the calculated potentials by 100 mV and, thus, is a heretofore unconsidered means of tuning the potential.« less
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Humphreys, John M.; Hemm, Matthew R.; Chapple, Clint
1999-01-01
The enzymes and genes of the lignin biosynthetic pathway have been studied for several decades, but the gene encoding ferulate 5-hydroxylase (F5H) was cloned only 3 years ago by T-DNA tagging in Arabidopsis. To characterize the enzyme in detail, we have expressed F5H in yeast. According to current models of the phenylpropanoid pathway, F5H catalyzes the hydroxylation of ferulate to 5-hydroxyferulate; however, our studies indicate that the enzyme also uses coniferaldehyde and coniferyl alcohol as substrates. Unexpectedly, the Km values measured for the latter two substrates are three orders of magnitude lower than that measured for ferulic acid, suggesting that in lignifying tissues, syringyl monomers may be derived from their guaiacyl counterparts by hydroxylation and subsequent methylation. Thus, F5H may function later in the lignin biosynthetic pathway than was originally proposed. To further test this model, recombinant F5H was incubated together with ferulic acid, coniferaldehyde, or coniferyl alcohol in the presence of native or recombinant Arabidopsis caffeic acid/5-hydroxyferulic acid O-methyltransferase and [14C]S-adenosylmethionine. In all cases, the corresponding radiolabeled sinapyl derivatives were synthesized, indicating that the necessary enzymes required for this pathway are present in Arabidopsis. Taken together, these data suggest that the previously accepted pathway for lignin biosynthesis is likely to be incorrect. PMID:10468559
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Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid
Hibi, Makoto; Mori, Ryosuke; Miyake, Ryoma; Kawabata, Hiroshi; Kozono, Shoko; Takahashi, Satomi
2016-01-01
Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities. PMID:26801577
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Ferrer-Sevillano, Francisco; Fernández-Cañón, José M
2007-03-01
Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).
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Ferrer-Sevillano, Francisco; Fernández-Cañón, José M.
2007-01-01
Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (ΔphacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B). PMID:17189487
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Seroogy, K; Schalling, M; Brené, S; Dagerlind, A; Chai, S Y; Hökfelt, T; Persson, H; Brownstein, M; Huan, R; Dixon, J
1989-01-01
The cellular localization of neurons expressing cholecystokinin (CCK) and tyrosine hydroxylase (TH) mRNAs was analysed in rat ventral mesencephalon using in situ hybridization techniques with both complementary DNA and synthetic oligonucleotide probes. Cell bodies distributed throughout the substantia nigra, ventral tegmental area, interfascicular nucleus, midline raphe nuclei, and central and ventral periaqueductal grey matter were found to contain CCK mRNA or TH mRNA as indicated by high densities of grains overlying the perikarya. The in situ hybridization technique was combined with immunocytochemistry on the same tissue section to localize the peptide or enzyme within its respective mRNA-containing somata. In addition, the presence of TH immunoreactivity was demonstrated within cell bodies labeled for CCK mRNA and immunostaining for CCK was detected within TH mRNA-containing neurons. In the medial geniculate nucleus a strong labeling for CCKmRNA was observed, in spite of the fact that so far no CCK-like immunoreactivity has been demonstrated in perikarya in this nucleus. The specificity of the probes was verified by RNA blot hybridization. These results confirm recent double-labeling immunocytochemical studies and further characterize the coexistence of CCK and TH at the level of their mRNAs as well as their post-translational products in a large population of mesencephalic dopamine neurons known to project to forebrain areas.
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MICROWAVE SPECTROSCOPY OF THE CALCIUM 4snf→4s(n+1)d, 4sng, 4snh, 4sni, AND 4snk TRANSITIONS
NASA Astrophysics Data System (ADS)
Nunkaew, Jirakan; Gallagher, Tom
2015-06-01
We use a delayed field ionization technique to observe the microwave transitions of calcium Rydberg states, from the 4snf states to the 4s(n+1)d, 4sng, 4snh, 4sni, and 4snk states for 18≤ n≤23. We analyze the observed intervals between the ℓ and (ℓ+1), ℓ≥5, states of the same n to determine the Ca^+ 4s dipole and quadrupole polarizabilities. We show that the adiabatic core polarization model is not adequate to extract the Ca^+ 4s dipole and quadrupole polarizabilities and a non adiabatic treatment is required. We use the non adiabatic core polarization model to determine the ionic dipole and quadrupole polarizabilities to be α_d=76.9(3);a_0^3 and α_q=206(9);a_0^5, respectively.
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Lijkwan, Maarten A.; Hellingman, Alwine A.; Bos, Ernst J.; van der Bogt, Koen E.A.; Huang, Mei; Kooreman, Nigel G.; de Vries, Margreet R.; Peters, Hendrika A.B.; Robbins, Robert C.; Quax, Paul H.A.
2014-01-01
Abstract In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×105±3.3×105 (MC-Luc) versus 0.4×105±0.3×105 (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31+ expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for
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Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T
1988-11-01
Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla.
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Schalling, M; Dagerlind, A; Brené, S; Hallman, H; Djurfeldt, M; Persson, H; Terenius, L; Goldstein, M; Schlesinger, D; Hökfelt, T
1988-01-01
Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla. Images PMID:2903502
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Parajes, Silvia; Loidi, Lourdes; Reisch, Nicole; Dhir, Vivek; Rose, Ian T.; Hampel, Rainer; Quinkler, Marcus; Conway, Gerard S.; Castro-Feijóo, Lidia; Araujo-Vilar, David; Pombo, Manuel; Dominguez, Fernando; Williams, Emma L.; Cole, Trevor R.; Kirk, Jeremy M.; Kaminsky, Elke; Rumsby, Gill; Arlt, Wiebke; Krone, Nils
2010-01-01
Context: Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency. Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations. Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein. Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11β-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively. Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling in 11OHD. PMID:20089618
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Brozek, Wolfgang; Manhardt, Teresa; Kállay, Enikö; Peterlik, Meinrad; Cross, Heide S
2012-07-26
Previous studies on the significance of vitamin D insufficiency and chronic inflammation in colorectal cancer development clearly indicated that maintenance of cellular homeostasis in the large intestinal epithelium requires balanced interaction of 1,25-(OH)2D3 and prostaglandin cellular signaling networks. The present study addresses the question how colorectal cancer pathogenesis depends on alterations of activities of vitamin D hydroxylases, i.e., CYP27B1-encoded 25-hydroxyvitamin D-1a-hydroxylase and CYP24A1-encoded 25-hydroxyvitamin D-24-hydroxylase, and inflammation-induced cyclooxygenase-2 (COX-2). Data from 105 cancer patients on CYP27B1, VDR, CYP24A1, and COX-2 mRNA expression in relation to tumor grade, anatomical location, gender and age were fit into a multivariate model of exploratory factor analysis. Nearly identical results were obtained by the principal factor and the maximum likelihood method, and these were confirmed by hierarchical cluster analysis: Within the eight mutually dependent variables studied four independent constellations were found that identify different features of colorectal cancer pathogenesis: (i) Escape of COX-2 activity from restraints by the CYP27B1/VDR system can initiate cancer growth anywhere in the colorectum regardless of age and gender; (ii) variations in COX-2 expression are mainly responsible for differences in cancer incidence in relation to tumor location; (iii) advancing age has a strong gender-specific influence on cancer incidence; (iv) progression from well differentiated to undifferentiated cancer is solely associated with a rise in CYP24A1 expression.
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Brozek, Wolfgang; Manhardt, Teresa; Kállay, Enikö; Peterlik, Meinrad; Cross, Heide S.
2012-01-01
Previous studies on the significance of vitamin D insufficiency and chronic inflammation in colorectal cancer development clearly indicated that maintenance of cellular homeostasis in the large intestinal epithelium requires balanced interaction of 1,25-(OH)2D3 and prostaglandin cellular signaling networks. The present study addresses the question how colorectal cancer pathogenesis depends on alterations of activities of vitamin D hydroxylases, i.e., CYP27B1-encoded 25-hydroxyvitamin D-1α-hydroxylase and CYP24A1-encoded 25-hydroxyvitamin D-24-hydroxylase, and inflammation-induced cyclooxygenase-2 (COX-2). Data from 105 cancer patients on CYP27B1, VDR, CYP24A1, and COX-2 mRNA expression in relation to tumor grade, anatomical location, gender and age were fit into a multivariate model of exploratory factor analysis. Nearly identical results were obtained by the principal factor and the maximum likelihood method, and these were confirmed by hierarchical cluster analysis: Within the eight mutually dependent variables studied four independent constellations were found that identify different features of colorectal cancer pathogenesis: (i) Escape of COX-2 activity from restraints by the CYP27B1/VDR system can initiate cancer growth anywhere in the colorectum regardless of age and gender; (ii) variations in COX-2 expression are mainly responsible for differences in cancer incidence in relation to tumor location; (iii) advancing age has a strong gender-specific influence on cancer incidence; (iv) progression from well differentiated to undifferentiated cancer is solely associated with a rise in CYP24A1 expression. PMID:24213465
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Federal Register 2010, 2011, 2012, 2013, 2014
2010-07-21
... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2010-N-0318... NDA 6-008 Mesantoin (mephenytoin) Tablets Novartis Pharmaceuticals Corp., One Health Plaza, East... Endo Pharmaceuticals NDA 17-255 MPI DTPA Chelate multidose (kit for Medi-Physics, Inc., d/b/a GE...
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Spectroscopic ellipsometry study of Cu2ZnSnS4 bulk poly-crystals
NASA Astrophysics Data System (ADS)
Levcenko, S.; Hajdeu-Chicarosh, E.; Garcia-Llamas, E.; Caballero, R.; Serna, R.; Bodnar, I. V.; Victorov, I. A.; Guc, M.; Merino, J. M.; Pérez-Rodriguez, A.; Arushanov, E.; León, M.
2018-04-01
The linear optical properties of Cu2ZnSnS4 bulk poly-crystals have been investigated using spectroscopic ellipsometry in the range of 1.2-4.6 eV at room temperature. The characteristic features identified in the optical spectra are explained by using the Adachi analytical model for the interband transitions at the corresponding critical points in the Brillouin zone. The experimental data have been modeled over the entire spectral range taking into account the lowest E0 transition near the fundamental absorption edge and E1A and E1B higher energy interband transitions. In addition, the spectral dependences of the refractive index, extinction coefficient, absorption coefficient, and normal-incidence reflectivity values have been accurately determined and are provided since they are essential data for the design of Cu2ZnSnS4 based optoelectronic devices.
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Crack, Jason C.; Thomson, Andrew J.
2017-01-01
The iron-sulfur cluster containing protein Fumarate and Nitrate Reduction (FNR) is the master regulator for the switch between anaerobic and aerobic respiration in Escherichia coli and many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O2 that leads to conversion to a [2Fe-2S] form with loss of high-affinity DNA binding. Here, we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a [3Fe-3S] cluster and persulfide-coordinated [2Fe-2S] clusters [[2Fe-2S](S)n, where n = 1 or 2]. Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion and not as a subsequent, secondary reaction to generate [2Fe-2S](S)n species. This methodology shows great potential for broad application to studies of protein cofactor–small molecule interactions. PMID:28373574
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Probing α-3(10) transitions in a voltage-sensing S4 helix.
Kubota, Tomoya; Lacroix, Jérôme J; Bezanilla, Francisco; Correa, Ana M
2014-09-02
The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 310 conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 310 conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 310 conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-310 helical conversions of the S4 in Ci-VSP associated with voltage activation. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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AdS/CFT beyond the N = 4 SYM paradigm
NASA Astrophysics Data System (ADS)
Pomoni, Elli
In this thesis we present studies in the AdS/CFT correspondence that intend to push the present knowledge beyond the N = 4 super Yang-Mills (SYM) paradigm. The first part is concerned with the study of non-supersymmetric deformations of N = 4 SYM (which still are in the N = 4 universality class). For non-supersymmetric CFT's at Large N we explore the correspondence between string theory tachyons in the bulk and instabilities on the boundary effective action. The operators dual to AdS tachyons have anomalous dimensions that are purely complex numbers. We give a prescription for calculating the mass of the tachyon from the field theory side. Moreover, we apply this general dictionary to the case of intersecting D7 flavor branes in AdS 5 x S5 and obtain the mass of the open string tachyon that is dual to the instability in the mesonic sector of the theory. In the second part we present work aiming at finding string theory duals for gauge theories beyond the N = 4 universality class, i.e. theories that have genuinely less supersymmetry and unquenched flavor. Arguably the next simplest example after N = 4 SYM is N = 2 SU(Nc) SYM coupled to Nf = 2Nc fundamental hypermultiplets. The theory admits a Veneziano expansion of large Nc and large Nf, with Nf/Nc and lambda = g2Nc kept fixed. The topological structure of large N diagrams invites a general conjecture: the flavor-singlet sector of a gauge theory in the Veneziano limit is dual to a closed string theory. We present the one-loop Hamiltonian for the scalar sector of N = 2 superconformal QCD and study this integrability of the theory. Furthermore, we explore the chiral spectrum of the protected operators of the theory using the one-loop anomalous dimensions and, additionally, by studying the index of the theory. We finally search for possible AdS dual trying to match the chiral spectrum. We conclude that the string dual is a sub-critical background containing both an AdS 5 and an S1 factor.
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Muro, Fumihito; Iimura, Shin; Sugimoto, Yuuichi; Yoneda, Yoshiyuki; Chiba, Jun; Watanabe, Toshiyuki; Setoguchi, Masaki; Iigou, Yutaka; Matsumoto, Keiko; Satoh, Atsushi; Takayama, Gensuke; Taira, Tomoe; Yokoyama, Mika; Takashi, Tohru; Nakayama, Atsushi; Machinaga, Nobuo
2009-12-24
We have focused on optimization of the inadequate pharmacokinetic profile of trans-4-substituted cyclohexanecarboxylic acid 5, which is commonly observed in many small molecule very late antigen-4 (VLA-4) antagonists. We modified the lipophilic moiety in 5 and found that reducing the polar surface area of this moiety results in improvement of the PK profile. Consequently, our efforts have led to the discovery of trans-4-[1-[[2,5-dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid (14e) with potent activity (IC(50) = 5.4 nM) and significantly improved bioavailability in rats, dogs, and monkeys (100%, 91%, 68%), which demonstrated excellent oral efficacy in murine and guinea pig models of asthma. Based on its overall profile, compound 14e was progressed into clinical trails. In a single ascending-dose phase I clinical study, compound 14e exhibited favorable oral exposure as expected and had no serious adverse events.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Konecke, Brian A.; Fiege, Adrian; Simon, Adam C.
In this study, we use micro-X-ray absorption near-edge structures (μ-XANES) spectroscopy at the S K-edge to investigate the oxidation state of S in natural magmatic-hydrothermal apatite (Durango, Mexico, and Mina Carmen, Chile) and experimental apatites crystallized from volatile-saturated lamproitic melts at 1000 °C and 300 MPa over a broad range of oxygen fugacities [( Embedded Image , FMQ+1.2, FMQ+3; FMQ = fayalite-magnetite-quartz solid buffer]. The data are used to test the hypothesis that S oxidation states other than S6+ may substitute into the apatite structure. Peak energies corresponding to sulfate S6+ (~2482 eV), sulfite S4+ (~2478 eV), and sulfide S2-more » (~2470 eV) were observed in apatite, and the integrated areas of the different sulfur peaks correspond to changes in Embedded Image and bulk S content. Here, multiple tests confirmed that the S oxidation state in apatite remains constant when exposed to the synchrotron beam, at least for up to 1 h exposure (i.e., no irradiation damages). To our knowledge, this observation makes apatite the first mineral to incorporate reduced (S2-), intermediate (S4+), and oxidized (S6+) S in variable proportions as a function of the prevailing Embedded Image of the system. Apatites crystallized under oxidizing conditions (FMQ+1.2 and FMQ+3), where the S6+/STotal peak area ratio in the coexisting glass (i.e., quenched melt) is ~1, are dominated by S6+ with a small contribution of S4+, whereas apatites crystallizing at reduced conditions (FMQ) contain predominantly S2-, lesser amounts of S6+, and possibly traces of S4+. A sulfur oxidation state vs. S concentration analytical line transect across hydrothermally altered apatite from the Mina Carmen iron oxide-apatite (IOA) deposit (Chile) demonstrates that apatite can become enriched in S4+ relative to S6+, indicating metasomatic overprinting via a SO2-bearing fluid or vapor phase. This XANES study demonstrates that as the Embedded Image increases from FQM to FMQ+1
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Chen, Haichao; Chen, Si; Shao, Hongyan; Li, Chao; Fan, Meiqiang; Chen, Da; Tian, Guanglei; Shu, Kangying
2016-01-01
Hierarchical NiCo2 S4 nanotube@NiCo2 S4 nanosheet arrays on Ni foam have been successfully synthesized. Owing to the unique hierarchical structure, enhanced capacitive performance can be attained. A specific capacitance up to 4.38 F cm(-2) is attained at 5 mA cm(-2) , which is much higher than the specific capacitance values of NiCo2 O4 nanosheet arrays, NiCo2 S4 nanosheet arrays and NiCo2 S4 nanotube arrays on Ni foam. The hierarchical NiCo2 S4 nanostructure shows superior cycling stability; after 5000 cycles, the specific capacitance still maintains 3.5 F cm(-2) . In addition, through the morphology and crystal structure measurement after cycling stability test, it is found that the NiCo2 S4 electroactive materials are gradually corroded; however, the NiCo2 S4 phase can still be well-maintained. Our results show that hierarchical NiCo2 S4 nanostructures are suitable electroactive materials for high performance supercapacitors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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McCoy, G D
1980-03-01
The aniline hydroxylase activity of microsomes isolated from hamster liver can be differentiated kinetically into high affinity (low K(m), form I) and low affinity (high K(m), form II) forms. Microsomes isolated from uninduced animals contain slightly more form I activity. The activity of the low affinity form (form II) is preferentially enhanced by Aroclor or 3-methylcholanthrene treatment, while phenobarbital treatment increases the activity of both forms. Chronic ethanol consumption results in enhancement of only the high affinity form (form I).
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yohannan, Jinu P.; Vidyasagar, Kanamaluru, E-mail: kvsagar@iitm.ac.in
Ten AInM′S{sub 4} (A=alkali metals, Tl; M′= Ge, Sn) compounds with diverse structure types have been synthesized and characterized by single crystal and powder X-ray diffraction and a variety of spectroscopic methods. They are wide band gap semiconductors. KInGeS{sub 4}(1-β), RbInGeS{sub 4}(2), CsInGeS{sub 4}(3-β), TlInGeS{sub 4}(4-β), RbInSnS{sub 4}(8-β) and CsInSnS{sub 4}(9) compounds with three-dimensional BaGa{sub 2}S{sub 4} structure and CsInGeS{sub 4}(3-α) and TlInGeS{sub 4}(4-α) compounds with a layered TlInSiS{sub 4} structure have tetrahedral [InM′S{sub 4}]{sup −} frameworks. On the other hand, LiInSnS{sub 4}(5) with spinel structure and NaInSnS{sub 4}(6), KInSnS{sub 4}(7), RbInSnS{sub 4}(8-α) and TlInSnS{sub 4}(10) compounds with layered structuremore » have octahedral [InM′S{sub 4}]{sup −} frameworks. NaInSnS{sub 4}(6) and KInSnS{sub 4}(7) compounds undergo facile topotactic ion-exchange, at room temperature, with various mono-, di- and tri-valent cations in aqueous medium to give rise to metastable layered phases. - Graphical abstract: NaInSnS{sub 4} and KInSnS{sub 4} compounds undergo, in aqueous medium at room temperature, facile topotactic ion-exchange with mono, di and trivalent cations. Display Omitted - Highlights: • Ten AInM′S{sub 4} compounds with diverse structure types were synthesized. • They are wide band gap semiconductors. • NaInSnS{sub 4} and KInSnS{sub 4} compounds undergo facile topotactic ion-exchange at room temperature.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Semari, F.; Khenata, R.; Depatment of Physics and Astronomy, King Saud University, PO Box 2455, Riyadh 11451
2010-12-15
The structural, elastic, electronic, and optical properties of cubic spinel MgIn{sub 2}S{sub 4} and CdIn{sub 2}S{sub 4} compounds have been calculated using a full relativistic version of the full-potential linearized-augmented plane wave with the mixed basis FP/APW+lo method. The exchange and correlation potential is treated by the generalized-gradient approximation (GGA). Moreover, the Engel-Vosko GGA formalism is also applied to optimize the corresponding potential for band structure calculations. The ground state properties, including the lattice constants, the internal parameter, the bulk modulus, and the pressure derivative of the bulk modulus are in reasonable agreement with the available data. Using the totalmore » energy-strain technique, we have determined the full set of first-order elastic constants C{sub ij} and their pressure dependence, which have not been calculated or measured yet. The shear modulus, Young's modulus, and Poisson's ratio are calculated for polycrystalline XIn{sub 2}S{sub 4} aggregates. The Debye temperature is estimated from the average sound velocity. Electronic band structures show a direct band gap ({Gamma}-{Gamma}) for MgIn{sub 2}S{sub 4} and an indirect band gap (K-{Gamma}) for CdIn{sub 2}S{sub 4}. The calculated band gaps with EVGGA show a significant improvement over the GGA. The optical constants, including the dielectric function {epsilon}({omega}), the refractive index n({omega}), the reflectivity R({omega}), and the energy loss function L({omega}) were calculated for radiation up to 30 eV. -- Graphical abstract: Calculated total and partial densities of states for MgIn{sub 2}S{sub 4} and CdIn{sub 2}S{sub 4}« less
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Sanchez-Sandoval, Ana Laura; Herrera Carrillo, Zazil; Díaz Velásquez, Clara Estela; Delgadillo, Dulce María; Rivera, Heriberto Manuel; Gomora, Juan Carlos
2018-01-01
Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30-40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers.
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Sanchez-Sandoval, Ana Laura; Herrera Carrillo, Zazil; Díaz Velásquez, Clara Estela; Delgadillo, Dulce María; Rivera, Heriberto Manuel
2018-01-01
Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30–40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers. PMID:29474447
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Sørensen, J.B.; Cha, A.; Latorre, R.; Rosenman, E.; Bezanilla, F.
2000-01-01
When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (ΔF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391–408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3–S4 linker. In the deletion mutant, the maximal ΔF/F seen was diminished 10-fold, and the ΔF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3–S4 linker. The residual ΔF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of −90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a ΔF at extreme hyperpolarizations (more negative than −90 mV) only. A signal from the interaction with this group in the wt S3–S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic ΔF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4
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Stage 4S neuroblastoma tumors show a characteristic DNA methylation portrait
Decock, Anneleen; Ongenaert, Maté; De Wilde, Bram; Brichard, Bénédicte; Noguera, Rosa; Speleman, Frank; Vandesompele, Jo
2016-01-01
ABSTRACT Stage 4S neuroblastoma (NB) is a special type of NB found in infants with metastases at diagnosis and is associated with an excellent outcome due to its remarkable capacity to undergo spontaneous regression. As genomics have not been able to explain this intriguing clinical presentation, we here aimed at profiling the DNA methylome of stage 4S NB to better understand this phenomenon. To this purpose, differential methylation analyses between International Neuroblastoma Staging System (INSS) stage 4S, stage 4 and stage 1/2 were performed, using methyl-CpG-binding domain (MBD) sequencing data of 14 stage 4S, 14 stage 4, and 13 stage 1/2 primary NB tumors (all MYCN non-amplified in order not to confound results). Stage 4S-specific hyper- and hypomethylated promoters were determined and further characterized for genomic localization and function by cytogenetic band enrichment, gene set enrichment, transcription factor target enrichment and differential RNA expression analyses. We show that specific chromosomal locations are enriched for stage 4S differentially methylated promoters and that stage 4S tumors show characteristic hypermethylation of specific subtelomeric promoters. Furthermore, genes involved in important oncogenic pathways, in neural crest development and differentiation, and in epigenetic processes are differentially methylated and expressed in stage 4S tumors. Based on these findings, we describe new biological mechanisms possibly contributing to the stage 4S-specific tumor biology and spontaneous regression. In conclusion, this study is the first to describe the highly characteristic stage 4S DNA methylome. These findings will open new avenues to further unravel the NB pathology in general and stage 4S disease specifically. PMID:27599161
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Stage 4S neuroblastoma tumors show a characteristic DNA methylation portrait.
Decock, Anneleen; Ongenaert, Maté; De Wilde, Bram; Brichard, Bénédicte; Noguera, Rosa; Speleman, Frank; Vandesompele, Jo
2016-09-06
Stage 4S neuroblastoma (NB) is a special type of NB found in infants with metastases at diagnosis and is associated with an excellent outcome due to its remarkable capacity to undergo spontaneous regression. As genomics have not been able to explain this intriguing clinical presentation, we here aimed at profiling the DNA methylome of stage 4S NB to better understand this phenomenon. To this purpose, differential methylation analyses between International Neuroblastoma Staging System (INSS) stage 4S, stage 4 and stage 1/2 were performed, using methyl-CpG-binding domain (MBD) sequencing data of 14 stage 4S, 14 stage 4, and 13 stage 1/2 primary NB tumors (all MYCN non-amplified in order not to confound results). Stage 4S-specific hyper- and hypo-methylated promoters were determined and further characterized for genomic localization and function by cytogenetic band enrichment, gene set enrichment, transcription factor target enrichment and differential RNA expression analyses. We show that specific chromosomal locations are enriched for stage 4S differentially methylated promoters and that stage 4S tumors show characteristic hypermethylation of subtelomeres. Furthermore, genes involved in important oncogenic pathways, in neural crest development and differentiation, and in epigenetic processes are differentially methylated and expressed in stage 4S tumors. Based on these findings, we describe new biological mechanisms possibly contributing to the stage 4S-specific tumor biology and spontaneous regression. In conclusion, this study is the first to describe the highly characteristic stage 4S DNA methylome. These findings will open new avenues to further unravel the NB pathology in general and stage 4S disease specifically.
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NASA Astrophysics Data System (ADS)
Rush, Larry E., Jr.; Holzwarth, N. A. W.
First principles simulations are used to examine the structural and physical properties of Na4P2S6 in comparison with its Li4P2S6 analog. Four model structures are considered including the C 2 / m structure recently reported by Kuhn and co-workers from their analysis of single crystals of Na4P2S6, and three structures related to the P63 / mcm structure with P site disorder found in 1982 by Mercier and co-workers from their analysis of single crystals of Li4P2S6. The computational results indicate that both Na4P2S6 and Li4P2S6 have the same disordered ground state structures consistent with the P63 / mcm space group, while the optimized C 2 / m structures have higher energies by 0.1 eV and 0.4 eV per formula unit for Na4P2S6 and Li4P2S6, respectively. Simulations of ion migration suggest that Na4P2S6 may have more favorable ionic conductivity compared to Li4P2S6. Supported by NSF Grant DMR-1105485 and DMR-1507942.
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Zhuang, XiaoMei; Zhang, TianHong; Yue, SiJia; Wang, Juan; Luo, Huan; Zhang, YunXia; Li, Zheng; Che, JinJing; Yang, HaiYing; Li, Hua; Zhu, MingShe; Lu, Chuang
2016-12-01
Icotinib (ICO), a novel small molecule and a tyrosine kinase inhibitor, was developed and approved recently in China for non-small cell lung cancer. During screening for CYP inhibition potential in human liver microsomes (HLM), heterotropic activation toward CYP3A5 was revealed. Activation by icotinib was observed with CYP3A-mediated midazolam hydroxylase activity in HLM (∼40% over the baseline) or recombinant human CYP3A5 (rhCYP3A5) (∼70% over the baseline), but not in the other major CYPs including rhCYP3A4. When co-incubated with selective CYP3A4 inhibitor CYP3cide or monoclonal human CYP3A4 inhibitory antibody in HLM, the activation was extended to ∼60%, suggesting CYP3A5 might be the isozyme involved. Further, the relative activation was enhanced to ∼270% in rhCYP3A5 in the presence of ketoconazole. The activation was substrate and pathway dependent and observed only in the formation of 1'-OH-midazolam, and not 4-OH-midazolam, 6β-OH-testosterone, or oxidized nifedipine. The activation requires the presence of cytochrome b5 and it is only observed in the liver microsomes of dogs, monkeys, and humans, but not in rats and mice. Kinetic analyses of 1'-OH-midazolam formation showed that ICO increased the V max values in HLM and rhCYP3A5 with no significant changes in K m values. By adding CYP3cide with ICO to the incubation, the V max values increased 2-fold over the CYP3cide control. Addition of ketoconazole with ICO alone or ICO plus CYP3cide resulted in an increase in V max values and decrease in K m values compared to their controls. This phenomenon may be attributed to a new mechanism of CYP3A5 heterotropic activation, which warrants further investigation. Copyright © 2016 Elsevier Inc. All rights reserved.
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Murad, S; Strycharz, G D; Kishimoto, Y
1976-09-10
Rat brain postnuclear preparations catalyzed the alpha-hydroxylation of nervonic acid with an apparent Km of 3 muM. Evidence has been presented which suggests that nervonic acid in the brain is hydroxylated by the same enzyme system which hydroxylates lignoceric acid. The hydroxylase activity in brains of normal (euthyroid) rats increased rapidly from a low in the period immediately following birth to a maximum at the 23rd day and then declined to a low level characteristic of the mature brain. Neonatal hypothyroidism retarded the development of the activity and shifted its peak to the 39th day after birth. Conversely, neonatal hyperthyroidism accelerated the entire developmental pattern and shifted the peak to the 16th day after birth. The hydroxylase activity in mouse brain was also increased by thyroid hormone administration from the 13th through the 18th day after birth. Unlike normal mice, the low activity in jimpy mice was not affected by this treatment. It is concluded that thyroid hormones play an important role in the control of brain fatty acid alpha-hydroxylation. The stimulation of alpha-hydroxy fatty acid synthesis in response to hyperthyroidism during the early postnatal period may be one of the major effects of thyroid hormones in accelerating myelination of the central nervous system.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Orefuwa, Samuel A.; Lai, Cheng-Yu; Dobson, Kevin D.
2014-05-12
Fe 2SiS 4 and Fe 2GeS 4 crystalline materials posses direct bandgaps of ~1.55 and ~1.4 eV respectively and an absorption coefficient larger than 10^5 cm–1; their theoretical potential as solar photovoltaic absorbers has been demonstrated. However, no solar devices that employ either Fe 2SiS 4 or Fe 2GeS 4 have been reported to date. In the presented work, nanoprecursors to Fe 2SiS 4 and Fe 2GeS 4 have been fabricated and employed to build ultra-thin-film layers via spray coating and rod coating methods. Temperature-dependent X-Ray diffraction analyses of nanoprecursors coatings show an unprecedented low temperature for forming crystalline Femore » 2SiS 4 and Fe 2GeS 4. Fabricating of ultra-thin-film photovoltaic devices utilizing Fe 2SiS 4 and Fe 2GeS 4 as solar absorber material is presented.« less
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Hamanaka, Yoshitaka; Minoura, Run; Nishino, Hiroshi; Miura, Toru; Mizunami, Makoto
2016-01-01
The catecholamine dopamine plays several vital roles in the central nervous system of many species, but its neural mechanisms remain elusive. Detailed neuroanatomical characterization of dopamine neurons is a prerequisite for elucidating dopamine’s actions in the brain. In the present study, we investigated the distribution of dopaminergic neurons in the brain of the American cockroach, Periplaneta americana, using two antisera: 1) an antiserum against dopamine, and 2) an antiserum against tyrosine hydroxylase (TH, an enzyme required for dopamine synthesis), and identified about 250 putatively dopaminergic neurons. The patterns of dopamine- and TH-immunoreactive neurons were strikingly similar, suggesting that both antisera recognize the same sets of “dopaminergic” neurons. The dopamine and TH antibodies intensively or moderately immunolabeled prominent brain neuropils, e.g. the mushroom body (memory center), antennal lobe (first-order olfactory center) and central complex (motor coordination center). All subdivisions of the mushroom body exhibit both dopamine and TH immunoreactivity. Comparison of immunolabeled neurons with those filled by dye injection revealed that a group of immunolabeled neurons with cell bodies near the calyx projects into a distal region of the vertical lobe, which is a plausible site for olfactory memory formation in insects. In the antennal lobe, ordinary glomeruli as well as macroglomeruli exhibit both dopamine and TH immunoreactivity. It is noteworthy that the dopamine antiserum labeled tiny granular structures inside the glomeruli whereas the TH antiserum labeled processes in the marginal regions of the glomeruli, suggesting a different origin. In the central complex, all subdivisions excluding part of the noduli and protocerebral bridge exhibit both dopamine and TH immunoreactivity. These anatomical findings will accelerate our understanding of dopaminergic systems, specifically in neural circuits underlying aversive memory
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NASA Astrophysics Data System (ADS)
Gambarov, E.; Bayramov, A.; Kato, A.; Iida, S.
2006-09-01
Ce-doped CaGa2S4 and SrGa2S4 thin film electroluminescent (TFEL) devices were prepared for the first time on the basis of films deposited by flash evaporation method. Significant crystallization, stoichiometry improvement of the films and increase of photoluminescence intensity were found after annealing in H2S and O2 gas stream. EL spectra of the cells exhibited the characteristic double-band emission similar to that seen for Ce3+ activated CaGa2S4 and SrGa2S4 films under photon excitation. Applied voltage and frequency dependences of the electroluminescence were studied. Low voltage operation as low as 20 V was observed for these cells. Luminance of about 4 cd/m2 at 100 V operating voltage with 2.5 kHz frequency was achieved for the TFEL cell with films annealed in O2 gas stream.
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Mechanistic Insight into the Nitrosylation of the [4Fe−4S] Cluster of WhiB-like Proteins
2010-01-01
The reactivity of protein bound iron−sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe−4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe−4S] cluster. The reaction is 104-fold faster than that observed with O2 and is by far the most rapid iron−sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe4S4(Cys)4]2− + 8NO → 2[FeI2(NO)4(Cys)2]0 + S2− + 3S0 has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [FeI4(NO)8(Cys)4]0 derived by dimerization of a pair of Roussin’s red ester (RRE) complexes. PMID:21182249
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Marsch, Elke; Demandt, Jasper A F; Theelen, Thomas L; Tullemans, Bibian M E; Wouters, Kristiaan; Boon, Mariëtte R; van Dijk, Theo H; Gijbels, Marion J; Dubois, Ludwig J; Meex, Steven J R; Mazzone, Massimiliano; Hung, Gene; Fisher, Edward A; Biessen, Erik A L; Daemen, Mat J A P; Rensen, Patrick C N; Carmeliet, Peter; Groen, Albert K; Sluimer, Judith C
2016-10-14
Normalization of hypercholesterolaemia, inflammation, hyperglycaemia, and obesity are main desired targets to prevent cardiovascular clinical events. Here we present a novel regulator of cholesterol metabolism, which simultaneously impacts on glucose intolerance and inflammation. Mice deficient for oxygen sensor HIF-prolyl hydroxylase 1 (PHD1) were backcrossed onto an atherogenic low-density lipoprotein receptor (LDLR) knockout background and atherosclerosis was studied upon 8 weeks of western-type diet. PHD1 -/- LDLR -/- mice presented a sharp reduction in VLDL and LDL plasma cholesterol levels. In line, atherosclerotic plaque development, as measured by plaque area, necrotic core expansion and plaque stage was hampered in PHD1 -/- LDLR -/- mice. Mechanistically, cholesterol-lowering in PHD1 deficient mice was a result of enhanced cholesterol excretion from blood to intestines and ultimately faeces. Additionally, flow cytometry of whole blood of these mice revealed significantly reduced counts of leucocytes and particularly of Ly6C high pro-inflammatory monocytes. In addition, when studying PHD1 -/- in diet-induced obesity (14 weeks high-fat diet) mice were less glucose intolerant when compared with WT littermate controls. Overall, PHD1 knockout mice display a metabolic phenotype that generally is deemed protective for cardiovascular disease. Future studies should focus on the efficacy, safety, and gender-specific effects of PHD1 inhibition in humans, and unravel the molecular actors responsible for PHD1-driven, likely intestinal, and regulation of cholesterol metabolism. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.
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CMB-S4 Technology Book, First Edition
DOE Office of Scientific and Technical Information (OSTI.GOV)
Abitbol, Maximilian H.
CMB-S4 is a proposed experiment to map the polarization of the Cosmic Microwave Background (CMB) to nearly the cosmic variance limit for the angular scales that are accessible from the ground. The science goals and capabilities of CMB-S4 in illuminating cosmic inflation, measuring the sum of neutrino masses, searching for relativistic relics in the early universe, characterizing dark energy and dark matter, and mapping the matter distribution in the universe have been described in the CMB-S4 Science Book. This Technology Book is a companion volume to the Science Book. The ambitious science goals of the proposed "Stage-IV" CMB-S4 will requiremore » a step forward in experimental capability from the current Stage-III experiments. To guide this process, the community summarized the current state of the technology and identify R&D efforts necessary to advance it for possible use in CMB-S4. The book focused on the technical challenges in four broad areas: Telescope Design; Receiver Optics; Focal-Plane Optical Coupling; and Focal-Plane Sensor and Readout.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Oshiro, Chikara; Takasu, Nobuyuki; Wakugami, Tamio
1995-08-01
Mutation of the cytochrome P450c17 (CYP17) gene causes 17{alpha}-hydroxylase deficiency (170HD). Recently, several researchers have elucidated the molecular basis of 170HD by gene analysis. We experienced a case of 170HD and intended to reveal the abnormality of the CYP17 gene in this Japanese female with 170HD. Leukocytes were obtained from the patient, her mother and sister, and normal control subjects. We amplified the CYP17 gene using polymerase chain reaction and performed the sequence analysis using the dideoxy terminator method and restriction enzyme analysis. We found that the patient had one base-pair deletion at the position of amino acid 438. Anmore » indentical result was obtained with restriction enzyme analysis. This G deletion altered the reading frame and resulted in a premature stop codon at position 443; the ligand of heme iron (Cys: cystine 442) was absent. This small mutation may account for the patient`s clinical manifestations of 170HD. This is the first case of 170HD with only one base pair deletion of the CYP17 gene. 18 refs., 3 figs.« less
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Reisch, Nicole; Rottenkolber, Marietta; Greifenstein, Anais; Krone, Nils; Schmidt, Heinrich; Reincke, Martin; Schwarz, Hans-Peter; Beuschlein, Felix
2013-11-01
Testicular adrenal rest tumors (TARTs) and hypogonadotropic hypogonadism are the two most common causes for male infertility in classic 21-hydroxylase deficiency. Current hypotheses suggest the quality of disease control to be one of the main pathogenic factors for TART development. The aim was to study long-term predictors for TART development in a retrospective longitudinal study. Fifty men with classic 21-hydroxylase deficiency (31 salt wasting, 19 simple virilizing) were investigated. Testicular ultrasound at a median age at investigation of 27 years detected TARTs in 28 of 50 subjects (19 salt wasting, 9 simple virilizing). TART presence was correlated with long-term parameters of disease control during childhood and adolescence obtained from patients' charts: 24-hour urine pregnanetriol, serum 17-hydroxyprogesterone, onset and stage of pubic hair development, testicular growth, and bone age in relation to chronological age. There was no difference in pregnanetriol excretion over lifetime between patients with and without TARTs. Similarly, neither development of pubic hair and testicular volume (Tanner) nor bone age in relation to chronological age differed between the two groups. Furthermore, the two groups had the same body mass index and the same impairment of final height in relation to midparental target height. Our longitudinal analysis demonstrates no association between TART presence and parameters of disease control. These data, therefore, argue for other mechanisms more relevant for TART induction including those occurring during fetal development.
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Cell wall modifications triggered by the down-regulation of Coumarate 3-hydroxylase-1 in maize.
Fornalé, Silvia; Rencoret, Jorge; Garcia-Calvo, Laura; Capellades, Montserrat; Encina, Antonio; Santiago, Rogelio; Rigau, Joan; Gutiérrez, Ana; Del Río, José-Carlos; Caparros-Ruiz, David
2015-07-01
Coumarate 3-hydroxylase (C3H) catalyzes a key step of the synthesis of the two main lignin subunits, guaiacyl (G) and syringyl (S) in dicotyledonous species. As no functional data are available in regards to this enzyme in monocotyledonous species, we generated C3H1 knock-down maize plants. The results obtained indicate that C3H1 participates in lignin biosynthesis as its down-regulation redirects the phenylpropanoid flux: as a result, increased amounts of p-hydroxyphenyl (H) units, lignin-associated ferulates and the flavone tricin were detected in transgenic stems cell walls. Altogether, these changes make stem cell walls more degradable in the most C3H1-repressed plants, despite their unaltered polysaccharide content. The increase in H monomers is moderate compared to C3H deficient Arabidopsis and alfalfa plants. This could be due to the existence of a second maize C3H protein (C3H2) that can compensate the reduced levels of C3H1 in these C3H1-RNAi maize plants. The reduced expression of C3H1 alters the macroscopic phenotype of the plants, whose growth is inhibited proportionally to the extent of C3H1 repression. Finally, the down-regulation of C3H1 also increases the synthesis of flavonoids, leading to the accumulation of anthocyanins in transgenic leaves. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator.
Cui, X; Pertile, R; Liu, P; Eyles, D W
2015-09-24
Vitamin D is a neuroactive steroid. Its genomic actions are mediated via the active form of vitamin D, 1,25(OH)2D3, binding to the vitamin D receptor (VDR). The VDR emerges in the rat mesencephalon at embryonic day 12, representing the peak period of dopaminergic cell birth. Our prior studies reveal that developmental vitamin D (DVD)-deficiency alters the ontogeny of dopaminergic neurons in the developing mesencephalon. There is also consistent evidence from others that 1,25(OH)2D3 promotes the survival of dopaminergic neurons in models of dopaminergic toxicity. In both developmental and toxicological studies it has been proposed that 1,25(OH)2D3 may modulate the differentiation and maturation of dopaminergic neurons; however, to date there is lack of direct evidence. The aim of the current study is to investigate this both in vitro using a human SH-SY5Y cell line transfected with rodent VDR and in vivo using a DVD-deficient model. Here we show that in VDR-expressing SH-SY5Y cells, 1,25(OH)2D3 significantly increased production of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. This effect was dose- and time-dependent, but was not due to an increase in TH-positive cell number, nor was it due to the production of trophic survival factors for dopamine neurons such as glial-derived neurotrophic factor (GDNF). In accordance with 1,25(OH)2D3's anti-proliferative actions in the brain, 1,25(OH)2D3 reduced the percentage of dividing cells from approximately 15-10%. Given the recently reported role of N-cadherin in the direct differentiation of dopaminergic neurons, we examined here whether it may be elevated by 1,25(OH)2D3. We confirmed this in vitro and more importantly, we showed DVD-deficiency decreases N-cadherin expression in the embryonic mesencephalon. In summary, in our in vitro model we have shown 1,25(OH)2D3 increases TH expression, decreases proliferation and elevates N-cadherin, a potential factor that mediates these processes
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Molecular Characterization of Ferulate 5-Hydroxylase Gene from Kenaf (Hibiscus cannabinus L.)
Park, Young-Hwan; Lim, Hyoun-Sub; Natarajan, Savithiry; Park, Sang-Un
2013-01-01
The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.). Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84), is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF) encoding 518 amino acids (GenBank Accession number JX524278). The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78%) with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old) of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h), wounding (24 h), cold (24 h), abscisic acid (24 h), and methyl jasmonate (24 h). PMID:24204204
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Persson, B C; Björk, G R
1993-01-01
The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present at position 37 (3' of the anticodon) of tRNAs that read codons beginning with U except tRNA(I,V Ser) in Escherichia coli. Salmonella typhimurium 2-methylthio-cis-ribozeatin (ms2io6A) is found in tRNA, probably in the corresponding species that have ms2i6A in E. coli. The gene (miaE) for the tRNA(ms2io6A)hydroxylase of S. typhimurium was isolated by complementation in E. coli. The miaE gene was localized close to the argI gene at min 99 of the S. typhimurium chromosomal map. Its DNA sequence and transcription pattern together with complementation studies revealed that the miaE gene is the second gene of a dicistronic operon. Southern blot analysis showed that the miaE gene is absent in E. coli, a finding consistent with the absence of the hydroxylated derivative of ms2i6A in this species. Mutants of S. typhimurium which have MudJ inserted in the miaE gene and which, consequently, are blocked in the ms2i6A hydroxylation reaction were isolated. Unexpectedly, such mutants cannot utilize the citric acid cycle intermediates malate, fumarate, and succinate as carbon sources. Images PMID:8253666
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USDA-ARS?s Scientific Manuscript database
The effects of azole-type P450 inhibitors and two metabolism-resistant ABA analogs on in vitro ABA 8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expr...
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DeBoer, Mark D; Scarlett, Jarrad M; Levasseur, Peter R; Grant, Wilmon F; Marks, Daniel L
2009-02-01
Inflammation-associated cachexia is associated with multiple chronic diseases and involves activation of appetite regulating centers in the arcuate nucleus of the hypothalamus (ARH). The nucleus of the solitary tract (NTS) in the brainstem has also been implicated as an important nucleus involved in appetite regulation. We set out to determine whether the NTS may be involved in inflammation-associated anorexia by injecting IL-1 beta into the 4th ventricle and assessing food intake and NTS neuronal activation. Injection of IL-1 beta produced a decrease in food intake at 3 and 12h after injection which was ameliorated at the 12h time point by a sub-threshold dose of agouti-related peptide (AgRP). Investigation into neuron types in the NTS revealed that IL-1 beta injection was associated with an increase in c-Fos activity in NTS neurons expressing tyrosine hydroxylase (TH). Additionally, injection of IL-1 beta into the 4th ventricle did not produce c-Fos activation of neurons expressing pro-opiomelanocortin (POMC) in the ARH, cells known to be involved in producing anorexia in response to systemic inflammation. Double-label in situ hybridization revealed that TH neurons did not express IL-1 receptor I (IL1-RI) transcript, demonstrating that c-Fos activation of TH neurons in this setting was not via direct stimulation of IL-1 beta on TH neurons themselves. We conclude that IL-1 beta injection into the 4th ventricle produces anorexia and is accompanied by an increase in activation in TH neurons in the NTS. This provides evidence that the brainstem may be an important mediator of anorexia in the setting of inflammation.
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DeBoer, Mark D.; Scarlett, Jarrad M.; Levasseur, Peter R.; Grant, Wilmon F.; Marks, Daniel L.
2010-01-01
Inflammation-associated cachexia is associated with multiple chronic diseases and involves activation of appetite regulating centers in the arcuate nucleus of the hypothalamus (ARH). The nucleus of the solitary tract (NTS) in the brainstem has also been implicated as an important nucleus involved in appetite regulation. We set out to determine whether the NTS may be involved in inflammation-associated anorexia by injecting IL-1β into the 4th ventricle and assessing food intake and NTS neuronal activation. Injection of IL-1β produced a decrease in food intake at 3 and 12 h after injection which was ameliorated at the 12 h time point by a sub-threshold dose of agouti-related peptide (AgRP). Investigation into neuron types in the NTS revealed that IL-1β injection was associated with an increase in c-Fos activity in NTS neurons expressing tyrosine hydroxylase (TH). Additionally, injection of IL-1β into the 4th ventricle did not produce c-Fos activation of neurons expressing pro-opiomelanocortin (POMC) in the ARH, cells known to be involved in producing anorexia in response to systemic inflammation. Double-label in situ hybridization revealed that TH neurons did not express IL-1 receptor I (IL1-RI) transcript, demonstrating that c-Fos activation of TH neurons in this setting was not via direct stimulation of IL-1β on TH neurons themselves. We conclude that IL-1β injection into the 4th ventricle produces anorexia and is accompanied by an increase in activation in TH neurons in the NTS. This provides evidence that the brainstem may be an important mediator of anorexia in the setting of inflammation. PMID:19028534
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1987-12-01
F T FILE I MEuSpecial Report 87-26 December 1987 US Army Corps of Engineers Cold Regions Research & Engineering Laboratory XYFREZ.4 User’s manual...Freeze/thaw User’s manual 19. ABSTRACT (Continue on reverse if necessary and identify by block number) - -- Using the program XYFREZ, version 4, one...may simulate two-dimensional conduction of heat, with or without phase change. The mathematical method employed uses finite elements in space and
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Hubácek, J A; Pistulková, H; Skodová, Z; Lánská, V; Poledne, R
2003-01-01
High plasma cholesterol is one of the risk factors of atherosclerosis. Both environmental (diet, physic activity) and genetic factors have been concerned in the development of hypercholesterolemia. Cholesterol 7 alpha hydroxylase (CYP-7A1) is a key enzyme in the bile acid synthesis and it plays an important role in cholesterol catabolism. The aim of the study was to establish the role of A-204-->C polymorphism in CYP-7A1 gene in plasma lipid determination in children. Using PCR and restriction analysis (BsaI) we have measured A-204-->C polymorphism in CYP-7A1 gene in two groups of children selected from opposite ends of the cholesterol distribution curve of 2000 children. Eighty-two children in high- (HCG) and eighty-six children in low- (LCG) cholesterolemic groups participated in the study. No significant difference was found in the frequencies of the genotypes or alleles of the A-204-->C polymorphism in the CYP-7A1 gene between HCG and LCG. In HCG, C/C-204 homozygotes have the highest and A/A homozygotes the lowest levels of LDL-cholesterol (4.21 +/- 0.68 mmol/l vers. 3.69 +/- 0.60 mmol/l, p < 0.05). No associations between lipid parameters and genotypes within the LCG group were found. The A-204-->C polymorphism in the gene for CYP-7A1 is not the major determinant of plasma lipid levels in childhood. Its impact is expressed only on high cholesterol background.
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ERIC Educational Resources Information Center
Steele, Sara M.; And Others
A study examined national participation in 4-H Club activities during the 1980s. The study population was a carefully drawn sample of 24,500 eighth graders from more than 1,000 public and private schools throughout the country as well as their parents and teachers. The study established that, as of 1988, about one of every six eighth-grade…
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Choi, Sul Ki; Kim, Hoe Suk; Jin, Tiefeng; Moon, Woo Kyung
2017-02-14
Lysyl oxidase (LOX) family genes catalyze collagen cross-link formation. To determine the effects of lysyl oxidase-like 4 (LOXL4) expression on breast tumor formation and metastasis, we evaluated primary tumor growth and lung metastasis in mice injected with LOXL4-knockdown MDA-MB-231 triple-negative human breast cancer cells. In addition, we analyzed overall survival in breast cancer patients based on LOXL4 expression using a public online database. In the mouse xenograft model, LOXL4 knockdown increased primary tumor growth and lung colonization as well as collagen I and IV, lysine hydroxylase 1 and 2, and prolyl 4-hydroxylase subunit alpha 1 and 2 levels. Second harmonic generation imaging revealed that LOXL4 knockdown resulted in the thickening of collagen bundles within tumors. In addition, weak LOXL4 expression was associated with poor overall survival in breast cancer patients from the BreastMark dataset, and this association was strongest in triple-negative breast cancer patients. These results demonstrate that weak LOXL4 expression leads to remodeling of the extracellular matrix through induction of collagen synthesis, deposition, and structural changes. These alterations in turn promote tumor growth and metastasis and are associated with poor clinical outcomes in triple-negative breast cancer.
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New insights into the structure, chemistry, and properties of Cu 4SnS 4
DOE Office of Scientific and Technical Information (OSTI.GOV)
Choudhury, Amitava; Mohapatra, Sudip; Yaghoobnejad Asl, Hooman
The ambient temperature structure of Cu 4SnS 4 has been revisited and the recently reported low temperature structure has been confirmed from single-crystal X-ray diffraction data. A structural phase transition from a large monoclinic unit cell at low temperature to a smaller orthorhombic unit cell at high temperature has been observed. The room temperature phase exhibited disorder in the two copper sites, which is a different finding from earlier reports. The low temperature monoclinic form crystallizes in P2 1/c space group, which is isostructural with Cu 4GeS 4. The phase transition has also been studied with variable temperature powder X-raymore » diffraction and 119Sn Mössbauer spectroscopy. The Seebeck coefficients and electrical resistivity of polycrystalline Cu 4SnS 4 are reported from 16 to 400 K on hot pressed pellets. Thermal conductivity measurements at high temperatures, 350 – 750 K exhibited very low thermal conductivities in the range 0.28 – 0.35 W K –1 m –1. In all the transport measurements the phase transition has been observed at around 232 K. Resistivity decreases, while Seebeck coefficient increases after the phase transition during warming up from low to high temperatures. This change in resistivity has been correlated with the results of first-principles electronic band structure calculations using highly-accurate screened-exchange local density approximation. It was found that both the low hole effective mass of 0.63 me for the Γ→Y crystallographic direction and small band gap, 0.49 eV, are likely to contribute to the observed higher conductivity of the orthorhombic phase. Cu 4SnS 4 is also electrochemically active and shows reversible reaction with lithium between 1.7 and 3.5 volts.« less
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New insights into the structure, chemistry, and properties of Cu 4SnS 4
Choudhury, Amitava; Mohapatra, Sudip; Yaghoobnejad Asl, Hooman; ...
2017-05-25
The ambient temperature structure of Cu 4SnS 4 has been revisited and the recently reported low temperature structure has been confirmed from single-crystal X-ray diffraction data. A structural phase transition from a large monoclinic unit cell at low temperature to a smaller orthorhombic unit cell at high temperature has been observed. The room temperature phase exhibited disorder in the two copper sites, which is a different finding from earlier reports. The low temperature monoclinic form crystallizes in P2 1/c space group, which is isostructural with Cu 4GeS 4. The phase transition has also been studied with variable temperature powder X-raymore » diffraction and 119Sn Mössbauer spectroscopy. The Seebeck coefficients and electrical resistivity of polycrystalline Cu 4SnS 4 are reported from 16 to 400 K on hot pressed pellets. Thermal conductivity measurements at high temperatures, 350 – 750 K exhibited very low thermal conductivities in the range 0.28 – 0.35 W K –1 m –1. In all the transport measurements the phase transition has been observed at around 232 K. Resistivity decreases, while Seebeck coefficient increases after the phase transition during warming up from low to high temperatures. This change in resistivity has been correlated with the results of first-principles electronic band structure calculations using highly-accurate screened-exchange local density approximation. It was found that both the low hole effective mass of 0.63 me for the Γ→Y crystallographic direction and small band gap, 0.49 eV, are likely to contribute to the observed higher conductivity of the orthorhombic phase. Cu 4SnS 4 is also electrochemically active and shows reversible reaction with lithium between 1.7 and 3.5 volts.« less
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High-Dose Hook Effect in 17-Hydroxyprogesterone Assay in a Patient with 21-Hydroxylase Deficiency.
Parlak, Mesut; Ellidağ, Hamit Yaşar; Türkkahraman, Doğa
2015-12-01
Congenital adrenal hyperplasia (CAH) describes a group of disorders characterized by enzyme defects in adrenal steroidogenesis. 21-hydroxylase deficiency (21-OHD) is the most commonly encountered form. The analysis of steroids in pediatric cases requires high-sensitivity assays. A 14-year-old Syrian girl was referred for evaluation of short stature, amenorrhea, and hirsutism. On physical examination, breast development was Tanner stage 1. She had a phallic clitoris with a single urogenital orifice. Laboratory findings revealed primary adrenal deficiency with high androgen levels and low levels of 17-hydroxyprogesterone (17-OHP), (<0.05 ng/mL) and estrogen. This unexpected result led to suspicion of a high-dose hook effect. The measurement was repeated after 1/10 dilution of serum, and a high level of 17-OHP (115.4 ng/mL) was detected with the same test-enzyme-linked immunosorbent assay (ELISA). Simple virilizing form of CAH (21-OHD) was suspected and confirmed with genetic analysis. After initiation of glucocorticoid therapy, breast development was noted along with a decrease in testosterone level and an increase in estrogen level. To our knowledge, this is the first case report of hook effect for 17-OHP immunoassay in a patient with 21-OHD. High-dose hook effect should be suspected in patients with CAH when the test results are incompatible with one another. Additionally, this case demonstrates that a high testosterone level can block aromatase activity and consequently also estrogen production and breast development.
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Cui, Lijie; Ni, Xiaoling; Ji, Qian; Teng, Xiaojuan; Yang, Yanru; Wu, Chao; Zekria, David; Zhang, Dasheng; Kai, Guoyin
2015-01-01
Camptothecin (CPT) belongs to a group of monoterpenoidindole alkaloids (TIAs) and its derivatives such as irinothecan and topothecan have been widely used worldwide for the treatment of cancer, giving rise to rapidly increasing market demands. Genes from Catharanthus roseus encoding strictosidine synthase (STR) and geraniol 10-hydroxylase (G10H), were separately and simultaneously introduced into Ophiorrhiza pumila hairy roots. Overexpression of individual G10H (G lines) significantly improved CPT production with respect to non-transgenic hairy root cultures (NC line) and single STR overexpressing lines (S lines), indicating that G10H plays a more important role in stimulating CPT accumulation than STR in O. pumila. Furthermore, co-overexpression of G10H and STR genes (SG Lines) caused a 56% increase on the yields of CPT compared to NC line and single gene transgenic lines, showed that simultaneous introduction of G10H and STR can produce a synergistic effect on CPT biosynthesis in O. pumila. The MTT assay results indicated that CPT extracted from different lines showed similar anti-tumor activity, suggesting that transgenic O. pumila hairy root lines could be an alternative approach to obtain CPT. To our knowledge, this is the first report on the enhancement of CPT production in O. pumila employing a metabolic engineering strategy. PMID:25648209
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Copper-containing monooxygenases: enzymatic and biomimetic studies of the O-atom transfer catalysis.
Blain, Ingrid; Slama, Patrick; Giorgi, Michel; Tron, Thierry; Réglier, Marius
2002-04-01
This review reports our recent studies or the mechanism of O-atom transfer to a benzylic C-H bond promoted by Dopamine beta-Hydroxylase (DBH) and its biomimetic models. We demonstrate that it is possible to carry out parallel and comparative studies on this enzyme (DBH) and its biomimetic models with the same substrate: 2-aminoindane (3). It was chosen because its two stereogenic centers, both in benzylic positions, make it very powerful for studying the stereochemistry of an O-atom transfer reaction. DBH-catalyzed hydroxylation of 3 produced exclusively 14% of trans-(1S,2S)-2-amino-1-indanol (4) (93% ee). Studies with stereospecifically deuterium-labeled 2-aminoindanes (1R,2S)-3b and (1S,2S)-3a showed that the formation of 4 was the rcsult of an overall process with retention of configuration where an O-atom is stereospecifically inserted in the trans pro-S position of the substrate. With copper(I) and (II) complexes of IndPY2 ligands we studied the reaction with dioxygen and observed an O-atom transfer to a benzylic C-H bond which was performed in the same manner as that of DBH. With the deuterium-labeled cis-2-d-IndPY2 ligand, we demonstrated that the reaction occurs by a stereospecific process with retention of configuration. In both cases (enzymatic vs. biomimetic) the O-atom transfers occur in a two-step process involving radical intermediates.
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Laćan, Goran; Dang, Hoa; Middleton, Blake; Horwitz, Marcus A.; Tian, Jide; Melega, William P.; Kaufman, Daniel L.
2018-01-01
We previously showed that, in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease (PD), vaccination with bacillus Calmette-Guerin (BCG) prior to MPTP exposure limited the loss of striatal dopamine (DA) and dopamine transporter (DAT) and prevented the activation of nigral microglia. Here, we conducted BCG dose studies and investigated the mechanisms underlying BCG vaccination’s neuroprotective effects in this model. We found that a dose of 1 × 106 cfu BCG led to higher levels of striatal DA and DAT ligand binding (28% and 42%, respectively) in BCG-vaccinated vs. unvaccinated MPTP-treated mice, but without a significant increase in substantia nigra tyrosine hydroxylase-staining neurons. Previous studies showed that BCG can induce regulatory T cells (Tregs) and that Tregs are neuroprotective in models of neurodegenerative diseases. However, MPTP is lymphotoxic, so it was unclear whether Tregs were maintained after MPTP treatment and whether a relationship existed between Tregs and the preservation of striatal DA system integrity. We found that, 21 days post-MPTP treatment, Treg levels in mice that had received BCG prior to MPTP were threefold greater than those in MPTP-only-treated mice and elevated above those in saline-only-treated mice, suggesting that the persistent BCG infection continually promoted Treg responses. Notably, the magnitude of the Treg response correlated positively with both striatal DA levels and DAT ligand binding. Therefore, BCG vaccine-mediated neuroprotection is associated with Treg levels in this mouse model. Our results suggest that BCG-induced Tregs could provide a new adjunctive therapeutic approach to ameliorating pathology associated with PD and other neurodegenerative diseases. PMID:23907992
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Yang, Yunwen; Yu, Xiaowen; Zhang, Yue; Ding, Guixia; Zhu, Chunhua; Huang, Songming; Jia, Zhanjun; Zhang, Aihua
2018-04-16
Renal hypoxia occurs in acute kidney injury (AKI) of various etiologies. Activation of hypoxia-inducible transcription factor (HIF) has been identified as an important mechanism of cellular adaptation to low oxygen. Preconditional HIF activation protects against AKI, suggesting a new approach in AKI treatment. HIF is degraded under normoxic conditions mediated by oxygen-dependent hydroxylation of specific prolyl residues of the regulative α-subunits by HIF prolyl hydroxylases (PHD). FG-4592 is a novel, orally active, small-molecule HIF PHD inhibitor for the treatment of anemia in patients with chronic kidney disease (CKD). The current study aimed to evaluate the effect of FG-4592 (Roxadustat) on cis -diamminedichloroplatinum (cisplatin)-induced kidney injury. In mice, pretreatment with FG-4592 markedly ameliorated cisplatin-induced kidney injury as shown by the improved renal function (blood urea nitrogen (BUN), serum creatinine (Scr), and cystatin C) and kidney morphology (periodic acid-Schiff (PAS) staining) in line with a robust blockade of renal tubular injury markers of kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Meanwhile, the renal apoptosis and inflammation induced by cisplatin were also strikingly attenuated in FG-4592-treated mice. Along with the protective effects shown above, FG-4592 pretreatment strongly enhanced HIF-1α in tubular cells, as well as the expressions of HIF target genes. FG-4592 alone did not affect the renal function and morphology in mice. In vitro , FG-4592 treatment significantly up-regulated HIF-1α and protected the tubular cells against cisplatin-induced apoptosis. In summary, FG-4592 treatment remarkably ameliorated the cisplatin-induced kidney injury possibly through the stabilization of HIF. Thus, besides the role in treating CKD anemia, the clinical use of FG-4592 also could be extended to AKI. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical
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Code of Federal Regulations, 2010 CFR
2010-07-01
... 32 National Defense 2 2010-07-01 2010-07-01 false U.S. guaranty. 274.4 Section 274.4 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS REGULATIONS GOVERNING COMPETITIVE BIDDING ON U.S. GOVERNMENT GUARANTEED MILITARY EXPORT LOAN AGREEMENTS § 274...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... 32 National Defense 2 2011-07-01 2011-07-01 false U.S. guaranty. 274.4 Section 274.4 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS REGULATIONS GOVERNING COMPETITIVE BIDDING ON U.S. GOVERNMENT GUARANTEED MILITARY EXPORT LOAN AGREEMENTS § 274...
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ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo.
Beilschmidt, Lena Kristina; Ollagnier de Choudens, Sandrine; Fournier, Marjorie; Sanakis, Ioannis; Hograindleur, Marc-André; Clémancey, Martin; Blondin, Geneviève; Schmucker, Stéphane; Eisenmann, Aurélie; Weiss, Amélie; Koebel, Pascale; Messaddeq, Nadia; Puccio, Hélène; Martelli, Alain
2017-05-11
Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe 4 S 4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe 2 S 2 -containing proteins that combine all features of Fe-S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe-S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe 4 S 4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1-ISCA2 complex seem to exist.
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ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo
Beilschmidt, Lena Kristina; Ollagnier de Choudens, Sandrine; Fournier, Marjorie; Sanakis, Ioannis; Hograindleur, Marc-André; Clémancey, Martin; Blondin, Geneviève; Schmucker, Stéphane; Eisenmann, Aurélie; Weiss, Amélie; Koebel, Pascale; Messaddeq, Nadia; Puccio, Hélène; Martelli, Alain
2017-01-01
Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron–sulfur cluster (Fe–S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe4S4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe2S2-containing proteins that combine all features of Fe–S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe–S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe4S4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1–ISCA2 complex seem to exist. PMID:28492233
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Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase.
Mitchell, Andrew J; Dunham, Noah P; Martinie, Ryan J; Bergman, Jonathan A; Pollock, Christopher J; Hu, Kai; Allen, Benjamin D; Chang, Wei-Chen; Silakov, Alexey; Bollinger, J Martin; Krebs, Carsten; Boal, Amie K
2017-10-04
Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orientation of the substrate C-H and ferryl Fe-O bonds, primarily by direction of the oxo group into one of two cis-related coordination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo position. Comparison of high-resolution X-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the l-arginine 3-hydroxylase, VioC, reveals coordinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.
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NASA Technical Reports Server (NTRS)
Sugano, Mami; Nakagawa, Yuriko; Nyunoya, Hiroshi; Nakamura, Teruko
2004-01-01
Expressions of the gibberellin biosynthesis gene were investigated in a normal upright type and a gravi-response mutant, a weeping type of Japanese flowering cherry (Prunus spachiana), that is unable to support its own weight and elongates downward. A segment of the gibberellin 3 beta-hydroxylase cDNA of Prunus spachiana (Ps3ox), which is responsible for active gibberellin synthesis, was amplified by using real-time RT-PCR. The content of Ps3ox mRNA in the weeping type was much greater than that in the upright type, while the endogenous gibberellin level was much higher in the elongating zone of the weeping type. These results suggest that the amount and distribution of synthesized gibberellin regulate secondary xylem formation, and the unbalanced distribution of gibberellin affects the gravi-response of the Prunus tree.
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Lacher, M D; Siegenthaler, A; Jäger, R; Yan, Xi; Hett, S; Xuan, L; Saurer, S; Lareu, R R; Dharmarajan, A M; Friis, R
2003-05-01
Using differential display, we isolated DDC-4, a secreted frizzled-related protein (sFRP), which is induced in the physiological apoptosis of hormonally regulated, reproductive tissues such as mammary gland, prostate, corpus luteum and uterus. The role of this gene in apoptosis was studied in animals overexpressing ectopic DDC-4/sFRP-4. Transgenic mice bearing the DDC-4/sFRP-4 cDNA under the control of the MMTV-LTR promoter showed lactational insufficiency and many apoptotic cells in the alveoli between day 19 of pregnancy and day 4 of lactation as demonstrated by TUNEL reaction and the presence of activated caspase-3. We performed a PKB/Akt kinase assay and studied several of its substrates using phosphorylation-specific antibodies to show reduced phosphorylation in PKB/Akt itself, as well as in glycogen synthetase kinase-3beta (GSK-3beta), BAD, and Forkhead. Taken together, our results show a role for DDC-4/sFRP-4 in abrogating an epithelial cell survival pathway at the onset of mammary gland involution.
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Conformational analysis of the N-terminal sequence Met1 Val60 of the tyrosine hydroxylase
NASA Astrophysics Data System (ADS)
Alieva, Irada N.; Mustafayeva, Narmina N.; Gojayev, Niftali M.
2006-03-01
Molecular mechanics method and molecular dynamics (MD) simulation techniques are used to study the behavior and the effect of the amino acids substitution on structure and molecular dynamics of the specific portion of Met1-Val60 amino acid residues from N-terminal regulatory domain of the tyrosine hydroxylase (TH) and its mutants in which the positively charged arginine residues at positions 37 and 38 were replaced by electrically neutral Gly and negatively charged Glu, and serine residue at position 40 was replaced by Ala or Asp residue. Our study allowed us to make the following conclusions: (i) the higher conformational flexibility of the Met1-Arg16 sequence is revealed in comparision to other part of the N-terminus; (ii) the stretch of amino acid residues Met30-Ser40 within the N-terminus forms β-turn so that two α-helices (residues 16-29 and residues 41-60) are paralel one another; (ii) the significant differences that are observed for the Arg37→Gly37, Arg37-Arg38→Glu37-Glu38 mutant segments indicates that the positive charge of the Arg37 and Arg38 residues is one of the main factor that maintains the characteristic of the turn; (ii) no major conformational changes are observed between Ser40→Ala40, and Ser40→Asp40 mutant segments.
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Grunt, Sebastian; Steinlin, Maja; Weisstanner, Christian; Schöning, Martin; Mullis, Primus E; Flück, Christa E
2013-01-01
Prenatal glucocorticoid (GC) treatment of the female fetus with 21-hydroxylase deficiency (21-OHD) may prevent genital virilization and androgen effects on the brain, but prenatal GC therapy is controversial because of possible adverse effects on fetal programming, the cardiovascular system and the brain. We report 2 patients with congenital adrenal hyperplasia (CAH) due to 21-OHD who were treated prenatally with dexamethasone, suffered from an acute encephalopathy and showed focal and multifocal cortical and subcortical diffusion restrictions in early MRI and signs of permanent alterations in the follow-up neuroimaging studies. Both patients recovered from the acute episode. Whereas the first patient recovered without neurological sequelae the second patient showed hemianopsia and spastic hemiplegia in the neurological follow-up examination. These are 2 children with CAH, both treated prenatally with high doses of dexamethasone to prevent virilization. The question arises whether prenatal high-dose GC treatment in patients with CAH might represent a risk factor for brain lesions in later life. Adverse effects/events should be reported systematically in patients undergoing prenatal GC treatment and long-term follow-up studies involving risk factors for cerebrovascular disease should be performed. Copyright © 2013 S. Karger AG, Basel.
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Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency
NASA Technical Reports Server (NTRS)
Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo
2002-01-01
Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.
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Chumarina, Margarita; Azevedo, Carla; Bigarreau, Julie; Vignon, Clémentine; Kim, Kwang-Soo; Li, Jia-Yi; Roybon, Laurent
2017-03-01
Mouse embryonic stem cell (mESC) lines were derived by crossing heterozygous transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase (TH) promoter, with homozygous alpha-synuclein (aSYN) mice expressing human mutant SNCA A53T under the control of the mouse Prion promoter (MoPrP), or wildtype (WT) mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Bhat, Rayees A.; Kumar, D.; Malla, Manzoor A.; Bhat, Sami U.; Khan, Md Shahzad; Manzoor, Ovais; Srivastava, Anurag; Naikoo, Rawoof A.; Mohsin, Mohd; Mir, Muzzaffar A.
2018-03-01
S-Benzyl-β-N-[3-(4-hydroxy-3-methoxy-phenylallylidene)]dithiocarbazate (HL1), Schiff base of S-benzyl dithiocarbazate, was synthesized by 1:1 condensation between S-benzyl dithiocarbazate and 4-hydroxy-3-methoxy cinnamaldehyde. The nitrogen-sulfur Schiff base (HL1) was characterized by Mass, FT-IR, H1-NMR, Raman, and UV-VIS spectroscopic techniques. Theoretical quantum chemical calculations were performed using DFT in combination with B3LYP exchange correlation functional and 6-311++ G (d, p) basis sets level. The calculated values of chemical potential (μ), HOMO-LUMO energy gap, chemical hardness, softness (S), ionization energy (IE), electron affinity (EA), dipole moment (D) and relative stabilization energy of the compound were 0.14881 eV, 0.12542 eV, 0.06271 eV, 3.37299 eV, -0.21152 eV, -0.08610 eV, 4.4090 Debye and -1753.350 eV respectively. Theoretically calculated parameters like H1-NMR, FT-IR, UV-VIS, Raman, electrostatic potential and HOMO-LUMO energy gap are in good agreement with experimental results. Also, in-vitro cytotoxicity studies were done against two habitually infection causing bacteria strains including gram-positive (S. aureus) and gram-negative (E. coli) for antibacterial activity. The results showed appreciable biological activity and the activity increased with increase in dose.
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S3/S4 Integrated Truss being moved into the Space Shuttle Payloa
2007-02-07
In the Space Station Processing Facility, an overhead crane moves the S3/S4 integrated truss to a payload canister. After it is stowed in the canister, the S3/S4 truss will be transported to the launch pad. The truss is the payload on mission STS-117, targeted for launch on March 15.
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S3/S4 Integrated Truss being moved into the Space Shuttle Payloa
2007-02-07
In the Space Station Processing Facility, an overhead crane settles the S3/S4 integrated truss into the payload canister. After it is stowed in the canister, the S3/S4 truss will be transported to the launch pad. The truss is the payload on mission STS-117, targeted for launch on March 15.
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Systematic Sensor Selection Strategy (S4) User Guide
NASA Technical Reports Server (NTRS)
Sowers, T. Shane
2012-01-01
This paper describes a User Guide for the Systematic Sensor Selection Strategy (S4). S4 was developed to optimally select a sensor suite from a larger pool of candidate sensors based on their performance in a diagnostic system. For aerospace systems, selecting the proper sensors is important for ensuring adequate measurement coverage to satisfy operational, maintenance, performance, and system diagnostic criteria. S4 optimizes the selection of sensors based on the system fault diagnostic approach while taking conflicting objectives such as cost, weight and reliability into consideration. S4 can be described as a general architecture structured to accommodate application-specific components and requirements. It performs combinational optimization with a user defined merit or cost function to identify optimum or near-optimum sensor suite solutions. The S4 User Guide describes the sensor selection procedure and presents an example problem using an open source turbofan engine simulation to demonstrate its application.
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Khoroshilova, Natalia; Popescu, Codrina; Münck, Eckard; Beinert, Helmut; Kiley, Patricia J.
1997-01-01
The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mössbauer spectroscopy. Our data showed that this form of FNR contained a [4Fe-4S]2+ cluster (δ = 0.45 mm/s; ΔEQ = 1.22 mm/s) and that the [4Fe-4S]2+ cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow–green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form. Little [3Fe-4S] cluster was, however, detected by EPR. According to Mössbauer spectroscopy, the [4Fe-4S]2+ cluster was converted in about 60% yield to a [2Fe-2S]2+ cluster (δ = 0.28 mm/s; ΔEQ = 0.58 mm/s) following 17 min of exposure to air. The [2Fe-2S]2+ cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding). However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S]2+ cluster could be largely restored from the [2Fe-2S]2+ form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [2Fe-2S]2+ cluster has any biological significance, e.g., as an in vivo intermediate that is more rapidly converted to the active form than the apoprotein. PMID:9177174
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Theilmann, J L; Skow, L C; Baker, J F; Womack, J E
1989-01-01
Genomic DNAs from animals representing six breeds of cattle (Angus, Brahman, Hereford, Holstein, Jersey and Texas Longhorn) were screened with cloned gene probes in a search for restriction fragment length polymorphisms (RFLPs). Eleven RFLPs were identified using seven different probes: growth hormone, prolactin, osteonectin, alpha A-crystallin, gamma crystallin, fibronectin and 21-steroid hydroxylase. The frequencies of the alleles identified by each probe were calculated and compared in a limited sampling of the six bovine breeds. These polymorphisms greatly enhance the pool of immunogenetic, biochemical and molecular markers available in cattle for linkage analysis, testing of parentage, and distinction of breeds.
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Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site
2015-01-01
Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain. PMID:25453233
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S3/S4 Integrated Truss being moved into the Space Shuttle Payloa
2007-02-07
In the Space Station Processing Facility, an overhead crane lowers the S3/S4 integrated truss into the open bay of the payload canister. After it is stowed in the canister, the S3/S4 truss will be transported to the launch pad. The truss is the payload on mission STS-117, targeted for launch on March 15.
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S3/S4 Integrated Truss being moved into the Space Shuttle Payloa
2007-02-07
In the Space Station Processing Facility, an overhead crane lowers the S3/S4 integrated truss toward the open doors of the payload canister. After it is stowed in the canister, the S3/S4 truss will be transported to the launch pad. The truss is the payload on mission STS-117, targeted for launch on March 15.
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S3/S4 Integrated Truss being moved into the Space Shuttle Payloa
2007-02-07
In the Space Station Processing Facility, workers attach an overhead crane to the S3/S4 integrated truss in order to move it to the payload canister. After it is stowed in the canister, the S3/S4 truss will be transported to the launch pad. The truss is the payload on mission STS-117, targeted for launch on March 15.
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Perche-Letuvée, Phanélie; Kathirvelu, Velavan; Berggren, Gustav; Clemancey, Martin; Latour, Jean-Marc; Maurel, Vincent; Douki, Thierry; Armengaud, Jean; Mulliez, Etienne; Fontecave, Marc; Garcia-Serres, Ricardo; Gambarelli, Serge; Atta, Mohamed
2012-01-01
Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called “Radical-SAM.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX3CX2C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed. PMID:23043105
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Role of the S4-S5 linker in CNG channel activation.
Kusch, Jana; Zimmer, Thomas; Holschuh, Jascha; Biskup, Christoph; Schulz, Eckhard; Nache, Vasilica; Benndorf, Klaus
2010-10-20
Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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RAGAN, C. M.; LONSTEIN, J. S.
2014-01-01
In female mammals, the postpartum period involves dramatic shifts in many socioemotional behaviors. This includes a suppression of anxiety-related behaviors that requires recent physical contact with offspring. Factors contributing to differences among females in their susceptibility to the anxiety-modulating effect of offspring contact are unknown, but could include their innate anxiety and brain monoaminergic activity. Anxiety behavior was assessed in a large group of nulliparous female rats and the least-anxious and most-anxious tertiles were mated. Anxiety was assessed again postpartum after females were permitted or prevented from contacting their offspring 4 h before testing. Levels of dopamine β-hydroxylase (DBH, norepinephrine synthesizing enzyme) and tryptophan hydroxylase- 2 (TPH2, serotonin synthesizing enzyme) were measured in the brainstem and dorsal raphe, respectively. It was found that anxiety-related behavior in the two groups did not differ when dams were permitted contact with offspring before testing. Removal of the offspring before testing, however, differentially affected anxiety based on dams’ innate anxiety. Specifically, dams reverted back to their pre-mating levels of anxiety such that offspring removal slightly increased anxiety in the most-anxious females but greatly lowered anxiety in the least-anxious females. This reduction in anxiety in the least-anxious females after litter removal was associated with lower brainstem DBH. There was no relationship between females’ anxiety and dorsal raphe TPH2. Thus, a primary effect of recent contact with offspring on anxiety-related behavior in postpartum rats is to shift females away from their innate anxiety to a more moderate level of responding. This effect is particularly true for females with the lowest anxiety, may be mediated by central noradrenergic systems, and has implications for their ability to attend to their offspring. PMID:24161285
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An additional substrate binding site in a bacterial phenylalanine hydroxylase
Ronau, Judith A.; Paul, Lake N.; Fuchs, Julian E.; Corn, Isaac R.; Wagner, Kyle T.; Liedl, Klaus R.; Abu-Omar, Mahdi M.; Das, Chittaranjan
2014-01-01
Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, existence of PAH regulation in evolutionarily distant organisms, such as certain bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 µM for phenylalanine. Under the same conditions, no detectable binding was observed in ITC for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) lead to impaired binding, consistent with the presence of distal site binding in solution. Kinetic analysis reveals that the distal site mutants suffer a discernible loss in their catalytic activity. However, x-ray structures of Y155A and F258A, two of the mutants showing more noticeable defect in their activity, show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PMID:23860686
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Induction of tyrosine hydroxylase mRNA by nicotine in rat midbrain is inhibited by mifepristone
Radcliffe, Pheona M.; Sterling, Carol R.; Tank, A. William
2009-01-01
Repeated nicotine administration induces tyrosine hydroxylase (TH) mRNA in rat midbrain. In this study we investigate the mechanisms responsible for this response using two models of midbrain dopamine neurons, rat ventral midbrain slice explant cultures and mouse MN9D cells. In both models nicotine stimulates TH gene transcription rate in a dose-dependent manner. However, this stimulation is short-lived, lasting for 1 hr, but less than 3 hr, and is not sufficient to induce TH mRNA or TH protein. Nicotine elevates circulating glucocorticoids, which induce TH expression in some model systems. We tested the hypothesis that the effect of nicotine on midbrain TH mRNA is mediated by the glucocorticoid receptor. When rats are administered the glucocorticoid receptor antagonist mifepristone, the induction of TH mRNA by nicotine in both substantia nigra and ventral tegmentum is inhibited. Furthermore, the glucocorticoid receptor agonist dexamethasone stimulates TH gene transcription for sustained periods of time in both midbrain slices and MN9D cells, leading to induction of TH mRNA and TH protein. Our results are consistent with the hypothesis that nicotine induces TH mRNA in midbrain by elevating glucocorticoids, which then act on glucocorticoid receptors in dopamine neurons leading to transcriptional activation of the TH gene. PMID:19476543
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Telotristat Ethyl, a Tryptophan Hydroxylase Inhibitor for the Treatment of Carcinoid Syndrome.
Kulke, Matthew H; Hörsch, Dieter; Caplin, Martyn E; Anthony, Lowell B; Bergsland, Emily; Öberg, Kjell; Welin, Staffan; Warner, Richard R P; Lombard-Bohas, Catherine; Kunz, Pamela L; Grande, Enrique; Valle, Juan W; Fleming, Douglas; Lapuerta, Pablo; Banks, Phillip; Jackson, Shanna; Zambrowicz, Brian; Sands, Arthur T; Pavel, Marianne
2017-01-01
Purpose Preliminary studies suggested that telotristat ethyl, a tryptophan hydroxylase inhibitor, reduces bowel movement (BM) frequency in patients with carcinoid syndrome. This placebo-controlled phase III study evaluated telotristat ethyl in this setting. Patients and Methods Patients (N = 135) experiencing four or more BMs per day despite stable-dose somatostatin analog therapy received (1:1:1) placebo, telotristat ethyl 250 mg, or telotristat ethyl 500 mg three times per day orally during a 12-week double-blind treatment period. The primary end point was change from baseline in BM frequency. In an open-label extension, 115 patients subsequently received telotristat ethyl 500 mg. Results Estimated differences in BM frequency per day versus placebo averaged over 12 weeks were -0.81 for telotristat ethyl 250 mg ( P < .001) and ‒0.69 for telotristat ethyl 500 mg ( P < .001). At week 12, mean BM frequency reductions per day for placebo, telotristat ethyl 250 mg, and telotristat ethyl 500 mg were -0.9, -1.7, and -2.1, respectively. Responses, predefined as a BM frequency reduction ≥ 30% from baseline for ≥ 50% of the double-blind treatment period, were observed in 20%, 44%, and 42% of patients given placebo, telotristat ethyl 250 mg, and telotristat ethyl 500 mg, respectively. Both telotristat ethyl dosages significantly reduced mean urinary 5-hydroxyindole acetic acid versus placebo at week 12 ( P < .001). Mild nausea and asymptomatic increases in gamma-glutamyl transferase were observed in some patients receiving telotristat ethyl. Follow-up of patients during the open-label extension revealed no new safety signals and suggested sustained BM responses to treatment. Conclusion Among patients with carcinoid syndrome not adequately controlled by somatostatin analogs, treatment with telotristat ethyl was generally safe and well tolerated and resulted in significant reductions in BM frequency and urinary 5-hydroxyindole acetic acid.
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Kan, Inna; Ben-Zur, Tali; Barhum, Yael; Levy, Yossef S; Burstein, Alex; Charlow, Tirza; Bulvik, Shlomo; Melamed, Eldad; Offen, Daniel
2007-05-23
Parkinson's disease (PD) is a neurodegenerative disorder, caused by a selective loss of dopaminergic neurons in the substantia nigra. In PD, the best therapeutic modalities cannot halt the degeneration. The selective hallmark pathology and the lack of effective treatment make PD an appropriate candidate for cell replacement therapy. Adult autologous bone-marrow-derived mesenchymal stem cells (MSCs) have been investigated as candidates for cell replacement strategies. Several laboratories, including ours, have induced MSCs into neuron-like cells demonstrating a variety of neuronal markers including dopaminergic characteristics, such as the expression of tyrosine hydroxylase (TH). This project aimed to induce MSCs into mature dopamine secreting cells and to generate a bioassay to evaluate the induction. For that purpose, we created a reporter vector containing a promoter of TH, the rate-limiting enzyme in the dopamine synthesis and red fluorescent protein DsRed2. Transfection of human neuroblastoma, dopamine synthesizing, SH-SY5Y cells confirmed the reliability of the constructed reporter plasmid. Following dopaminergic differentiation of the transfected human MSCs cells, TH expressing cells were identified and quantified using flow cytometry. Further study revealed that not only did the differentiated cells activate TH promoter but they also expressed TH protein and secreted dopamine. The reported results indicate that MSCs may be primed in vitro towards a dopaminergic fate offering the promise of innovative therapy for currently incurable human disorders, including PD.
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Walker, Linda C; Overstreet, Mayra A; Yeowell, Heather N
2005-01-01
Lysyl hydroxylases 1, 2, and 3 catalyse the hydroxylation of specific lysines in collagen. A small percentage of these hydroxylysine residues are precursors for the cross-link formation essential for the tensile strength of collagen. Lysyl hydroxylase 2 (LH2) exists as two alternatively-spliced forms; the long transcript (the major ubiquitously-expressed form) includes a 63 bp exon (13A) that is spliced out in the short form (expressed, together with the long form, in human kidney, spleen, liver, and placenta). This study shows that this alternative splicing event can be regulated by both cell density and cycloheximide (CHX). Although only the long form of LH2 is detected in untreated confluent human skin fibroblasts, after 24 h treatment with CHX the short LH2 transcript is also expressed. In kidney cells, in which both LH2 transcripts are equally expressed, the long LH2 transcript is significantly decreased after 24 h CHX treatment, whereas expression of the short transcript is slightly increased. This suggests that, in kidney cells, the splicing mechanism for the inclusion of exon 13A in LH2 requires a newly-synthesized protein factor that is suppressed by CHX, whereas, in skin fibroblasts in which levels of LH2 (long) are unaffected, CHX appears to suppress a factor that inhibits exclusion of exon 13A, thereby promoting expression of LH2 (short). As these alternate transcripts of LH2 may have specificity for hydroxylation of lysines in either telopeptide or helical collagen domains, their relative expression determines the type of cross-links formed, thereby affecting collagen strength. Therefore, any perturbation of the regulation of LH2 splicing could influence the stability of the extracellular matrix and contribute to specific connective tissue disorders.
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Effects of Zb states and bottom meson loops on ϒ (4 S )→ϒ (1 S ,2 S )π+π- transitions
NASA Astrophysics Data System (ADS)
Chen, Yun-Hua; Cleven, Martin; Daub, Johanna T.; Guo, Feng-Kun; Hanhart, Christoph; Kubis, Bastian; Meißner, Ulf-G.; Zou, Bing-Song
2017-02-01
We study the dipion transitions ϒ (4 S )→ϒ (n S )π+π- (n =1 ,2 ) . In particular, we consider the effects of the two intermediate bottomoniumlike exotic states Zb(10610 ) and Zb(10650 ) as well as bottom meson loops. The strong pion-pion final-state interactions, especially including channel coupling to K K ¯ in the S wave, are taken into account model independently by using dispersion theory. Based on a nonrelativistic effective field theory we find that the contribution from the bottom meson loops is comparable to those from the chiral contact terms and the Zb-exchange terms. For the ϒ (4 S )→ϒ (2 S )π+π- decay, the result shows that including the effects of the Zb exchange and the bottom meson loops can naturally reproduce the two-hump behavior of the π π mass spectra. Future angular distribution data are decisive for the identification of different production mechanisms. For the ϒ (4 S )→ϒ (1 S )π+π- decay, we show that there is a narrow dip around 1 GeV in the π π invariant mass distribution, caused by the final-state interactions. The distribution is clearly different from that in similar transitions from lower ϒ states, and needs to be verified by future data with high statistics. Also we predict the decay width and the dikaon mass distribution of the ϒ (4 S )→ϒ (1 S )K+K- process.
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Hyperinsulinemia and Insulin Resistance in Dopamine β-Hydroxylase Deficiency
Arnold, Amy C.; Garland, Emily M.; Celedonio, Jorge E.; Raj, Satish R.; Abumrad, Naji N.; Biaggioni, Italo; Robertson, David; Luther, James M.
2017-01-01
Context: Dopamine β-hydroxylase (DBH) deficiency is a rare genetic disorder characterized by failure to convert dopamine to norepinephrine. DBH-deficient patients lack sympathetic adrenergic function and are therefore predisposed to orthostatic hypotension. DBH-deficient mice exhibit hyperinsulinemia, lower plasma glucose levels, and insulin resistance due to loss of tonic sympathetic inhibition of insulin secretion. The impact of DBH deficiency on glucose homeostasis in humans is unknown. Case Description: We describe the metabolic profile of an adolescent female DBH-deficient patient. The patient underwent genetic testing, cardiovascular autonomic function testing, and evaluation of insulin secretion and sensitivity with hyperglycemic clamp under treatment-naive conditions. All procedures were repeated after 1 year of treatment with the norepinephrine prodrug droxidopa (300 mg, 3 times a day). Genetic testing showed a homozygous mutation in the DBH gene (rs74853476). Under treatment-naive conditions, she had undetectable plasma epinephrine and norepinephrine levels, resulting in sympathetic noradrenergic failure and orthostatic hypotension (−32 mm Hg supine to seated). She had high adiposity (41%) and fasting plasma insulin levels (25 μU/mL), with normal glucose (91 mg/dL). Hyperglycemic clamp revealed increased glucose-stimulated insulin secretion and insulin resistance. Droxidopa restored plasma norepinephrine and improved orthostatic tolerance, with modest effects on glucose homeostasis. Conclusions: We provide evidence for impairment in cardiovascular autonomic regulation, hyperinsulinemia, enhanced glucose-stimulated insulin secretion, and insulin resistance in a DBH-deficient patient. These metabolic derangements were not corrected by chronic droxidopa treatment. These findings provide insight into the pathophysiology and treatment of DBH deficiency and into the importance of catecholaminergic mechanisms to resting metabolism. PMID:27778639
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Hyperinsulinemia and Insulin Resistance in Dopamine β-Hydroxylase Deficiency.
Arnold, Amy C; Garland, Emily M; Celedonio, Jorge E; Raj, Satish R; Abumrad, Naji N; Biaggioni, Italo; Robertson, David; Luther, James M; Shibao, Cyndya A
2017-01-01
Dopamine β-hydroxylase (DBH) deficiency is a rare genetic disorder characterized by failure to convert dopamine to norepinephrine. DBH-deficient patients lack sympathetic adrenergic function and are therefore predisposed to orthostatic hypotension. DBH-deficient mice exhibit hyperinsulinemia, lower plasma glucose levels, and insulin resistance due to loss of tonic sympathetic inhibition of insulin secretion. The impact of DBH deficiency on glucose homeostasis in humans is unknown. We describe the metabolic profile of an adolescent female DBH-deficient patient. The patient underwent genetic testing, cardiovascular autonomic function testing, and evaluation of insulin secretion and sensitivity with hyperglycemic clamp under treatment-naive conditions. All procedures were repeated after 1 year of treatment with the norepinephrine prodrug droxidopa (300 mg, 3 times a day). Genetic testing showed a homozygous mutation in the DBH gene (rs74853476). Under treatment-naive conditions, she had undetectable plasma epinephrine and norepinephrine levels, resulting in sympathetic noradrenergic failure and orthostatic hypotension (-32 mm Hg supine to seated). She had high adiposity (41%) and fasting plasma insulin levels (25 μU/mL), with normal glucose (91 mg/dL). Hyperglycemic clamp revealed increased glucose-stimulated insulin secretion and insulin resistance. Droxidopa restored plasma norepinephrine and improved orthostatic tolerance, with modest effects on glucose homeostasis. We provide evidence for impairment in cardiovascular autonomic regulation, hyperinsulinemia, enhanced glucose-stimulated insulin secretion, and insulin resistance in a DBH-deficient patient. These metabolic derangements were not corrected by chronic droxidopa treatment. These findings provide insight into the pathophysiology and treatment of DBH deficiency and into the importance of catecholaminergic mechanisms to resting metabolism. Copyright © 2017 by the Endocrine Society
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Catalytic water dissociation by greigite Fe3S4 surfaces: density functional theory study
Roldan, A.; de Leeuw, N. H.
2016-01-01
The iron sulfide mineral greigite, Fe3S4, has shown promising capability as a hydrogenating catalyst, in particular in the reduction of carbon dioxide to produce small organic molecules under mild conditions. We employed density functional theory calculations to investigate the {001},{011} and {111} surfaces of this iron thiospinel material, as well as the production of hydrogen ad-atoms from the dissociation of water molecules on the surfaces. We systematically analysed the adsorption geometries and the electronic structure of both bare and hydroxylated surfaces. The sulfide surfaces presented a higher flexibility than the isomorphic oxide magnetite, Fe3O4, allowing perpendicular movement of the cations above or below the top atomic sulfur layer. We considered both molecular and dissociative water adsorption processes, and have shown that molecular adsorption is the predominant state on these surfaces from both a thermodynamic and kinetic point of view. We considered a second molecule of water which stabilizes the system mainly by H-bonds, although the dissociation process remains thermodynamically unfavourable. We noted, however, synergistic adsorption effects on the Fe3S4{001} owing to the presence of hydroxyl groups. We concluded that, in contrast to Fe3O4, molecular adsorption of water is clearly preferred on greigite surfaces. PMID:27274698
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hincal, F.
Aryl hydrocarbon hydroxylase (AHH) activities were determined in placental tissues of 152 nonsmoker or exsmoker women who live in Ankara and 125 nonsmoker women who live in areas surrounding Ankara. Levels of AHH were also determined in the placentas of 52 cigarette smokers. The mean AHH activity in the Ankara group was 11.17 +/- 5.41; in the control group, 6.44 +/- 5.48; and for smokers, 45.68 +/- 53.36, which indicates significant differences (p < .001). There was a strong correlation (r = 0.89) between the AHH activities of individuals who live in Ankara and smoke content of the air. Placentalmore » AHH activity did not show any relation to the age, nutritional and dietary habits, factors of indoor pollution, duration of pregnancy, nor did the weight, length and Apgar score of the babies.« less
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Linewidth studies on the the NI(4S-4P) resonance multiplet. [applicable to analysis of dayglow
NASA Technical Reports Server (NTRS)
Erdman, P. W.; Zipf, E. C.
1983-01-01
Doppler broadening of the 8691, 8212, and 1200-A multiplet lines of N I is investigated experimentally, and its implications for the interpretation of the earth's 1200-A UV dayglow are considered. A regulated 100-eV, 1-mA electron beam is passed through N2 at 300 K and about 0.0005 torr flowing through a collision chamber within a UHV system, and the radiation emitted is observed with a temperature-stabilized short-focal length monochromator with a bandpass of 0.2 A in the IR and an effective UV resolution (in second-order operation with a 3600-groove/mm plane grating) of about 0.04 A. Both the IR and VUV lines are found to be broadened to about 25 times the thermal Doppler linewidth, with the IR transitions accounting for more than half of the total N(4P) cross section at 100 eV. The kinetic energy of the N(4P) atoms produced by dissociative excitation is such that their 1200-A resonance radiation (2p2 3s4P - 2p3 4SO) would be optically thin in the upper atmosphere, contrary to what has been observed. A need to revise some aspects of current UV-dayglow models is identified.
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Sucharitakul, Jeerus; Tongsook, Chanakan; Pakotiprapha, Danaya; van Berkel, Willem J. H.; Chaiyen, Pimchai
2013-01-01
3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 106 m−1 s−1 (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s−1. This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s−1. 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent kcat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s−1. Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s−1) and hydroxylation (36 ± 2 s−1) partially control the overall turnover. PMID:24129570
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Finite-action solutions of Yang-Mills equations on de Sitter dS4 and anti-de Sitter AdS4 spaces
NASA Astrophysics Data System (ADS)
Ivanova, Tatiana A.; Lechtenfeld, Olaf; Popov, Alexander D.
2017-11-01
We consider pure SU(2) Yang-Mills theory on four-dimensional de Sitter dS4 and anti-de Sitter AdS4 spaces and construct various solutions to the Yang-Mills equations. On de Sitter space we reduce the Yang-Mills equations via an SU(2)-equivariant ansatz to Newtonian mechanics of a particle moving in R^3 under the influence of a quartic potential. Then we describe magnetic and electric-magnetic solutions, both Abelian and non-Abelian, all having finite energy and finite action. A similar reduction on anti-de Sitter space also yields Yang-Mills solutions with finite energy and action. We propose a lower bound for the action on both backgrounds. Employing another metric on AdS4, the SU(2) Yang-Mills equations are reduced to an analytic continuation of the above particle mechanics from R^3 to R^{2,1} . We discuss analytical solutions to these equations, which produce infinite-action configurations. After a Euclidean continuation of dS4 and AdS4 we also present self-dual (instanton-type) Yang-Mills solutions on these backgrounds.
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A novel prolyl hydroxylase inhibitor protects against cell death after hypoxia.
Kontani, Satoru; Nagata, Eiichiro; Uesugi, Tsuyoshi; Moriya, Yusuke; Fujii, Natsuko; Miyata, Toshio; Takizawa, Shunya
2013-12-01
Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.
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EPA’s SPECIATE 4.4 Database: Bridging Data Sources and Data Users
SPECIATE is the U.S. Environmental Protection Agency's (EPA)repository of volatile organic gas and particulate matter (PM) speciation profiles for air pollution sources. EPA released SPECIATE 4.4 in early 2014 and, in total, the SPECIATE 4.4 database includes 5,728 PM, VOC, total...
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A study on effects of glutathione s-transferase from silkworm on CCL4-induced mouse liver injury.
Yan, Hui; Gui, Zhongzheng; Wang, Bochu
2011-01-01
To assess the hepatoprotective activity of Glutathione S-transferase(GSTsw), extracted and purified from silkworm, in experimental acute mice liver injury and explore mechanisms. Mice were divided into five groups: control group, carbon tetrachloride (CCl4) group, and three treatment groups that received CCl4 and GSTsw at doses of 0.083 mg•g(-1), 0.0415 mg•g(-1) and 0.0207 mg•g(-1) for 3 days. ALT in serum, GST, SOD and T-AOC in liver tissue homogenate, and changes in liver pathology in the five groups were studied. CCl4 administration led to pathological and biochemical evidence of liver injury as compared to untreated controls. GSTsw administration led to significant protection against CCl4-induced changes in liver pathology. It was also associatedwith significantly lower serum ALT levels, higher GST-SOD and T-AOC level in live tissue homogenate. Thus, GSTsw showed protective activity against CCl4-induced hepatotoxicity in mice.
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Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase*
Zhang, Shengnan; Fitzpatrick, Paul F.
2016-01-01
Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25–117) is the regulatory domain of PheH lacking residues 1–24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25–117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25–117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. PMID:26823465
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NASA Astrophysics Data System (ADS)
Alekseeva, O. S.; Devdariani, A. Z.; Grigorian, G. M.; Lednev, M. G.; Zagrebin, A. L.
2017-02-01
This study is devoted to the theoretical investigation of the quasimolecular emission of Xe*-He and Kr*-He collision pairs near the Xe (5p 56s 1,3 P 1 - 5p 6 1 S 0) and Kr (4p 55s 1,3 P 1 - 4p 6 1 S 0) resonance atomic lines. The potential curves of the quasimolecules Xe(5p 56s) + He and Kr(4p 55s) + He have been obtained with the use of the effective Hamiltonian and pseudopotential methods. Based on these potential curves the processes of quasimolecular emission of Xe*+He and Kr*+He mixtures have been considered and the spectral distributions I(ħΔω) of photons emitted have been obtained in the framework of quasistatic approximation.
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Bank, Ruud A.; Robins, Simon P.; Wijmenga, Cisca; Breslau-Siderius, Liesbeth J.; Bardoel, Alfons F. J.; Van der Sluijs, Hans A.; Pruijs, Hans E. H.; TeKoppele, Johan M.
1999-01-01
Bruck syndrome is characterized by the presence of osteoporosis, joint contractures, fragile bones, and short stature. We report that lysine residues within the telopeptides of collagen type I in bone are underhydroxylated, leading to aberrant crosslinking, but that the lysine residues in the triple helix are normally modified. In contrast to bone, cartilage and ligament show unaltered telopeptide hydroxylation as evidenced by normal patterns of crosslinking. The results provide compelling evidence that collagen crosslinking is regulated primarily by tissue-specific enzymes that hydroxylate only telopeptide lysine residues and not those destined for the helical portion of the molecule. This new family of enzymes appears to provide the primary regulation for controlling the different pathways of collagen crosslinking and explains why crosslink patterns are tissue specific and not related to a genetic collagen type. A genome screen identified only a single region on chromosome 17p12 where all affected sibs shared a cluster of haplotypes identical by descent; this might be the BS (Bruck syndrome) locus and consequently the region where bone telopeptidyl lysyl hydroxylase is located. Further knowledge of this enzyme has important implications for conditions where aberrant expression of telopeptide lysyl hydroxylase occurs, such as fibrosis and scar formation. PMID:9927692
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A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay
Guo, Hou-Fu; Cho, Eun Jeong; Devkota, Ashwini K.; Chen, Yulong; Russell, William; Phillips, George N.; Yamauchi, Mitsuo; Dalby, Kevin; Kurie, Jonathan M.
2017-01-01
Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems. PMID:28216326
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FoxO1 in dopaminergic neurons regulates energy homeostasis and targets tyrosine hydroxylase
Doan, Khanh V.; Kinyua, Ann W.; Yang, Dong Joo; Ko, Chang Mann; Moh, Sang Hyun; Shong, Ko Eun; Kim, Hail; Park, Sang-Kyu; Kim, Dong-Hoon; Kim, Inki; Paik, Ji-Hye; DePinho, Ronald A.; Yoon, Seul Gi; Kim, Il Yong; Seong, Je Kyung; Choi, Yun-Hee; Kim, Ki Woo
2016-01-01
Dopaminergic (DA) neurons are involved in the integration of neuronal and hormonal signals to regulate food consumption and energy balance. Forkhead transcriptional factor O1 (FoxO1) in the hypothalamus plays a crucial role in mediation of leptin and insulin function. However, the homoeostatic role of FoxO1 in DA system has not been investigated. Here we report that FoxO1 is highly expressed in DA neurons and mice lacking FoxO1 specifically in the DA neurons (FoxO1 KODAT) show markedly increased energy expenditure and interscapular brown adipose tissue (iBAT) thermogenesis accompanied by reduced fat mass and improved glucose/insulin homoeostasis. Moreover, FoxO1 KODAT mice exhibit an increased sucrose preference in concomitance with higher dopamine and norepinephrine levels. Finally, we found that FoxO1 directly targets and negatively regulates tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of the catecholamine synthesis, delineating a mechanism for the KO phenotypes. Collectively, these results suggest that FoxO1 in DA neurons is an important transcriptional factor that directs the coordinated control of energy balance, thermogenesis and glucose homoeostasis. PMID:27681312
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Long-term hepatic outcomes in survivors of stage 4S and 4 neuroblastoma in infancy.
French, Amy E; Irwin, Meredith S; Navarro, Oscar M; Greenberg, Mark; Nathan, Paul C
2012-02-01
Infants with stage 4 and 4S neuroblastoma (NB) have a superior prognosis to older children. However, they often require intensive therapy including abdominal radiation. We aimed to investigate the long-term hepatic outcomes in infants with stage 4S and 4 NB. We reviewed the charts of 38 infants diagnosed with stage 4S and 4 NB between 1984 and 2002. We included only those with available follow-up 5 years following diagnosis. We assessed hepatic imaging and function (transaminases, bilirubin, alkaline phosphatase) at all available time points from diagnosis. Abnormalities present at more than 5 years from diagnosis were considered persistent late changes. We identified 15 stage 4S and 12 stage 4 patients. Twelve of 15 stage 4S patients had hepatic involvement at diagnosis, 8 of whom required abdominal radiation. Five of eight demonstrated late imaging changes. Two of four with hepatic metastases but no radiation demonstrated late imaging changes. The late imaging changes resolved over time and without intervention in 3/7 survivors. The persistent lesions included liver fibrosis (1) and focal nodular hyperplasia (FNH) (3). Five of 12 stage 4 NB patients had hepatic involvement at diagnosis; none required radiation or had late hepatic imaging changes. In stage 4S NB, adverse hepatic effects are infrequent, may resolve over time, and occur with or without radiation. FNH should be considered in those with persistent late imaging changes. Adverse hepatic outcomes after liver involvement or radiation in infants with stage 4 NB rarely occur. Copyright © 2011 Wiley Periodicals, Inc.