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Sample records for hydrocarbon hydroxylase ahh

  1. Absence of aryl hydrocarbon hydroxylase (AHH) in three marine bivalves

    Energy Technology Data Exchange (ETDEWEB)

    Vandermeulen, J.H. (Bedford Inst. of Oceanography, Dartmouth, Nova Scotia); Penrose, W.R.

    1978-05-01

    Bivalves exposed to short-term (4 d) and long-term (6 yr) oil pollution were assayed for aryl hydrocarbon hydroxylase (AHH) and N-demethylase activity. Short-term induction studies were carried out on Mya arenaria, Mytilus edulis, and Ostrea edulis incubated in aqueous extracts of Kuwait crude oil or Bunker C (fuel) oil. For the chronic-induction studies Mya arenaria and Mytilus edulis were collected from oiled clam beds (Arrow Bunker C) in Chedabucto Bay, Nova Scotia. None of the bivalves showed any basal or petroleum-hydrocarbon-induced aryl hydrocarbon hydroxylase or N-demethylase activity, as shown by their inability to metabolize benzopyrene or imipramine. In contrast, oil-free control trout and trout taken from a polluted lake readily metabolized both these compounds. The inability of these bivalves to degrade petroleum aromatic hydrocarbons and the tendency of these compounds to accumulate in their tissues present an opportunity for transfer of unaltered hydrocarbons into the food chain.

  2. Effects of exposure to air pollution and smoking on the placental aryl hydrocarbon hydroxylase (AHH) activity

    Energy Technology Data Exchange (ETDEWEB)

    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. Placental 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.

  3. 七种染料对鲤鱼肝微粒体芳烃羟化酶的诱导%THE INDUCTION OF ARYL HYDROCARBON HYDROXYLASE(AHH)OF CARP(CYPRINUS CARPIO) LIVER MICROSOME BY SEVEN DYES

    Institute of Scientific and Technical Information of China (English)

    徐楠; 王春霞; 莫争; 呼世斌

    2001-01-01

    The aryl hydrocarbon hydroxylase(AHH)activity of carp(Cyprinus Carpio)liver microsome induced by seven dyes was examined.The all tested dyes induced AHH activity to different extent.The AHH activity increased according to the increase of the dye concentration.The intense of AHH activity induced by seven dyes was as following order:acid red B>acid complex blue RRN>weak acid brilliant red B>reactive brilliant red K-2BP>reactive brilliant red K-2G,acid mordant red S-80>disperse red E-4B.The intense of AHH activity was related to the toxicity of the chemicals.%以鲤鱼肝微粒体为实验体系,研究了七种染料化合物对其芳烃羟化酶(AHH)的诱导,发现七种染料都可以诱导AHH的活性,随染料浓度增大AHH的活性升高.七种染料对AHH活性诱导能力大小为:酸性红B>派拉丁蓝RRN>普拉红B>活性艳红K-2BP>活性艳红K-2G,媒介大红S-80>分散红E-4B,与其毒性大小相关.

  4. Comparative effects of indole and aminoacetonitrile derivatives on dimethylnitrosamine-demethylase and aryl hydrocarbon hydroxylase activities.

    Science.gov (United States)

    Arcos, J C; Myers, S C; Neuburger, B J; Argus, M F

    1980-04-01

    The effect of in vivo administration of indole and five 3-indolyl derivatives including L-tryptophan, as well as of aminoacetonitrile and 3 of its derivatives, were studied on the carcinogen-metabolizing hepatic mixed-function oxidases dimethylnitrosamine (DMN)-demethylase I and II and aryl hydrocarbon hydroxylase (AHH). Indole, 3-indolylmethanol, 3-indolyl-acetonitrile, 3-indolylacetone and L-tryptophan induce AHH activity from 3- to 6-fold of the control level, whereas beta-3-indolylethanol has no effect; the latter compound produces a 21% decrease of the endoplasmic reticulum content in the tissue. Only L-tryptophan induces DMN-demethylase I and only L-tryptophan and 3-indolylmethanol induce DMN-demethylase II, representing a doubling of enzyme activity in all 3 instances. Aminoacetonitrile is a potent repressor of DMN-demethylase I. Substitutions on the amino group bring about strong decrease or abolishment of mixed-function oxidase repressor activity; thus, iminodiacetonitrile has only about 1/5th the repressor activity of the parent compound, whereas nitrilotriacetonitrile and dimethylaminoacetonitrile appear to be inactive. Aminoacetonitrile and its derivatives studied have no effect on DMN-demethylase II and AHH activities. The mixed-function oxidase-modifying effects of the indole compounds and of aminoacetonitrile and its derivatives illustrate the potential complexity of effects of dietary constituents on the carcinogenic responses.

  5. Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse.

    Science.gov (United States)

    Nebert, D W; Robinson, J R; Niwa, A; Kumaki, K; Poland, A P

    1975-04-01

    Monooxygenases require NADPH and molecular oxygen during the metabolism of numerous endogenous hydrophobic substrates and carcinogenic and toxic exogenous chemicals. The complexity of these membrane-bound multicomponent drug-metabolizing enzyme systems is reviewed. What "aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity" actually represents is reviewed and discussed. At least two forms of the hydroxylase activity exist and we suggest that they are associated with different molecular species of membrane-bound CO-binding hemoprotein (i.e., they are associated with different enzyme active-sties). At least two, and probably more than two, nonlinked loci are responsible for the genetic expression of new cytochrome P1450 formation and aryl hydrocarbon hydroxylase induction--and the stimulation of 10 other monooxygenase "activities"--in the mouse treated with certain aromatic hydrocarbons. The individual variability of hydroxylase activity in an inbred and in a random-bred strain of micr is illustrated. The basal hydroxylase activity appears to be inherited differently from the aromatic hydrocarbon-inducible hydroxylase activity. The potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin can stimulate increases in these hepatic monooxygenase activities and p1450 formation in so-called "nonresponsive" mice, whereas inducers such as beta-naphthoflavone and 3-methylcholanthrene cannot. Thus, the genetically "nonresponsive" micr apparently possess the structural and regulatory genes necessary for expression of these inducible monooxygenase activities and associated new formation of cytochrome P1450. We suggest that a mutation has occurred in the "nonresponsive" inbred strains that results in production of an inducer-binding receptor having a diminished affinity for aromatic hydrocarbons.

  6. Influence of some anti-inflammatory drugs on the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content

    Energy Technology Data Exchange (ETDEWEB)

    Mostafa, M.H.; Sheweita, S.A.; Abdel-Moneam, N.M. (Alexandria Univ. (Egypt))

    1990-06-01

    The metabolism of benzo({alpha})pyrene is mediated by the mixed function oxidase system including the cytochrome P450-dependent aryl hydrocarbon hydroxylase. The data of the present study revealed the ability of various commonly used anti-inflammatory drugs to alter the activity of this enzyme system, where all the tested drugs, namely phenyl butazone, ketoprofen, piroxicam, and acetaminophen, caused an increase in both the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content whether administered as a single dose or as a repeated dose for 6 consecutive days. The percentage of change for all drugs except phenyl butazone was proportional to the duration of drug administration. On the other hand, pyrazole which is chemically related to phenyl butazone, had no significant effect when administered as a single dose but caused a decrease in both studied parameters when administered as a repeated dose for 6 consecutive days. The mechanisms by which these commonly used drugs modify the aryl hydrocarbon hydroxylase activity and the cytochrome p450 content are discussed in the text.

  7. Leucoplakia of the oral cavity, smoking and arylhydrocarbon-hydroxylase inducibility

    Science.gov (United States)

    Andréasson, Lars; Korsgaard, Rolf; Trell, Erik; Björlin, Gunnar; Mattiasson, Ingrid; Trell, Lena

    1982-01-01

    In a consecutive series of 53 cases of oral leucoplakia, smoking history, oro-dental status and arylhydrocarbon-hydroxylase (AHH) inducibility were assessed. There was an increased association of leucoplakia with smoking, particularly in the male cases, but no remarkable findings as to dental status. Overall, there was a slightly higher frequency of cases with high AHH inducibility than expected from a normal control population. This difference was confined to the smokers, where there was a statistically highly significant over-representation of high AHH inducibility and under-representation of low AHH inducibility. The figures in the non-smoking patients were close to the expected. PMID:7100034

  8. Effectiveness of a Prudhoe Bay crude oil and its aliphatic, aromatic and heterocyclic fractions in inducing mortality and aryl hydrocarbon hydroxylase in chick embryo in ovo

    Energy Technology Data Exchange (ETDEWEB)

    Walters, P.; Khan, S.; O' Brien, P.J.O.; Rahimtula, A.T.; Payne, J.F.

    1987-08-01

    Prudhoe Bay crude oil (PBCO) and its aliphatic, aromatic and heterocyclic fractions were tested on the developing chick embryo for (i) embryotoxicity (ii) their ability to induce hepatic and renal cytochrome P450 levels as well as hepatic, renal and pulmonary aryl hydrocarbon hydroxylase activities. On the basis of its concentration in PBCO, the aromatic fraction was responsible for most of the embryotoxicity as well as for the enzyme inducing ability. The NOS fraction constituted less than 7% (w/v) of PbCO but, on a weight equivalent basis, was roughly as potent as the aromatic fraction in causing embryotoxicity and in inducing cytochrome P450 levels and aryl hydrocarbon hydroxylase. The aliphatic fraction was found to be essentially inactive. The results are consistent with the concept that elevation of aryl hydrocarbon hydroxylase levels by certain components of PBCO may lead to increased embroyotoxicity.

  9. Embryotoxicity, teratogenicity, and aryl hydrocarbon hydroxylase activity in Forster's terns on Green Bay, Lake Michigan

    Energy Technology Data Exchange (ETDEWEB)

    Hoffman, D.J.; Rattner, B.A.; Sileo, L.; Docherty, D.; Kubiak, T.J.

    1987-02-01

    Known reproductive problems, including congenital malformations and poor hatching success, exist for the state endangered Forster's tern (Sterna forsteri) in Green Bay, Wisconsin. Twenty Forster's tern eggs were collected from separate nests at (i) a natural colony with documented reproductive problems, situated at Green Bay, Lake Michigan, and (ii) an inland colony at Lake Poygan (control) where reproduction was documented as normal. Eggs from the two locations were placed in the same laboratory incubator and candled throughout incubation. Hatching success of Green Bay eggs was 52% of that for controls. Several early embryonic deaths occurred, but most mortality occurred close to the time of hatching. Liver microsomal aryl hydrocarbon hydroxylase activity was elevated approximately threefold in Green Bay hatchlings compared to controls. Green Bay terns that hatched weighed less than controls, had an increased liver to body weight ratio, and had a shorter femur length. Two Green Bay embryos that failed to hatch had anomalies, one with a crossed beak and one with poor ossification of the foot. One Green Bay hatchling had an abnormally ossified ilium. These effects were observed in eggs where there were measurable levels of aryl hydrocarbon hydroxylase inducers including polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins.

  10. X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase.

    Science.gov (United States)

    Sazinsky, Matthew H; Dunten, Pete W; McCormick, Michael S; DiDonato, Alberto; Lippard, Stephen J

    2006-12-26

    Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket

  11. Acquired Hypogonadotropic Hypogonadism (AHH) in Thalassaemia Major Patients: An Underdiagnosed Condition?

    Science.gov (United States)

    De Sanctis, Vincenzo; Elsedfy, Heba; Soliman, Ashraf T; Elhakim, Ihab Zaki; Pepe, Alessia; Kattamis, Christos; Soliman, Nada A.; Elalaily, Rania; El Kholy, Mohamed; Yassin, Mohamed

    2016-01-01

    Introduction In males, acquired hypogonadotropic hypogonadism (AHH) includes all disorders that damage or alter the function of gonadotropin-releasing hormone (GnRH) neurons and/or pituitary gonadotroph cells. The clinical characteristics of AHH are androgen deficiency and lack, delay or halt of pubertal sexual maturation. AHH lead to decreased libido, impaired erectile function, and strength, a worsened sense of well-being and degraded quality of life (QOL). Patients and methods We studied 11 adult men with thalassemia major (TM) aged between 26 to 54 years (mean ± SD: 34.3 ± 8.8 years) with AHH. Twelve age- and sex-matched TM patients with normal pubertal development were used as a control group. All patients were on regular transfusions and iron chelation therapy. Fasting venous blood samples were collected two weeks after transfusion to measure serum concentrations of IGF-1, free thyroxine (FT4), thyrotropin (TSH), cortisol, luteinizing hormone (LH), follicle stimulating hormone (FSH), total testosterone (TT), prolactin and estradiol (E2), glucose, urea, creatinine and electrolytes (including calcium and phosphate). Liver functions and screening for hepatitis C virus seropositivity (HCVab and HCV-RNA) were performed. Iron status was assessed by measuring serum ferritin levels, and evaluation of iron concentrations in the liver (LIC) and heart using MRI- T2*. Bone mineral density was measured at the lumbar spine (L1–L4) for all patients with AHH by dual energy X-ray absorptiometry (DXA) using Hologic QDR 4000 machine. Results The mean basal serum LH and FSH concentrations in AHH patients were 2.4 ± 2.2 IU/L and 1.2 ± 0.9 IU/L respectively; these, values were significantly lower compared to the control group. Semen analysis in 5 patients with AHH showed azoospermia in 3 and oligoasthenozoospermia in 2. The percentage of patients with serum ferritin level >2000 ng/ml (severe iron load) was significantly higher in AHH patients compared to controls, 5/11 (45

  12. Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study

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    Akahoshi Eiichi

    2009-06-01

    Full Text Available Abstract Background Dioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR. This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR. Methods N2a-Rβ cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA. Results Induction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE, though AHRE-III was not identical to XRE, the conventional AhR-binding motif. Conclusion Our results suggest TCDD directly regulate the dopamine system by TH gene

  13. Activity Inducement of Oil Wastewater on AHH of Cypinus Carpio Hepatic Microsome%含油废水对鲤鱼肝脏微粒体芳烃羟化酶的活性诱导研究

    Institute of Scientific and Technical Information of China (English)

    邵涛

    2012-01-01

    The aryl hydrocarbon hydroxylase(AHH) activities of cyprinus carpio liver microsome induced by three oil wastewaters have been examined in Vitro.It was found that all the tested chemicals show different degrees of inducement on AHH activity.The AHH activity becomes higher with the increase of oil quantity.The AHH activity of fish liver microsome can be used as a biomarker to evaluate the oil wastewater in waters.%以鲤鱼肝脏微粒体为实验体系,运用体外实验方法,研究三种含油废水对芳烃羟化酶的活性影响,结果表明,三种含油废水对芳烃羟化酶活性都表现出不同程度的诱导,随着含油量的增大,AHH活性升高,并有相似的变化趋势。芳烃羟化酶活性升高可以作为监测含油废水对水体污染的一种生物指标。

  14. Nutrients can enhance the abundance and expression of alkane hydroxylase CYP153 gene in the rhizosphere of ryegrass planted in hydrocarbon-polluted soil.

    Science.gov (United States)

    Arslan, Muhammad; Afzal, Muhammad; Amin, Imran; Iqbal, Samina; Khan, Qaiser M

    2014-01-01

    Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination.

  15. Nutrients can enhance the abundance and expression of alkane hydroxylase CYP153 gene in the rhizosphere of ryegrass planted in hydrocarbon-polluted soil.

    Directory of Open Access Journals (Sweden)

    Muhammad Arslan

    Full Text Available Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination.

  16. Protective activity of C-geranylflavonoid analogs from Paulownia tomentosa against DNA damage in 137Cs irradiated AHH-1 cells.

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    Moon, Hyung-In; Jeong, Min Ho; Jo, Wol Soon

    2014-09-01

    Radiotherapy is an important form of treatment for a wide range of cancers, but it can damage DNA and cause adverse effects. We investigated if the diplacone analogs of P. tomentosa were radio-protective in a human lymphoblastoid cell line (AHH-1). Four geranylated flavonoids, diplacone, 3'-O-methyl-5'-hydroxydiplacone, 3'-O-methyl-5'-O-methyldiplacone and 3'-O-methyldiplacol, were tested for their antioxidant and radio-protective effects. Diplacone analogs effectively scavenged free radicals and inhibited radiation-induced DNA strand breaks in vitro. They significantly decreased levels of reactive oxygen species and cellular DNA damage in 2 Gy-irradiated AHH-1 cells. Glutathione levels and superoxide dismutase activity in irradiated AHH-1 cells increased significantly after treatment with these analogs. The enhanced biological anti-oxidant activity and radioprotective activity of diplacone analogs maintained the survival of irradiated AHH-1 cells in a clonogenic assay. These data suggest that diplacone analogs may protect healthy tissue surrounding tumor cells during radiotherapy to ensure better control of radiotherapy and allow higher doses of radiotherapy to be employed.

  17. Aryl hydrocarbon hydroxylase represents CYP1B1, and not CYP1A1, in human freshly isolated white cells: trimodal distribution of Japanese population according to induction of CYP1B1 mRNA by environmental dioxins.

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    Toide, Kenji; Yamazaki, Hiroshi; Nagashima, Rikako; Itoh, Keisuke; Iwano, Shunsuke; Takahashi, Yoshiki; Watanabe, Shaw; Kamataki, Tetsuya

    2003-03-01

    The expression level of mRNAs for cytochrome P450 (CYP) 1A1 and 1B1 in freshly prepared white cells from 72 subjects exposed to dioxins at waste incinerators was investigated. The amounts of CYP1B1 mRNA ranged from 0.16 to 671 molecules/10(7) molecules of 18S rRNA, whereas the amounts of CYP1A1 mRNA were dioxins. The inducibility of CYP1B1 mRNA in leukocytes, defined as the ratio of CYP1B1 mRNA to the plasma concentration of dioxins, varied among the subjects. It was found that the subjects showed trimodal distribution according to inducibility: 39 (54.2%), 25 (34.7%), and 8 (11.1%) of 72 subjects were judged as poor, intermediate, and high responders to environmental dioxins, respectively. The amounts of CYP1B1 mRNA in leukocytes of the intermediate and high responders were highly correlated with the plasma concentrations of dioxins (P dioxins is involved in aromatic hydrocarbon hydroxylase activities in human lymphocytes.

  18. Search for the Neutral MSSM Higgs Bosons in the Decay Channel A/H/h to mu+ mu-

    CERN Document Server

    The ATLAS collaboration

    2009-01-01

    Motivated by the high muon momentum resolution and identification efficiency achievable with the ATLAS detector, the observability of A/H/h to mu+ mu- channel is explored. The high experimental resolution in this decay mode compensates to some extent for the suppression of the branching ratio, with respect to the A/H/h to tau+ tau- decays. The analyses are performed in the Higgs mass range from 100 to 500 GeV. Two main analysis strategies are applied - the search for the dimuon final states resulting from the direct A/H/h production is combined with the search for the associated bbA/H/h production mode. The studies are optimized for the early stage of data taking up to an integrated luminosity of 30 fb−1 . All results are obtained combining the fast and the full simulation of the ATLAS detector with the nominal detector layout and the trigger efficiencies included. In addition, dedicated data samples are produced to study the impact of the pile-up and cavern background on the analysis performance. The estim...

  19. Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin transactivates aryl hydrocarbon receptor-responsive element III in the tyrosine hydroxylase immunoreactive neurons of the mouse midbrain.

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    Tanida, Takashi; Tasaka, Ken; Akahoshi, Eiichi; Ishihara-Sugano, Mitsuko; Saito, Michiko; Kawata, Shigehisa; Danjo, Megumi; Tokumoto, Junko; Mantani, Youhei; Nagahara, Daichi; Tabuchi, Yoshiaki; Yokoyama, Toshifumi; Kitagawa, Hiroshi; Kawata, Mitsuhiro; Hoshi, Nobuhiko

    2014-02-01

    Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000 ng kg⁻¹ body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg⁻¹ TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD).

  20. Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase.

    Science.gov (United States)

    Faletto, M B; Koser, P L; Battula, N; Townsend, G K; Maccubbin, A E; Gelboin, H V; Gurtoo, H L

    1988-09-01

    Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

  1. 1,4-benzoquinone-induced STAT-3 hypomethylation in AHH-1 cells: Role of oxidative stress

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    Jing Yang

    2015-01-01

    Full Text Available Benzene, a known occupational and environmental contaminant, is associated with increased risk of leukemia. The objectives of this study were to elucidate the regulatory mechanism of the hypomethylated STAT3 involved in benzene toxicity in vitro. As 1,4-benzoquinone (1,4-BQ is one of benzene’s major toxic metabolites, AHH-1 cells were treated by 1,4-BQ for 24 h with or without pretreatment of the antioxidant a-LA or the methyltransferase inhibitor, 5-aza-2′ deoxycytidine (5-aza. The cell viability was investigated using the 3-(4, 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. ROS was determined via 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA flow cytometric assays. The level of oxidative stress marker 8-OHdG was measured by enzyme-linked immunosorbent assay. Methylation-specific PCR was used to detect the methylation status of STAT3. Results indicated the significantly increasing expression of ROS and 8-OHdG which accompanied with STAT3 hypomethylation in 1,4-BQ-treated AHH-1 cells. α-LA suppressed the expression of both ROS and 8-OHdG, simultaneously reversed 1,4-BQ-induced STAT3 hypomethylation. However, although the methylation inhibitor, 5-aza reduced the expression level of ROS and 8-OHdG, but had no obvious inhibiting effect on STAT3 methylation level. Taken together, oxidative stress are involved 1,4-BQ-induced STAT3 methylation expression.

  2. Phenylalanine hydroxylase deficiency.

    Science.gov (United States)

    Mitchell, John J; Trakadis, Yannis J; Scriver, Charles R

    2011-08-01

    Phenylalanine hydroxylase deficiency is an autosomal recessive disorder that results in intolerance to the dietary intake of the essential amino acid phenylalanine. It occurs in approximately 1:15,000 individuals. Deficiency of this enzyme produces a spectrum of disorders including classic phenylketonuria, mild phenylketonuria, and mild hyperphenylalaninemia. Classic phenylketonuria is caused by a complete or near-complete deficiency of phenylalanine hydroxylase activity and without dietary restriction of phenylalanine most children will develop profound and irreversible intellectual disability. Mild phenylketonuria and mild hyperphenylalaninemia are associated with lower risk of impaired cognitive development in the absence of treatment. Phenylalanine hydroxylase deficiency can be diagnosed by newborn screening based on detection of the presence of hyperphenylalaninemia using the Guthrie microbial inhibition assay or other assays on a blood spot obtained from a heel prick. Since the introduction of newborn screening, the major neurologic consequences of hyperphenylalaninemia have been largely eradicated. Affected individuals can lead normal lives. However, recent data suggest that homeostasis is not fully restored with current therapy. Treated individuals have a higher incidence of neuropsychological problems. The mainstay of treatment for hyperphenylalaninemia involves a low-protein diet and use of a phenylalanine-free medical formula. This treatment must commence as soon as possible after birth and should continue for life. Regular monitoring of plasma phenylalanine and tyrosine concentrations is necessary. Targets of plasma phenylalanine of 120-360 μmol/L (2-6 mg/dL) in the first decade of life are essential for optimal outcome. Phenylalanine targets in adolescence and adulthood are less clear. A significant proportion of patients with phenylketonuria may benefit from adjuvant therapy with 6R-tetrahydrobiopterin stereoisomer. Special consideration must be

  3. Allosteric regulation of phenylalanine hydroxylase.

    Science.gov (United States)

    Fitzpatrick, Paul F

    2012-03-15

    The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.

  4. Allosteric Regulation of Phenylalanine Hydroxylase

    OpenAIRE

    Fitzpatrick, Paul F.

    2011-01-01

    The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.

  5. Hydrocarbon pneumonia

    Science.gov (United States)

    Pneumonia - hydrocarbon ... Coughing Fever Shortness of breath Smell of a hydrocarbon product on the breath Stupor (decreased level of ... Most children who drink or inhale hydrocarbon products and develop ... hydrocarbons may lead to rapid respiratory failure and death.

  6. National Status and Trends, Benthic Surveillance Project Aryl Hydrocarbon Hydrolase (AHH) Data, 1988-1992, National Centers for Coastal Ocean Science

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — In order to determine the current status of and detect any long-term trends in the environmental quality of U.S. nearshore waters, NOAA initiated the National Status...

  7. National Status and Trends, Benthic Surveillance Project Aryl Hydrocarbon Hydrolase (AHH) Data, 1988-1992, National Centers for Coastal Ocean Science

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — In order to determine the current status of and detect any long-term trends in the environmental quality of U.S. nearshore waters, NOAA initiated the National...

  8. Seventeen Alpha-hydroxylase Deficiency

    Directory of Open Access Journals (Sweden)

    Siew-Lee Wong

    2006-01-01

    Full Text Available Seventeen a-hydroxylase deficiency (17OHD is a rare form of congenital adrenal hyperplasia in which defects in the biosynthesis of cortisol and sex steroid result in mineralocorticoid excess, hypokalemic hypertension and sexual abnormalities such as pseudohermaphroditism in males, and sexual infantilism in females. The disease is inherited in an autosomal recessive pattern, and is caused by mutations in the gene encoding cytochrome P450c17 (CYP17, which is the single polypeptide that mediates both 17α-hydroxylase and 17,20-lyase activities. We report the case of a 15-year-old patient with 17OHD who had a female phenotype but male karyotype (46,XY. The diagnosis was made based on classical clinical features, biochemical data and molecular genetic study. Two mutations were identified by polymerase chain reaction amplification and sequencing, including a S106P point mutation in exon 2 and a 9-bp (GACTCTTTC deletion from nucleotide position 1519 in exon 8 of CYP17. The first of these mutations was found in the father and the second in the mother, and both have been previously reported in Asia. The patient's hypertension and hypokalemia resolved after glucocorticoid replacement and treatment with potassium-sparing diuretics. Sex hormone replacement was prescribed for induction of sexual development and reduction of the final height. Prophylactic gonadectomy was scheduled. In summary, 17OHD should be suspected in patients with hypokalemic hypertension and lack of secondary sexual development so that appropriate therapy can be implemented.

  9. Assay of deoxyhypusine hydroxylase activity.

    Science.gov (United States)

    Park, Jong Hwan; Wolff, Edith C; Park, Myung Hee

    2011-01-01

    The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains an unusual amino acid, hypusine [N (ε)-(4-amino-2-hydroxybutyl)-lysine]. eIF5A and its hypusine/deoxyhypusine modification are vital for eukaryotic cell proliferation. Hypusine is formed posttranslationally by two enzymatic steps catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. Deoxyhypusine hydroxylase catalyzes a stereo-specific hydroxylation of the deoxyhypusine residue in the eIF5A intermediate protein, eIF5A(Dhp). The enzyme is totally specific for this protein and does not act on short peptides (hypusine residue in eIF5A. Optimum conditions for the reaction and two detection methods for the product, hypusine-containing eIF5A, are described in this chapter. The first, and most reliable, method is the measurement of the amount of [(3)H]hypusine in the protein hydrolysate after its separation from [(3)H]deoxyhypusine, by ion exchange chromatography. This method does require specialized equipment. The second method is based on counting the total TCA soluble radioactivity after sodium periodate oxidation of the reaction mixture, since the radiolabeled 4-amino-2-hydroxy butyl moiety of the hypusine residue is cleaved and is released from protein as radiolabeled β-propionaldehyde and formaldehyde by periodate oxidation.

  10. Dopamine beta-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Senard Jean-Michel

    2006-03-01

    Full Text Available Abstract Dopamine beta-hydroxylase (DβH deficiency is a very rare form of primary autonomic failure characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased dopamine plasma levels. The prevalence of DβH deficiency is unknown. Only a limited number of cases with this disease have been reported. DβH deficiency is mainly characterized by cardiovascular disorders and severe orthostatic hypotension. First symptoms often start during a complicated perinatal period with hypotension, muscle hypotonia, hypothermia and hypoglycemia. Children with DβH deficiency exhibit reduced ability to exercise because of blood pressure inadaptation with exertion and syncope. Symptoms usually worsen progressively during late adolescence and early adulthood with severe orthostatic hypotension, eyelid ptosis, nasal stuffiness and sexual disorders. Limitation in standing tolerance, limited ability to exercise and traumatic morbidity related to falls and syncope may represent later evolution. The syndrome is caused by heterogeneous molecular alterations of the DBH gene and is inherited in an autosomal recessive manner. Restoration of plasma noradrenaline to the normal range can be achieved by therapy with the synthetic precursor of noradrenaline, L-threo-dihydroxyphenylserine (DOPS. Oral administration of 100 to 500 mg DOPS, twice or three times daily, increases blood pressure and reverses the orthostatic intolerance.

  11. 21-Hydroxylase deficiency in Brazil

    Directory of Open Access Journals (Sweden)

    T.A.S.S. Bachega

    2000-10-01

    Full Text Available We determined the frequency of large rearrangements and point mutations in 130 Brazilian patients with 21-hydroxylase deficiency and correlated genotype with phenotype. The frequency of CYP21 deletions was lower (4.4% than in most of the previous series described, whereas the frequency of large gene conversions was similar to the frequency reported in the literature (6.6%. The most frequent point mutations were I2 splice (41.8% in salt wasting - SW, I172N (32.6% in simple virilizing - SV and V281L (40.2% in the late onset form - LO. The frequency of the nine most common point mutations was similar to that reported for other countries. The 93 fully genotyped patients were classified into 3 mutation groups based on the degree of enzymatic activity (A@ 2%, C>20%. In group A, 62% of cases presented the SW form; in group B, 96% the SV form, and in group C, 88% the LO form. We diagnosed 80% of the affected alleles after screening for large rearrangements and 15 point mutations. To diagnose these remaining alleles we sequenced the CYP21 gene of one patient with the SV form and identified a heterozygous G->A transition in codon 424. This mutation leads to a substitution of glycine by serine in a conserved region and was also found in a compound heterozygous state in 4 other patients. The mutation G424S presented a linkage disequilibrium with CYP21P and C4A gene deletions and HLA DR17, suggesting a probable founder effect. Search for the G424S mutation in other populations will reveal if it is restricted to the Brazilian patients or if it has a wider ethnic distribution.

  12. Phenylalanine hydroxylase deficiency: diagnosis and management guideline.

    Science.gov (United States)

    Vockley, Jerry; Andersson, Hans C; Antshel, Kevin M; Braverman, Nancy E; Burton, Barbara K; Frazier, Dianne M; Mitchell, John; Smith, Wendy E; Thompson, Barry H; Berry, Susan A

    2014-02-01

    Phenylalanine hydroxylase deficiency, traditionally known as phenylketonuria, results in the accumulation of phenylalanine in the blood of affected individuals and was the first inborn error of metabolism to be identified through population screening. Early identification and treatment prevent the most dramatic clinical sequelae of the disorder, but new neurodevelopmental and psychological problems have emerged in individuals treated from birth. The additional unanticipated recognition of a toxic effect of elevated maternal phenylalanine on fetal development has added to a general call in the field for treatment for life. Two major conferences sponsored by the National Institutes of Health held >10 years apart reviewed the state of knowledge in the field of phenylalanine hydroxylase deficiency, but there are no generally accepted recommendations for therapy. The purpose of this guideline is to review the strength of the medical literature relative to the treatment of phenylalanine hydroxylase deficiency and to develop recommendations for diagnosis and therapy of this disorder. Evidence review from the original National Institutes of Health consensus conference and a recent update by the Agency for Healthcare Research and Quality was used to address key questions in the diagnosis and treatment of phenylalanine hydroxylase deficiency by a working group established by the American College of Medical Genetics and Genomics. The group met by phone and in person over the course of a year to review these reports, develop recommendations, and identify key gaps in our knowledge of this disorder. Above all, treatment of phenylalanine hydroxylase deficiency must be life long, with a goal of maintaining blood phenylalanine in the range of 120-360 µmol/l. Treatment has predominantly been dietary manipulation, and use of low protein and phenylalanine medical foods is likely to remain a major component of therapy for the immediate future. Pharmacotherapy for phenylalanine

  13. Enzyme induction in rat lung and liver by condensates and fractions from main-stream and side-stream cigarette smoke

    Energy Technology Data Exchange (ETDEWEB)

    Pasquini, R.; Sforzolini, G.S.; Savino, A.; Angeli, G.; Monarca, S.

    1987-12-01

    Aryl hydrocarbon hydroxylase (AHH) and dimethylnitrosamine demethylase (DMND) activities in pulmonary and hepatic tissues of male Sprague-Dawley rats were assayed following pretreatment with known inducers (benzo(a)pyrene, 3-methylcholanthrene, Aroclor 1254, phenobarbital) and with main-stream (MS) and side-stream (SS) cigarette smoke condensates and their related fractions. Biochemical assays by spectrophotofluorimetry (AHH activity) and spetrophotometry (DMND activity) and by a biological assay (Ames test) were performed to detect AHH and DMND induction. Ames test proved to be much less sensitive than the spectrophotofluorimetric analysis for AHH determination. Both main-stream and side-stream cigarette smoke condensates and some fractions, containing water-soluble bases, water-insoluble bases, and polycyclic aromatic hydrocarbons, were found to induce AHH activity in lung and liver, the lung being induced to the greatest extent. The highest levels of AHH inducibility were found for the SS-smoke condensate and related fractions. In particular, the insoluble bases fractions gave the highest induction. On the contrary, pulmonary DMND activity was not affected by pretreatment with the same materials, while hepatic DMND response was only minimally induced by Aroclor and phenobarbital treatment.

  14. Phenylalanine hydroxylase misfolding and pharmacological chaperones.

    Science.gov (United States)

    Underhaug, Jarl; Aubi, Oscar; Martinez, Aurora

    2012-01-01

    Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant phenylalanine hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH(4)) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH(4). Furthermore, PAH shares a number of ligands, such as BH(4), amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.

  15. Alkane Hydroxylase Gene (alkB Phylotype Composition and Diversity in Northern Gulf of Mexico Bacterioplankton

    Directory of Open Access Journals (Sweden)

    Conor Blake Smith

    2013-12-01

    Full Text Available Natural and anthropogenic activities introduce alkanes into marine systems where they are degraded by alkane hydroxylases expressed by phylogenetically diverse bacteria. Partial sequences for alkB, one of the structural genes of alkane hydroxylase, have been used to assess the composition of alkane-degrading communities, and to determine their responses to hydrocarbon inputs. We present here the first spatially extensive analysis of alkB in bacterioplankton of the northern Gulf of Mexico (nGoM, a region that experiences numerous hydrocarbon inputs. We have analyzed 401 partial alkB gene sequences amplified from genomic extracts collected during March 2010 from 17 water column samples that included surface waters and bathypelagic depths. Previous analyses of 16S rRNA gene sequences for these and related samples have shown that nGoM bacterial community composition and structure stratify strongly with depth, with distinctly different communities above and below 100 m. Although we hypothesized that alkB gene sequences would exhibit a similar pattern, PCA analyses of operational protein units (OPU indicated that community composition did not vary consistently with depth or other major physical-chemical variables. We observed 22 distinct OPUs, one of which was ubiquitous and accounted for 57% of all sequences. This OPU clustered with alkB sequences from known hydrocarbon oxidizers (e.g., Alcanivorax and Marinobacter. Some OPUs could not be associated with known alkane degraders, however, and perhaps represent novel hydrocarbon-oxidizing populations or genes. These results indicate that the capacity for alkane hydrolysis occurs widely in the nGoM, but that alkane degrader diversity varies substantially among sites and responds differently than bulk communities to physical-chemical variables.

  16. Obesity in 21-hydroxylase deficient patients

    OpenAIRE

    Cornean, R.; Hindmarsh, P; Brook, C.

    1998-01-01

    OBJECTIVES—To evaluate the natural history and timing of adiposity rebound (nadir of body mass index (BMI)) in children with congenital adrenal hyperplasia 21-hydroxylase deficiency (CYP21). 
STUDY DESIGN—A retrospective mixed longitudinal study.
METHODS—Height and changes in body composition (BMI; weight (kg)/height2 (m)), triceps and subscapular skinfolds) were analysed in 22(14 girls, eight boys) prepubertal patients with CYP21 for whom continuous anthropometric data were ...

  17. Are Striatal Tyrosine Hydroxylase Interneurons Dopaminergic?

    OpenAIRE

    Xenias, Harry S.; Ibáñez-Sandoval, Osvaldo; Koós, Tibor; Tepper, James M.

    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...

  18. Expression and purification of the metal-containing monooxygenases tryptophan hydroxylase and dopamine β-hydroxylase

    DEFF Research Database (Denmark)

    Karlsen, Pernille Efferbach

    to abnormal levels of the neurotransmitters serotonin, dopamine and norepinephrine and the regulation of tryptophan hydroxylase and dopamine β-hydroxylase. These include depression, anxiety disorders, obsessive compulsive disorder (OCD), schizophrenia, Parkinson's disease and attention deficit......-containing enzyme which belongs to the aromatic amino acid hydroxylase (AAAH) family. It exist in two isoforms, TPH1 and TPH2, which are expressed in different tissues and have different properties. TPH is known as a very diffcult protein to work with especially due to instability and only truncated forms of TPH1...... to the family of ascorbate dependent type II Cu monooxygenases. Very little knowledge exist on DβH and most of it comes from investigations of related proteins. Attempts to express human DβH in bacterial systems have been done in the Metalloprotein Chemistry and Engineering Group, but at present no system...

  19. Characterization of hydrocarbon utilizing fungi from hydrocarbon ...

    African Journals Online (AJOL)

    Prof. Ogunji

    hydrocarbon polluted sediments and water .... ecosystem may result in selective increase or decrease in microbial population (Okpokwasili ... been implicated in degradation of hydrocarbons such as crude oil, polyaromatic hydrocarbons and.

  20. Genetics Home Reference: congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency

    Science.gov (United States)

    ... due to 11-beta-hydroxylase deficiency congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency Enable Javascript ... PDF Open All Close All Description Congenital adrenal hyperplasia (CAH) due to 11-beta-hydroxylase deficiency is ...

  1. Mutagenicity and Induction of Drug—metabolizing Enzyme Activity by LPG Combustion Particulates in Rats

    Institute of Scientific and Technical Information of China (English)

    YINXue-Jun; LIUJun-Zhuo; 等

    1994-01-01

    Methylene chloride extracts of particulates from liquefied petroleum gas(LPG)combustion appliance were studied by using Ames test,micronucleus test and inducibility of pulmonary and hepatic aryl hydrocarbon hydroxylase(AHH)and glutathione S-transferase(GST)in rats.The extracts showed mutagenicity for Salmonella typhimurium strain TA98 and its derivatives TA98NR and TA98/1,8-DNP6 with or without S9 mix.The revertants in strains TA98NR and TA98/1,8-DNP6 were less than 40%and 50% of than in strain TA98 without S9 mix,respectively.Positive results were obtained in mouse bone marrow micronucleus assay.Intratracheal instillation of the extracts led to increase in pulmonary(but not hepatic)AHH and GST activities in rats.In was seen that AHH was more sensitive than GST to induction by the extracts.

  2. Apparatus for hydrocarbon extraction

    Science.gov (United States)

    Bohnert, George W.; Verhulst, Galen G.

    2013-03-19

    Systems and methods for hydrocarbon extraction from hydrocarbon-containing material. Such systems and methods relate to extracting hydrocarbon from hydrocarbon-containing material employing a non-aqueous extractant. Additionally, such systems and methods relate to recovering and reusing non-aqueous extractant employed for extracting hydrocarbon from hydrocarbon-containing material.

  3. Enhancement of toxicity and enzyme-repressing activity of p-dioxane by chlorination: stereoselective effects.

    Science.gov (United States)

    Woo, Y T; Neuburger, B J; Arcos, J C; Argus, M F; Nishiyama, K; Griffin, G W

    1980-01-01

    The acute toxicity of p-dioxane may be enhanced up to 1000-fold by chlorination of the compound. The effect was stereoselective. Of the stereoisomers tested, tetrachloro-p-dioxane, isomer I (2r, 3t, 5t, 6c) was over 80 times more toxic than isomer II (2r, 3c, 5t, 6t). The latter compound was also a potent repressor of hepatic dimethylnitrosamine-demethylase I (DMN-d) and aryl hydrocarbon hydroxylase (AHH).

  4. Heterogeneous expression of cholesterol 7α-hydroxylase and sterol 27- hydroxylase genes in the rat liver lobulus

    NARCIS (Netherlands)

    Twisk, J.; Hoekman, M.F.M.; Mager, W.H.; Moorman, A.F.M.; Boer, P.A.J. de; Scheja, L.; Princen, H.M.G.; Gebhardt, R.

    1995-01-01

    We investigated the lobular localization and molecular level of expression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene tr

  5. The phylogeny of the aromatic amino acid hydroxylases revisited by characterizing phenylalanine hydroxylase from Dictyostelium discoideum.

    Science.gov (United States)

    Siltberg-Liberles, Jessica; Steen, Ida Helene; Svebak, Randi M; Martinez, Aurora

    2008-12-31

    The social amoeba Dictyostelium discoideum contains only one aromatic amino acid hydroxylase (AAAH) gene compared to at least three in metazoans. As shown in this work this gene codes for a phenylalanine hydroxylase (DictyoPAH) and phylogenetic analysis places this enzyme close to the precursor AAAHs, aiding to define the evolutionary history of the AAAH family. DictyoPAH shows significant similarities to other eukaryote PAH, but it exhibits higher activity with tetrahydrodictyopterin (DH4) than with tetrahydrobiopterin (BH4) as cofactor. DH4 is an abundant tetrahydropterin in D. discoideum while BH4 is the natural cofactor of the AAAHs in mammals. Moreover, DictyoPAH is devoid of the characteristic regulatory mechanisms of mammalian PAH such as positive cooperativity for L-Phe and activation by preincubation with the substrate. Analysis of the few active site substitutions between DictyoPAH and mammalian PAH, including mutant expression analysis, reveals potential structural determinants for allosteric regulation.

  6. Purification and Characterization of Tryptophan Hydroxylase

    DEFF Research Database (Denmark)

    Haahr, Lærke Tvedebrink

    This thesis deals with the purification and characterization of the iron-containing enzyme tryptophan hydroxylase (TPH). TPH exists in two isoforms, called TPH1 and TPH2. Each isoform consists of threestructural distinct domains: the regulatory, the catalytic and the tetramerization domain. TPH...... of this project was to developpurification methods for full-length TPH1 and TPH2 as well as to characterize purified TPH variants. A successful purification method for full-length human TPH1 (hTPH1) was developed, which resulted in pure, active and stable protein. The method includes affinity-purification using....... The crystallization procedure for the catalytic domain of gallus gallus TPH1 (cgTPH1) was optimized to faster crystal growth by addition of tryptophan and incubation at room temperature. Crystals without imidazole in the crystallization conditions could be obtained. The solved structures were however of poor quality...

  7. Unresponsiveness to tetrahydrobiopterin of phenylalanine hydroxylase deficiency.

    Science.gov (United States)

    Ponzone, Alberto; Porta, Francesco; Mussa, Alessandro; Alluto, Alessandra; Ferraris, Silvio; Spada, Marco

    2010-05-01

    Conflicting results have been reported concerning the efficacy of tetrahydrobiopterin (BH4), the cofactor of phenylalanine hydroxylase, for reducing phenylalanine (Phe) concentration in phenylketonuria (PKU). We aimed to test quantitatively the effects of BH4 in PKU patients. Seven fully characterized patients were selected among a population of 130 PKU subjects as harboring PKU mutations predicted as BH4 responsive and previously considered responsive to a cofactor challenge. They received a simple Phe (100 mg/kg) and 2 combined Phe (100 mg/kg) and BH4 (20 mg/kg) oral loading tests. Cofactor was administered either before or after the amino acid. The concentrations of Phe, tyrosine (Tyr), and biopterin were measured over 24 hours after loading. The comparative analysis of the loading tests showed that in all patients plasma Phe concentrations peaked within 3 hours, and fell within 24 hours by about 50% in benign, 20% in mild, and 15% in severe phenylalanine hydroxylase deficiency regardless of BH4 administration. A consistent or moderate increase of plasma Tyr, again independent of the cofactor challenge, was observed only in the less severe forms of PAH deficiency. Mean blood biopterin concentration increased 6 times after simple Phe and 34 to 39 times after combined loading tests. The administration of BH4 does not alter Phe and Tyr metabolism in PKU patients. The clearance of plasma Phe after oral loading and, as well as Tyr production, is not related to cofactor challenge but to patient's phenotype. The assessment of BH4 responsiveness by the methods so far used is not reliable, and the occurrence of BH4-responsive forms of PKU still has to be definitely proven.

  8. Effects of embryonic and adult exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic microsomal testosterone hydroxylase activities in great blue herons (Ardea herodias)

    Energy Technology Data Exchange (ETDEWEB)

    Sanderson, J.T.; Giesy, J.P. [Michigan State Univ., East Lansing, MI (United States); Janz, D.M.; Bellward, G.D. [Univ. of British Columbia, Vancouver, British Columbia (Canada)

    1997-06-01

    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}, 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.

  9. Treatment of Nonclassic 11-Hydroxylase Deficiency with Ashwagandha Root

    Directory of Open Access Journals (Sweden)

    Daniel Powell

    2017-01-01

    Full Text Available An elderly woman presented with acne and male pattern alopecia, which upon diagnostic evaluation was found to be due to nonclassic 11-hydroxylase deficiency. We previously reported that Ashwagandha root ameliorates nonclassic 3-β-ol dehydrogenase and aldosterone synthase deficiencies. This is the first report of its use being associated with amelioration of nonclassic 11-hydroxylase deficiency, where its apparent effects appear to be dose-related.

  10. The hydrocarbon sphere

    Energy Technology Data Exchange (ETDEWEB)

    Mandev, P.

    1984-01-01

    The hydrocarbon sphere is understood to be the area in which hydrocarbon compounds are available. It is believed that the lower boundary on the hydrocarbon sphere is most probably located at a depth where the predominant temperatures aid in the destruction of hydrocarbons (300 to 400 degrees centigrade). The upper limit on the hydrocarbon sphere obviously occurs at the earth's surface, where hydrocarbons oxidize to H20 and CO2. Within these ranges, the occurrence of the hydrocarbon sphere may vary from the first few hundred meters to 15 kilometers or more. The hydrocarbon sphere is divided into the external (mantle) sphere in which the primary gas, oil and solid hydrocarbon fields are located, and the internal (metamorphic) sphere containing primarily noncommercial accumulations of hydrocarbon gases and solid carbon containing compounds (anthraxilite, shungite, graphite, etc.) based on the nature and scale of hydrocarbon compound concentrations (natural gas, oil, maltha, asphalt, asphaltite, etc.).

  11. Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase.

    Science.gov (United States)

    Thöny, Beat; Calvo, Ana C; Scherer, Tanja; Svebak, Randi M; Haavik, Jan; Blau, Nenad; Martinez, Aurora

    2008-07-01

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l-DOPA responsive and non-responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription-translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH(4) supplementation to mice increased TH activity and protein levels in brain extracts, while the Th-mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding-aid effect of BH(4) and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH(4) crosses the blood-brain barrier and therapeutic regimes based on BH(4) supplementation should thus consider the effect on TH. Furthermore, BH(4) supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.

  12. Phenylalanine hydroxylase: function, structure, and regulation.

    Science.gov (United States)

    Flydal, Marte I; Martinez, Aurora

    2013-04-01

    Mammalian phenylalanine hydroxylase (PAH) catalyzes the rate-limiting step in the phenylalanine catabolism, consuming about 75% of the phenylalanine input from the diet and protein catabolism under physiological conditions. In humans, mutations in the PAH gene lead to phenylketonuria (PKU), and most mutations are mainly associated with PAH misfolding and instability. The established treatment for PKU is a phenylalanine-restricted diet and, recently, supplementation with preparations of the natural tetrahydrobiopterin cofactor also shows effectiveness for some patients. Since 1997 there has been a significant increase in the understanding of the structure, catalytic mechanism, and regulation of PAH by its substrate and cofactor, in addition to improved correlations between genotype and phenotype in PKU. Importantly, there has also been an increased number of studies on the structure and function of PAH from bacteria and lower eukaryote organisms, revealing an additional anabolic role of the enzyme in the synthesis of melanin-like pigments. In this review, we discuss these recent studies, which contribute to define the evolutionary adaptation of the PAH structure and function leading to sophisticated regulation for effective catabolic processing of phenylalanine in mammalian organisms.

  13. Genetic diversity of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) genes in cattle breeds

    Science.gov (United States)

    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

  14. Release kinetics of prolyl hydroxylase inhibitors from collagen barrier membranes.

    Science.gov (United States)

    Hamid, Omar; Pensch, Manuela; Agis, Hermann

    2015-03-01

    Collagen barrier membranes are used in guided tissue regeneration to support healing. This strategy, however, relies on the healing capacity of the tissue. Pharmacological inhibitors of prolyl hydroxylases can support regeneration by enhancing angiogenesis and are therefore a promising tool for periodontology. Here we evaluate the release kinetics of the prolyl hydroxylase inhibitors dimethyloxalylglycine and L-mimosine from collagen barrier membranes. Dimethyloxalylglycine and L-mimosine were lyophilized onto the collagen barrier membranes. The morphology of the collagen barrier membranes was analysed using scanning electron microscopy. The release of prolyl hydroxylase inhibitors was assessed by colorimetric and spectroscopic methods. Their ability to induce a cellular response was assessed in bioassays with gingival and periodontal ligament fibroblasts based on vascular endothelial growth factor production, proliferation, and metabolic activity of the cells. We found that loading of collagen barrier membranes with prolyl hydroxylase inhibitors did not change the overall membrane morphology. Assessment of the release kinetics by direct measurements and based on vascular endothelial growth factor production showed that supernatants obtained from the collagen barrier membranes in the first 6 hours had a sufficient level of prolyl hydroxylase inhibitors to induce vascular endothelial growth factor production. A similar kinetic was found when cell proliferation was assessed. Changes in metabolic activity did not reach the level of significance in the MTT assay. In conclusion, collagen barrier membranes can release prolyl hydroxylase inhibitors thereby increasing the pro-angiogenic capacity of periodontal cells in vitro. These findings provide the basis for preclinical studies to evaluate the regenerative capacity of prolyl hydroxylase inhibitors in periodontology and oral surgery.

  15. Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

    Science.gov (United States)

    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

  16. Are striatal tyrosine hydroxylase interneurons dopaminergic?

    Science.gov (United States)

    Xenias, Harry S; Ibáñez-Sandoval, Osvaldo; Koós, Tibor; Tepper, James M

    2015-04-22

    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.

  17. Raman characteristics of hydrocarbon and hydrocarbon inclusions

    Institute of Scientific and Technical Information of China (English)

    ZHANG Nai; TIAN ZuoJi; LENG YingYing; WANG HuiTong; SONG FuQing; MENG JianHua

    2007-01-01

    The Raman spectrograms of hydrocarbon standard samples show that: (1) the Raman spectrogram of normal paraffin has very strong peaks of methyl and methylene (from 2700 cm-1 to 2970 cm-1); (2)branch methyl has the particular peak of 748 cm-1±; (3) six cyclic has the particular peak of 804 cm-1±; (4)phenyl has two particular peaks of 988 cm-1± and 3058 cm-1± and the 988 cm-1± peak is stronger than the 3058 cm-1± peak; and (5) hexene has three alkenyl spectrum peaks of 1294 cm-1±, 1635 cm-1± and 2996 cm-1±, with the 1635 cm-1± peak being the strongest, showing that the number of carbon in hydrocarbon does not affect its Raman spectrogram, and the hydrocarbon molecular structure and base groups affect its Raman spectrogram, the same hydrocarbons (such as normal paraffin) have the same Raman spectrogram; the types (such as CH4, C2H6, C3H8) and the content of hydrocarbon in oil inclusions are not estimated by their characteristic Raman peaks. According to the Raman spectrograms of hydrocarbon compositions, the Raman spectrogram of hydrocarbon inclusion can be divided into five types: saturated hydrocarbon Raman spectrogram, fluoresce Raman spectrogram, saturated hydrocarbon bitumen Raman spectrogram, bitumen Raman spectrogram, and ethane Raman spectrogram.And according to the characteristics of Raman spectrogram, hydrocarbon inclusions can be divided into five types: saturated hydrocarbon inclusion, less saturated hydrocarbon (oil or gas) inclusion,saturated hydrocarbon bitumen inclusion, bitumen inclusion, and methane water inclusion.

  18. Raman characteristics of hydrocarbon and hydrocarbon inclusions

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The Raman spectrograms of hydrocarbon standard samples show that: (1) the Raman spectrogram of normal paraffin has very strong peaks of methyl and methylene (from 2700 cm-1 to 2970 cm-1); (2) branch methyl has the particular peak of 748 cm-1±; (3) six cyclic has the particular peak of 804 cm-1±; (4) phenyl has two particular peaks of 988 cm-1± and 3058 cm-1± and the 988 cm-1± peak is stronger than the 3058 cm-1± peak; and (5) hexene has three alkenyl spectrum peaks of 1294 cm-1±, 1635 cm-1± and 2996 cm-1±, with the 1635 cm-1± peak being the strongest, showing that the number of carbon in hy-drocarbon does not affect its Raman spectrogram, and the hydrocarbon molecular structure and base groups affect its Raman spectrogram, the same hydrocarbons (such as normal paraffin) have the same Raman spectrogram; the types (such as CH4, C2H6, C3H8) and the content of hydrocarbon in oil inclu-sions are not estimated by their characteristic Raman peaks. According to the Raman spectrograms of hydrocarbon compositions, the Raman spectrogram of hydrocarbon inclusion can be divided into five types: saturated hydrocarbon Raman spectrogram, fluoresce Raman spectrogram, saturated hydro-carbon bitumen Raman spectrogram, bitumen Raman spectrogram, and ethane Raman spectrogram. And according to the characteristics of Raman spectrogram, hydrocarbon inclusions can be divided into five types: saturated hydrocarbon inclusion, less saturated hydrocarbon (oil or gas) inclusion, saturated hydrocarbon bitumen inclusion, bitumen inclusion, and methane water inclusion.

  19. A case of 17 alpha-hydroxylase deficiency.

    Science.gov (United States)

    Kim, Sung Mee; Rhee, Jeong Ho

    2015-06-01

    17α-hydroxylase and 17,20-lyase are enzymes encoded by the CYP17A1 gene and are required for the synthesis of sex steroids and cortisol. In 17α-hydroxylase deficiency, there are low blood levels of estrogens, androgens, and cortisol, and resultant compensatory increases in adrenocorticotrophic hormone that stimulate the production of 11-deoxycorticosterone and corticosterone. In turn, the excessive levels of mineralocorticoids lead to volume expansion and hypertension. Females with 17α-hydroxylase deficiency are characterized by primary amenorrhea and delayed puberty, with accompanying hypertension. Affected males usually have female external genitalia, a blind vagina, and intra-abdominal testes. The treatment of this disorder is centered on glucocorticoid and sex steroid replacement. In patients with 17α-hydroxylase deficiency who are being raised as females, estrogen should be supplemented, while genetically female patients with a uterus should also receive progesterone supplementation. Here, we report a case of a 21-year-old female with 17α-hydroxylase deficiency who had received inadequate treatment for a prolonged period of time. We also include a brief review of the recent literature on this disorder.

  20. Ovarian intratumoral 21-hydroxylase deficiency in a postmenopausal hirsute woman.

    Science.gov (United States)

    Souto, Selma B; Baptista, Pedro V; Barreto, Filomena; Sousa, Pedro F; Braga, Daniel C; Carvalho, Davide

    2012-12-01

    Virilising ovarian tumours are a rare cause of hyperandrogenism in women, accounting for less than 5% of all ovarian neoplasms. It occurs most often in - and postmenopausal women. We report a case of a 64 year-old woman with signs of virilisation that had started 3 years before. Blood hormone analysis revealed increased levels of testosterone, and 17-hydroxyprogesterone. The tetracosactin test revealed 21-hydroxylase deficiency. Radiological imaging demonstrated a nodule in her left ovary. The patient was submitted to bilateral laparoscopic oophorectomy, and histopathological examination revealed a luteoma of the left ovary. Postoperative serum testosterone level and 17-hydroxyprogesterone returned to normal levels in one month. Virilism regressed within six months. Our patient also showed an elevation in 17-OHP serum levels. Normalization of 17-OHP after oophorectomy suggests a case of intratumoral 21-hydroxylase deficiency. To our knowledge, this is the first description of ovarian intratumoral 21-hydroxylase deficiency in a postmenopausal woman.

  1. Sleep patterns in congenital dopamine beta-hydroxylase deficiency

    NARCIS (Netherlands)

    J.H.M. Tulen (Joke); A.J. Man in't Veld (A.); K. Mechelse (Karel); F. Boomsma (Frans)

    1990-01-01

    textabstractSleep patterns of two young female patients with congenital dopamine beta-hydroxylase deficiency are described. In this orthostatic syndrome central and peripheral noradrenergic failure occurs as a result of impaired beta-hydroxylation of dopamine. Consequently, the levels of dopamine an

  2. Association between Tryptophan Hydroxylase 2 Gene Polymorphism and Completed Suicide

    Science.gov (United States)

    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…

  3. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency.

    Science.gov (United States)

    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

    2014-02-01

    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.

  4. Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Dutch neonates

    NARCIS (Netherlands)

    Spaapen, LJM; Bakker, JA; Velter, C; Loots, W; Rubio-Gonzalbo, ME; Forget, PP; Dorland, L; De Koning, TJ; Poll-The, BT; Van Amstel, HKP; Bekhof, J; Blau, N; Duran, M

    2001-01-01

    Four neonates with a positive phenylalanine screening test (Phe concentrations between 258 and 1250 mu mol/L) were investigated further to differentiate between phenylalanine hydroxylase (PAH) deficiency and variant hyperphenylalaninaemia (HPA) forms. In patients 1 and 2 a tetrahydrobiopterin (BH4)

  5. Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes.

    Science.gov (United States)

    Cafaro, Valeria; Izzo, Viviana; Scognamiglio, Roberta; Notomista, Eugenio; Capasso, Paola; Casbarra, Annarita; Pucci, Piero; Di Donato, Alberto

    2004-04-01

    Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.

  6. The P450-type carotene hydroxylase PuCHY1 from Porphyra suggests the evolution of carotenoid metabolism in red algae

    Institute of Scientific and Technical Information of China (English)

    Li-En Yang; Xing-Qi Huang; Yu Hang; Yin-Yin Deng; Qin-Qin Lu; Shan Lu

    2014-01-01

    Carotene hydroxylases catalyze the hydroxylation of a-and b-carotene hydrocarbons into xanthophylls. In red algae, b-carotene is a ubiquitously distributed carotenoid, and hydroxylated carotenoids such as zeaxanthin and lutein are also found. However, no enzyme with carotene hydroxy-lase activity had been previously identified in red algae. Here, we report the isolation of a gene encoding a cytochrome P450-type carotene hydroxylase (PuCHY1) from Porphyra umbilicalis, a red alga with an ancient origin. Sequence comparisons found PuCHY1 belongs to the CYP97B subfamily, which has members from different photosynthetic organisms ranging from red algae to land plants. Functional comple-mentation in Escherichia coli suggested that PuCHY1 catalyzed the conversion from b-carotene to zeaxanthin. When we overexpressed PuCHY1 in the Arabidopsis thaliana chy2 mutant, pigment analysis showed a significant accumu-lation of hydroxylated carotenoids, including neoxanthin, violaxanthin, and lutein in the leaves of transgenic plants. These results confirmed a b-hydroxylation activity of PuCHY1, and also suggested a possible e-hydroxylation function. The pigment profile and gene expression analyses of the algal thallus under high-light stress suggested that P. umbilicalis is unlikely to operate a partial xanthophyll cycle for photoprotection.

  7. Mutagenesis of a specificity-determining residue in tyrosine hydroxylase establishes that the enzyme is a robust phenylalanine hydroxylase but a fragile tyrosine hydroxylase.

    Science.gov (United States)

    Daubner, S Colette; Avila, Audrey; Bailey, Johnathan O; Barrera, Dimitrios; Bermudez, Jaclyn Y; Giles, David H; Khan, Crystal A; Shaheen, Noel; Thompson, Janie Womac; Vasquez, Jessica; Oxley, Susan P; Fitzpatrick, Paul F

    2013-02-26

    The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites; however, PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme [Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652-9661]. Alanine-scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 10(4)) decreases in the V(max) and V(max)/K(tyr) values for tyrosine hydroxylation, but only small decreases or even increases in the V(max) and V(max)/K(phe) values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the extent of coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the V(max) value with tyrosine as the substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.

  8. Expression of the vitamin D receptor, 25-hydroxylases, 1alpha-hydroxylase and 24-hydroxylase in the human kidney and renal clear cell cancer

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Andersen, Claus B.; Nielsen, John E

    2010-01-01

    The vitamin D receptor (VDR), CYP27B1 and CYP24A1 are expressed in the human kidney, but the segmental expression of the 25-hydroxylases is unknown. A comprehensive analysis of CYP2R1, CYP27A1, CYP27B1, VDR and CYP24A1 expression in normal kidney and renal clear cell cancer (CCc) would reveal...... the segmental location of expression, and clarify whether the reported loss of VDR in CCc is coincident with alterations of vitamin D metabolism....

  9. Expression of the vitamin D receptor, 25-hydroxylases, 1alpha-hydroxylase and 24-hydroxylase in the human kidney and renal clear cell cancer

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Andersen, Claus B.; Nielsen, John E;

    2010-01-01

    The vitamin D receptor (VDR), CYP27B1 and CYP24A1 are expressed in the human kidney, but the segmental expression of the 25-hydroxylases is unknown. A comprehensive analysis of CYP2R1, CYP27A1, CYP27B1, VDR and CYP24A1 expression in normal kidney and renal clear cell cancer (CCc) would reveal...... the segmental location of expression, and clarify whether the reported loss of VDR in CCc is coincident with alterations of vitamin D metabolism....

  10. HIF hydroxylase pathways in cardiovascular physiology and medicine.

    Science.gov (United States)

    Bishop, Tammie; Ratcliffe, Peter J

    2015-06-19

    Hypoxia inducible factors (HIFs) are α/β heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate-dependent dioxygenases that catalyze post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFα subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here, we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities, and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease.

  11. Identification of phenylalanine 3-hydroxylase for meta-tyrosine biosynthesis.

    Science.gov (United States)

    Zhang, Wenjun; Ames, Brian D; Walsh, Christopher T

    2011-06-21

    Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (L-Phe) to l-tyrosine (L-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe. Subsequent mutagenesis studies revealed that two residues in the active site of Phe3H (Cys187 and Thr202) contribute to C-3 rather than C-4 hydroxylation of the phenyl ring. This work sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.

  12. Perinatal kynurenine 3-hydroxylase inhibition in rodents: pathophysiological implications.

    Science.gov (United States)

    Ceresoli-Borroni, Gianpiera; Guidetti, Paolo; Amori, Laura; Pellicciari, Roberto; Schwarcz, Robert

    2007-03-01

    The kynurenine pathway (KP) of tryptophan degradation contains three neuroactive metabolites: the neuroinhibitory agent kynurenic acid (KYNA) and, in a competing branch, the free radical generator 3-hydroxykynurenine (3-HK) and the excitotoxin quinolinic acid (QUIN). These three "kynurenines" derive from a common precursor, L-kynurenine, and are recognized for their role in brain physiology and pathophysiology. Inhibition of kynurenine 3-hydroxylase, the enzyme responsible for 3-HK formation, shifts KP metabolism in the mature brain toward enhanced KYNA formation. We now tested the cerebral effects of kynurenine 3-hydroxylase inhibition in immature rodents. Rat pups treated with the kynurenine 3-hydroxylase inhibitor UPF 648 (30 mg/kg, i.p.) 10 min after birth showed substantial increases in cerebral and liver kynurenine and KYNA levels up to 24 hr later, whereas 3-HK and QUIN levels were simultaneously decreased. Administered to pregnant rats or mice on the last day of gestation, UPF 648 (50 mg/kg, i.p.) produced qualitatively similar changes (i.e., large increases in kynurenine and KYNA and reductions in 3-HK and QUIN) in the brain and liver of the offspring. Rat pups delivered by UPF 648-treated mothers and immediately exposed to neonatal asphyxia showed further enhanced brain KYNA levels. These studies demonstrate that acute kynurenine 3-hydroxylase inhibition effectively shifts cerebral KP metabolism in neonatal rodents toward increased KYNA formation. Selective inhibitors of this enzyme may therefore provide neuroprotection in newborns and will also be useful for the experimental evaluation of the long-term effects of perinatal KP impairment.

  13. Sleep patterns in congenital dopamine beta-hydroxylase deficiency

    OpenAIRE

    Tulen, Joke; Man in't Veld, A.; Mechelse, Karel; Boomsma, Frans

    1990-01-01

    textabstractSleep patterns of two young female patients with congenital dopamine beta-hydroxylase deficiency are described. In this orthostatic syndrome central and peripheral noradrenergic failure occurs as a result of impaired beta-hydroxylation of dopamine. Consequently, the levels of dopamine and its metabolites are elevated. The relative importance of noradrenaline deficit in the face of dopamine excess for sleep-regulatory mechanisms can be inferred from the sleep pattern of these patie...

  14. A unique dual activity amino acid hydroxylase in Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Gaskell

    Full Text Available The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces L-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to L-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s of these bi-functional enzymes during host infection are discussed.

  15. Oxygenated Derivatives of Hydrocarbons

    Science.gov (United States)

    For the book entitled “Insect Hydrocarbons: Biology, Biochemistry and Chemical Ecology”, this chapter presents a comprehensive review of the occurrence, structure and function of oxygenated derivatives of hydrocarbons. The book chapter focuses on the occurrence, structural identification and functi...

  16. Hydrocarbon Spectral Database

    Science.gov (United States)

    SRD 115 Hydrocarbon Spectral Database (Web, free access)   All of the rotational spectral lines observed and reported in the open literature for 91 hydrocarbon molecules have been tabulated. The isotopic molecular species, assigned quantum numbers, observed frequency, estimated measurement uncertainty and reference are given for each transition reported.

  17. Plasma devices for hydrocarbon reformation

    KAUST Repository

    Cha, Min Suk

    2017-02-16

    Plasma devices for hydrocarbon reformation are provided. Methods of using the devices for hydrocarbon reformation are also provided. The devices can include a liquid container to receive a hydrocarbon source, and a plasma torch configured to be submerged in the liquid. The plasma plume from the plasma torch can cause reformation of the hydrocarbon. The device can use a variety of plasma torches that can be arranged in a variety of positions in the liquid container. The devices can be used for the reformation of gaseous hydrocarbons and/or liquid hydrocarbons. The reformation can produce methane, lower hydrocarbons, higher hydrocarbons, hydrogen gas, water, carbon dioxide, carbon monoxide, or a combination thereof.

  18. Plant hydrocarbon recovery process

    Energy Technology Data Exchange (ETDEWEB)

    Dzadzic, P.M.; Price, M.C.; Shih, C.J.; Weil, T.A.

    1982-01-26

    A process for production and recovery of hydrocarbons from hydrocarbon-containing whole plants in a form suitable for use as chemical feedstocks or as hydrocarbon energy sources which process comprises: (A) pulverizing by grinding or chopping hydrocarbon-containing whole plants selected from the group consisting of euphorbiaceae, apocynaceae, asclepiadaceae, compositae, cactaceae and pinaceae families to a suitable particle size, (B) drying and preheating said particles in a reducing atmosphere under positive pressure (C) passing said particles through a thermal conversion zone containing a reducing atmosphere and with a residence time of 1 second to about 30 minutes at a temperature within the range of from about 200* C. To about 1000* C., (D) separately recovering the condensable vapors as liquids and the noncondensable gases in a condition suitable for use as chemical feedstocks or as hydrocarbon fuels.

  19. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu

    2009-06-01

    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  20. Identification and characterization of phenol hydroxylase from phenol-degrading Candida tropicalis strain JH8.

    Science.gov (United States)

    Long, Yan; Yang, Sheng; Xie, Zhixiong; Cheng, Li

    2014-09-01

    The gene phhY encoding phenol hydroxylase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The gene phhY contained an open reading frame of 2130 bp encoding a polypeptide of 709 amino acid residues. From its sequence analysis, it is a member of a family of flavin-containing aromatic hydroxylases and shares 41% amino acid identity with phenol hydroxylase from Trichosporon cutaneum. The recombinant phenol hydroxylase exists as a homotetramer structure with a native molecular mass of 320 kDa. Recombinant phenol hydroxylase was insensitive to pH treatment; its optimum pH was at 7.6. The optimum temperature for the enzyme was 30 °C, and its activity was rapidly lost at temperatures above 60 °C. Under the optimal conditions with phenol as substrate, the K(m) and V(max) of recombinant phenol hydroxylase were 0.21 mmol·L(-1) and 0.077 μmol·L(-1)·min(-1), respectively. This is the first paper presenting the cloning and expression in E. coli of the phenol hydroxylase gene from C. tropicalis and the characterization of the recombinant phenol hydroxylase.

  1. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;

    2009-01-01

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed...

  2. Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal ion and tetrahydrobiopterin dependence.

    Science.gov (United States)

    Watschinger, Katrin; Keller, Markus A; Hermetter, Albin; Golderer, Georg; Werner-Felmayer, Gabriele; Werner, Ernst R

    2009-01-01

    Glyceryl ether monooxygenase is a tetrahydrobiopterin-dependent membrane-bound enzyme which catalyses the cleavage of lipid ethers into glycerol and the corresponding aldehyde. Despite many different characterisation and purification attempts, so far no gene and primary sequence have been assigned to this enzyme. The seven other tetrahydrobiopterin-dependent enzymes can be divided in the family of aromatic amino acid hydroxylases - comprising phenylalanine hydroxylase, tyrosine hydroxylase and the two tryptophan hydroxylases - and into the three nitric oxide synthases. We tested the influences of different metal ions and metal ion chelators on glyceryl ether monooxygenase, phenylalanine hydroxylase and nitric oxide synthase activity to elucidate the relationship of glyceryl ether monooxygenase to these two families. 1,10-Phenanthroline, an inhibitor of non-heme iron-dependent enzymes, was able to potently block glyceryl ether monooxygenase as well as phenylalanine hydroxylase, but had no effect on inducible nitric oxide synthase. Two tetrahydrobiopterin analogues, N(5)-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin, had a similar impact on glyceryl ether monooxygenase activity, as has already been shown for phenylalanine hydroxylase. These observations point to a close analogy of the role of tetrahydrobiopterin in glyceryl ether monooxygenase and in aromatic amino acid hydroxylases and suggest that glyceryl ether monooxygenase may require a non-heme iron for catalysis.

  3. Thraustochytrid protists degrade hydrocarbons

    Digital Repository Service at National Institute of Oceanography (India)

    Raikar, M.T.; Raghukumar, S.; Vani, V.; David, J.J.; Chandramohan, D.

    Although thraustochytrid protists are known to be of widespread occurrence in the sea, their hydrocarbon-degrading abilities have never been investigated. We isolated thraustochytrids from coastal waters and sediments of Goa coast by enriching MPN...

  4. Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?

    Energy Technology Data Exchange (ETDEWEB)

    Paisse, Sandrine; Duran, Robert; Goni-Urriza, Marisol [Pau Univ. (France). Equipe Environnement et Microbiologie-UMR IPREM5254; Coulon, Frederic [Cranfield Univ. (United Kingdom). Centre for Resource Management and Efficiency

    2011-11-15

    The diversity of alkB-related alkane hydroxylase sequences and the relationship between alkB gene expression and the hydrocarbon contamination level have been investigated in the chronically polluted Etang-de-Berre sediments. For this purpose, these sediments were maintained in microcosms and submitted to a controlled oil input miming an oil spill. New degenerated PCR primers targeting alkB-related alkane hydroxylase sequences were designed to explore the diversity and the expression of these genes using terminal restriction fragment length polymorphism fingerprinting and gene library analyses. Induction of alkB genes was detected immediately after oil addition and their expression detected only during 2 days, although the n-alkane degradation was observed throughout the 14 days of incubation. The alkB gene expression within triplicate microcosms was heterogeneous probably due to the low level of alkB transcripts. Moreover, the alkB gene expression of dominant OTUs has been observed in unoiled microcosms indicating that the expression of this gene cannot be directly related to the oil contamination. Although the dominant alkB genes and transcripts detected were closely related to the alkB of Marinobacter aquaeolei isolated from an oil-producing well, and to alkB genes related to the obligate alkanotroph Alcanivorax borkumensis, no clear relationship between the oil contamination and the expression of the alkB genes could be established. This finding suggests that in such coastal environments, alkB gene expression is not a function relevant enough to monitor bacterial response to oil contamination. (orig.)

  5. Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments?

    Science.gov (United States)

    Paisse, Sandrine; Duran, Robert; Coulon, Frédéric; Goñi-Urriza, Marisol

    2011-11-01

    The diversity of alkB-related alkane hydroxylase sequences and the relationship between alkB gene expression and the hydrocarbon contamination level have been investigated in the chronically polluted Etang-de-Berre sediments. For this purpose, these sediments were maintained in microcosms and submitted to a controlled oil input miming an oil spill. New degenerated PCR primers targeting alkB-related alkane hydroxylase sequences were designed to explore the diversity and the expression of these genes using terminal restriction fragment length polymorphism fingerprinting and gene library analyses. Induction of alkB genes was detected immediately after oil addition and their expression detected only during 2 days, although the n-alkane degradation was observed throughout the 14 days of incubation. The alkB gene expression within triplicate microcosms was heterogeneous probably due to the low level of alkB transcripts. Moreover, the alkB gene expression of dominant OTUs has been observed in unoiled microcosms indicating that the expression of this gene cannot be directly related to the oil contamination. Although the dominant alkB genes and transcripts detected were closely related to the alkB of Marinobacter aquaeolei isolated from an oil-producing well, and to alkB genes related to the obligate alkanotroph Alcanivorax borkumensis, no clear relationship between the oil contamination and the expression of the alkB genes could be established. This finding suggests that in such coastal environments, alkB gene expression is not a function relevant enough to monitor bacterial response to oil contamination.

  6. Glucocorticoid-induced osteoporosis in children with 21-hydroxylase deficiency.

    Science.gov (United States)

    Ventura, Annamaria; Brunetti, Giacomina; Colucci, Silvia; Oranger, Angela; Ladisa, Filomena; Cavallo, Luciano; Grano, Maria; Faienza, Maria Felicia

    2013-01-01

    21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), resulting from deletions or mutations of the P450 21-hydroxylase gene (CYP21A2). Children with 21-OHD need chronic glucocorticoid (cGC) therapy, both to replace congenital deficit in cortisol synthesis and to reduce androgen secretion by adrenal cortex. GC-induced osteoporosis (GIO) is the most common form of secondary osteoporosis that results in an early, transient increase in bone resorption accompanied by a decrease in bone formation, maintained for the duration of GC therapy. Despite the conflicting results in the literature about the bone status on GC-treated patients with 21-OHD, many reports consider these subjects to be at risk for osteoporosis and fractures. In bone cells, at the molecular level, GCs regulate various functions including osteoblastogenesis, osteoclastogenesis, and the apoptosis of osteoblasts and osteocytes. In this paper, we focus on the physiology and biosynthesis of endogenous steroid hormones as well as on the effects of GCs on bone cells, highlighting the pathogenetic mechanism of GIO in children with 21-OHD.

  7. Characterization of two carnation petal prolyl 4 hydroxylases.

    Science.gov (United States)

    Vlad, Florina; Tiainen, Päivi; Owen, Carolyn; Spano, Thodhoraq; Daher, Firas Bou; Oualid, Fatiha; Senol, Namik Ozer; Vlad, Daniela; Myllyharju, Johanna; Kalaitzis, Panagiotis

    2010-10-01

    Prolyl 4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification, of hydroxyproline-rich glycoproteins. Two carnation petal P4H cDNAs, (Dianthus caryophyllus prolyl 4-hydroxylase) DcP4H1 and DcP4H2, were identified and characterized at the gene expression and biochemical level in order to investigate their role in flower senescence. Both mRNAs showed similar patterns of expression with stable transcript abundance during senescence progression and differential tissue-specific expression with DcP4H1 and DcP4H2 strongly expressed in ovaries and stems, respectively. Recombinant DcP4H1 and DcP4H2 proteins were produced and their catalytic properties were determined. Pyridine 2,4-dicarboxylate (PDCA) was identified as a potent inhibitor of the in vitro enzyme activity of both P4Hs and used to determine whether inhibition of proline hydroxylation in petals is involved in senescence progression of cut carnation flowers. PDCA suppressed the climacteric ethylene production indicating a strong correlation between the inhibition of DcP4H1 and DcP4H2 activity in vitro by PDCA and the suppression of climacteric ethylene production in cut carnation flowers.

  8. Glucocorticoid-Induced Osteoporosis in Children with 21-Hydroxylase Deficiency

    Directory of Open Access Journals (Sweden)

    Annamaria Ventura

    2013-01-01

    Full Text Available 21-Hydroxylase deficiency (21-OHD is the most common cause of congenital adrenal hyperplasia (CAH, resulting from deletions or mutations of the P450 21-hydroxylase gene (CYP21A2. Children with 21-OHD need chronic glucocorticoid (cGC therapy, both to replace congenital deficit in cortisol synthesis and to reduce androgen secretion by adrenal cortex. GC-induced osteoporosis (GIO is the most common form of secondary osteoporosis that results in an early, transient increase in bone resorption accompanied by a decrease in bone formation, maintained for the duration of GC therapy. Despite the conflicting results in the literature about the bone status on GC-treated patients with 21-OHD, many reports consider these subjects to be at risk for osteoporosis and fractures. In bone cells, at the molecular level, GCs regulate various functions including osteoblastogenesis, osteoclastogenesis, and the apoptosis of osteoblasts and osteocytes. In this paper, we focus on the physiology and biosynthesis of endogenous steroid hormones as well as on the effects of GCs on bone cells, highlighting the pathogenetic mechanism of GIO in children with 21-OHD.

  9. Spectrum of mutations in Lebanese patients with phenylalanine hydroxylase deficiency.

    Science.gov (United States)

    Karam, Pascale E; Alhamra, Rasha Shahabeddeen; Nemer, Georges; Usta, Julnar

    2013-02-15

    Phenylketonuria is an autosomal recessive inborn error of metabolism resulting from phenylalanine hydroxylase deficiency. Genetic basis of phenylalanine hydroxylase deficiency has been reported in various European and Asian countries with few reports available in Arab populations of the Mediterranean region. This is the first pilot study describing phenotype and genotype of 23 Lebanese patients with phenylketonuria. 48% of the patients presented mainly with neurological signs at a mean age of 2 years 9 months, as newborn screening is not yet a nationwide policy. 56.5% of the patients had classical phenylketonuria. Thirteen different mutations were identified: splice site 52%, frameshift 31%, and missense 17% with no nonsense mutations. IVS10-11G>A was found mainly in Christians at high relative frequency whereas Muslims carried the G352fs and R261Q mutations. A rare splice mutation IVS7+1G>T, not described before, was identified in the homozygous state in one family with moderate phenylketonuria phenotype. Genotype-phenotype correlation using Guldberg arbitrary value method showed high consistency between predicted and observed phenotypes. Calculated homozygosity rate was 0.07 indicating the genetic heterogeneity in our patients. Our findings underline the admixture of different ethnicities and religions in Lebanon that might help tracing back the PAH gene flux history across the Mediterranean region.

  10. Adult human liver mesenchymal progenitor cells express phenylalanine hydroxylase.

    Science.gov (United States)

    Baruteau, Julien; Nyabi, Omar; Najimi, Mustapha; Fauvart, Maarten; Sokal, Etienne

    2014-09-01

    Phenylketonuria (PKU) is one of the most prevalent inherited metabolic diseases and is accountable for a severe encephalopathy by progressive intoxication of the brain by phenylalanine. This results from an ineffective L-phenylalanine hydroxylase enzyme (PAH) due to a mutated phenylalanine hydroxylase (PAH) gene. Neonatal screening programs allow an early dietetic treatment with restrictive phenylalanine intake. This diet prevents most of the neuropsychological disabilities but remains challenging for lifelong compliance. Adult-derived human liver progenitor cells (ADHLPC) are a pool of precursors that can differentiate into hepatocytes. We aim to study PAH expression and PAH activity in a differenciated ADHLPC. ADHLPC were isolated from human hepatocyte primary culture of two different donors and differenciated under specific culture conditions. We demonstrated the high expression of PAH and a large increase of PAH activity in differenciated LPC. The age of the donor, the cellular viability after liver digestion and cryopreservation affects PAH activity. ADHLPC might therefore be considered as a suitable source for cell therapy in PKU.

  11. Dynamic regulation of phenylalanine hydroxylase by simulated redox manipulation.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    Full Text Available Recent clinical studies revealed increased phenylalanine levels and phenylalanine to tyrosine ratios in patients suffering from infection, inflammation and general immune activity. These data implicated down-regulation of activity of phenylalanine hydroxylase by oxidative stress upon in vivo immune activation. Though the structural damage of oxidative stress is expected to be comparably small, a structural rationale for this experimental finding was lacking. Hence, we investigated the impact of side chain oxidation at two vicinal cysteine residues on local conformational flexibility in the protein by comparative molecular dynamics simulations. Analysis of backbone dynamics revealed a highly flexible loop region (Tyr138-loop in proximity to the active center of phenylalanine hydroxylase. We observed elevated loop dynamics in connection with a loop movement towards the active site in the oxidized state, thereby partially blocking access for the substrate phenylalanine. These findings were confirmed by extensive replica exchange molecular dynamics simulations and serve as a first structural explanation for decreased enzyme turnover in situations of oxidative stress.

  12. Dynamic regulation of phenylalanine hydroxylase by simulated redox manipulation.

    Science.gov (United States)

    Fuchs, Julian E; Huber, Roland G; von Grafenstein, Susanne; Wallnoefer, Hannes G; Spitzer, Gudrun M; Fuchs, Dietmar; Liedl, Klaus R

    2012-01-01

    Recent clinical studies revealed increased phenylalanine levels and phenylalanine to tyrosine ratios in patients suffering from infection, inflammation and general immune activity. These data implicated down-regulation of activity of phenylalanine hydroxylase by oxidative stress upon in vivo immune activation. Though the structural damage of oxidative stress is expected to be comparably small, a structural rationale for this experimental finding was lacking. Hence, we investigated the impact of side chain oxidation at two vicinal cysteine residues on local conformational flexibility in the protein by comparative molecular dynamics simulations. Analysis of backbone dynamics revealed a highly flexible loop region (Tyr138-loop) in proximity to the active center of phenylalanine hydroxylase. We observed elevated loop dynamics in connection with a loop movement towards the active site in the oxidized state, thereby partially blocking access for the substrate phenylalanine. These findings were confirmed by extensive replica exchange molecular dynamics simulations and serve as a first structural explanation for decreased enzyme turnover in situations of oxidative stress.

  13. Plasma dopamine beta hydroxylase activity in affective disorders.

    Science.gov (United States)

    Levitt, M; Dunner, D L; Mendlewicz, J; Frewin, D B; Lawlor, W; Fleiss, J L; Stallone, F; Fieve, R R

    1976-03-16

    Plasma dopamine beta hydroxylase (DBH) activity was measured in patients with affective disorders and in their relatives. The groups studied had wide distributions of values for plasma DBH activity. No significant difference of plasma DBH activity was found between unipolar and bipolar patients, nor between patients given lithium or placebo. Exercise on a treadmill at 40 degrees or 10 degrees C elicited a different pattern of response for plasma DBH activity in three patients as compared to control subjects. In familial studies we found the values of plasma DBH activity to be almost identical in monozygotic twin pairs and quite similar in dizygotic twin pairs. All pairs, however, were discordant for affective illness. There was also a marked similarity of plasma DBH activity in 15 pairs of the same sex sibs discordant for affective illness. These studies suggest that the resting level of plasma DBH activity is not related to affective illness but is genetically determined.

  14. A New Tyrosine Hydroxylase Genotype with Orofacial Dyskinaesia

    Directory of Open Access Journals (Sweden)

    Ahood M. Al-Muslamani

    2014-08-01

    Full Text Available Tyrosine hydroxylase (TH deficiency is a rare autosomal recessive and often treatable neurometabolic disorder with variable phenotypes. More than 20 pathological mutations have been identified in patients with TH deficiency. We report the case of a 10-month-old male patient who presented with developmental delay, hypotonia and oculogyric crises to the Salmaniya Medical Complex in Manama, Bahrain. At a later stage, he developed orofacial dyskinaesia and tremors with hyper-reflexia and clonus. A magnetic resonance imaging scan of the brain showed mild atrophy with widened ventricles and genetic testing revealed a novel homozygous mutation (c.938G>T; p.Arg313Leu in exon 9 of the TH gene. The patient showed a remarkable response to treatment using combined levodopa-carbidopa. In this case, the orofacial dyskinaesia may be a specific clinical association unique to this novel mutation, which is the first to be described in Bahrain and the Middle East.

  15. Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine.

    Science.gov (United States)

    Kim, Hye-Lim; Park, Mi-Bee; Kim, Yumin; Yang, Yun Gyeong; Lee, Soo-Woong; Zhuang, Ningning; Lee, Kon Ho; Park, Young Shik

    2012-10-19

    We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

  16. Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major.

    Science.gov (United States)

    Lye, Lon-Fye; Kang, Song Ok; Nosanchuk, Joshua D; Casadevall, Arturo; Beverley, Stephen M

    2011-01-01

    Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H(4)B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H(4)B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H(4)B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah(-) null mutant and an episomal complemented overexpressing derivative (pah-/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah(-) parasites showed reactivity with an antibody to melanin when grown with l-3,4-dihydroxyphenylalanine (L-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah(-) showed an increased sensitivity to Tyr deprivation, while the pah(-)/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah(-) showed no alterations in H(4)B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin.

  17. Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism

    Directory of Open Access Journals (Sweden)

    Yang M

    2014-08-01

    Full Text Available Ming Yang,1 Huizhong Su,1 Tomoyoshi Soga,2 Kamil R Kranc,3 Patrick J Pollard1 1Cancer Biology and Metabolism Group, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; 2Institute for Advanced Biosciences, Keio University, Mizukami, Tsuruoka, Yamagata, Japan; 3MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK Abstract: The hypoxia-inducible factor (HIF prolyl hydroxylase domain enzymes (PHDs regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2 availability enables its association with the von Hippel-Lindau (VHL tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFβ and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism. Keywords: prolyl hydroxylase domain (PHD

  18. Quantitative Hydrocarbon Surface Analysis

    Science.gov (United States)

    Douglas, Vonnie M.

    2000-01-01

    The elimination of ozone depleting substances, such as carbon tetrachloride, has resulted in the use of new analytical techniques for cleanliness verification and contamination sampling. The last remaining application at Rocketdyne which required a replacement technique was the quantitative analysis of hydrocarbons by infrared spectrometry. This application, which previously utilized carbon tetrachloride, was successfully modified using the SOC-400, a compact portable FTIR manufactured by Surface Optics Corporation. This instrument can quantitatively measure and identify hydrocarbons from solvent flush of hardware as well as directly analyze the surface of metallic components without the use of ozone depleting chemicals. Several sampling accessories are utilized to perform analysis for various applications.

  19. Miscellaneous hydrocarbon solvents.

    Science.gov (United States)

    Bebarta, Vikhyat; DeWitt, Christopher

    2004-08-01

    The solvents discussed in this article are common solvents not categorized as halogenated, aromatic, or botanical. The solvents discussed are categorized into two groups: hydrocarbon mixtures and single agents. The hydrocarbon mixtures discussed are Stoddard solvent, naphtha, and kerosene. The remaining solvents described are n-hexane, methyl n-butyl ketone, dimethylformamide, dimethyl sulfoxide, and butyl mercaptans. Effects common to this group of agents and their unique effects are characterized. Treatment of exposures and toxic effects of these solvents is described, and physiochemical properties and occupational exposure levels are listed.

  20. Apparatus and methods for hydrocarbon extraction

    Science.gov (United States)

    Bohnert, George W.; Verhulst, Galen G.

    2016-04-26

    Systems and methods for hydrocarbon extraction from hydrocarbon-containing material. Such systems and methods relate to extracting hydrocarbon from hydrocarbon-containing material employing a non-aqueous extractant. Additionally, such systems and methods relate to recovering and reusing non-aqueous extractant employed for extracting hydrocarbon from hydrocarbon-containing material.

  1. Optrode for sensing hydrocarbons

    Science.gov (United States)

    Miller, Holly; Milanovich, Fred P.; Hirschfeld, Tomas B.; Miller, Fred S.

    1987-01-01

    A two-phase system employing the Fujiwara reaction is provided for the fluorometric detection of halogenated hydrocarbons. A fiber optic is utilized to illuminate a column of pyridine trapped in a capillary tube coaxially attached at one end to the illuminating end of the fiber optic. A strongly alkaline condition necessary for the reaction is maintained by providing a reservoir of alkali in contact with the column of pyridine, the surface of contact being adjacent to the illuminating end of the fiber optic. A semipermeable membrane caps the other end of the capillary tube, the membrane being preferentially permeable to the halogenated hydrocarbon and but preferentially impermeable to water and pyridine. As the halogenated hydrocarbon diffuses through the membrane and into the column of pyridine, fluorescent reaction products are formed. Light propagated by the fiber optic from a light source, excites the fluorescent products. Light from the fluorescence emission is also collected by the same fiber optic and transmitted to a detector. The intensity of the fluorescence gives a measure of the concentration of the halogenated hydrocarbons.

  2. Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Woo, S.L.C.; Dilella, A.G.

    1990-10-23

    This patent describes a method of detecting a mutation in a phenylalanine hydroxylase gene of human genomic DNA. Also described is an automated method of detecting PKU affected, PKU helerozgotes and normals in fetal to adult human samples.

  3. Mantle hydrocarbons: abiotic or biotic?

    Science.gov (United States)

    Sugisaki, R; Mimura, K

    1994-06-01

    Analyses of 227 rocks from fifty localities throughout the world showed that mantle derived rocks such as tectonized peridotites in ophiolite sequences (tectonites) arid peridotite xenoliths in alkali basalts contain heavier hydrocarbons (n-alkanes), whereas igneous rocks produced by magmas such as gabbro arid granite lack them. The occurrence of hydrocarbons indicates that they were not derived either from laboratory contamination or from held contamination; these compounds found in the mantle-derived rocks are called here "mantle hydrocarbons." The existence of hydrocarbons correlates with petrogenesis. For example, peridotite cumulates produced by magmatic differentiation lack hydrocarbons whereas peridotite xenoliths derived from the mantle contain them. Gas chromatographic-mass spectrometric records of the mantle hydrocarbons resemble those of aliphatics in meteorites and in petroleum. Features of the hydrocarbons are that (a) the mantle hydrocarbons reside mainly along grain boundaries and in fluid inclusions of minerals; (b) heavier isoprenoids such as pristane and phytane are present; and (c) delta 13C of the mantle hydrocarbons is uniform (about -27%). Possible origins for the mantle hydrocarbons are as follows. (1) They were in organically synthesized by Fischer-Tropsch type reaction in the mantle. (2) They were delivered by meteorites and comets to the early Earth. (3) They were recycled by subduction. The mantle hydrocarbons in the cases of (1) and (2) are abiogenic and those in (3) are mainly biogenic. It appears that hydrocarbons may survive high pressures and temperatures in the mantle, but they are decomposed into lighter hydrocarbon gases such as CH4 at lower pressures when magmas intrude into the crust; consequently, peridotite cumulates do not contain heavier hydrocarbons but possess hydrocarbon gases up to C4H10.

  4. Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases

    OpenAIRE

    Yurui eJi; Guannan eMao; Yingying eWang; Mark eBartlam

    2013-01-01

    Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini) depending on the type of enzymes. In this review, we summarize th...

  5. Direct Evidence for a Phenylalanine Site in the Regulatory Domain of Phenylalanine Hydroxylase

    OpenAIRE

    Li, Jun; Ilangovan, Udayar; Daubner, S. Colette; Hinck, Andrew P.; Fitzpatrick, Paul F.

    2010-01-01

    The hydroxylation of phenylalanine to tyrosine by the liver enzyme phenylalanine hydroxylase is regulated by the level of phenylalanine. Whether there is a distinct allosteric binding site for phenylalanine outside of the active site has been unclear. The enzyme contains an N-terminal regulatory domain that extends through Thr117. The regulatory domain of rat phenylalanine hydroxylase was expressed in E. coli. The purified protein behaves as a dimer on a gel filtration column. In the presence...

  6. Bacterial sources for phenylalkane hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Ellis, L.; Winans, R.E. [Argonne National Lab., IL (United States); Langworthy, T. [Univ. of South Dakota, Vermillion, SD (United States)

    1996-10-01

    The presence of phenylalkane hydrocarbons in geochemical samples has been the source of much controversy. Although an anthropogenic input from detergent sources always appears likely, the distribution of phenylalkane hydrocarbons in some cases far exceeding that attributed to detergent input has led to a reappraisal of this view. Indeed, recent work involving analysis of the lipid hydrocarbon extracts from extant Thermoplasma bacteria has revealed the presence of phenylalkane hydrocarbons. The presence of phenylalkane hydrocarbons in sedimentary organic matter may therefore represent potential biological markers for thermophilic bacteria.

  7. Microbial degradation of petroleum hydrocarbons.

    Science.gov (United States)

    Varjani, Sunita J

    2017-01-01

    Petroleum hydrocarbon pollutants are recalcitrant compounds and are classified as priority pollutants. Cleaning up of these pollutants from environment is a real world problem. Bioremediation has become a major method employed in restoration of petroleum hydrocarbon polluted environments that makes use of natural microbial biodegradation activity. Petroleum hydrocarbons utilizing microorganisms are ubiquitously distributed in environment. They naturally biodegrade pollutants and thereby remove them from the environment. Removal of petroleum hydrocarbon pollutants from environment by applying oleophilic microorganisms (individual isolate/consortium of microorganisms) is ecofriendly and economic. Microbial biodegradation of petroleum hydrocarbon pollutants employs the enzyme catalytic activities of microorganisms to enhance the rate of pollutants degradation. This article provides an overview about bioremediation for petroleum hydrocarbon pollutants. It also includes explanation about hydrocarbon metabolism in microorganisms with a special focus on new insights obtained during past couple of years. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Epigenetic Control of Prolyl and Asparaginyl Hydroxylases in Prostate Cancer

    Science.gov (United States)

    2011-07-01

    that PHD3 may have a direct impact on cellular metabolism, as opposed to an indirect effect through the hypoxia inducible factors (HIFs). To query if...hydrocarbon nuclear translocator receptor (ARNT/HIF-b)HIF-1a activates the transcription of EPO, VEGF, heme oxygenase-1 and several other critical...containing proteins (PHD/EGLN/HPH) which utilize iron , oxygen and 2-oxoglutarate as co-factors to enzymatically catalyze hydroxylation on the oxygen-dependent

  9. Superconductivity in aromatic hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Kubozono, Yoshihiro, E-mail: kubozono@cc.okayama-u.ac.jp [Research Laboratory for Surface Science, Okayama University, Okayama 700-8530 (Japan); Research Center of New Functional Materials for Energy Production, Storage and Transport, Okayama University, Okayama 700-8530 (Japan); Japan Science and Technology Agency, ACT-C, Kawaguchi 332-0012 (Japan); Goto, Hidenori; Jabuchi, Taihei [Research Laboratory for Surface Science, Okayama University, Okayama 700-8530 (Japan); Yokoya, Takayoshi [Research Laboratory for Surface Science, Okayama University, Okayama 700-8530 (Japan); Research Center of New Functional Materials for Energy Production, Storage and Transport, Okayama University, Okayama 700-8530 (Japan); Kambe, Takashi [Department of Physics, Okayama University, Okayama 700-8530 (Japan); Sakai, Yusuke; Izumi, Masanari; Zheng, Lu; Hamao, Shino; Nguyen, Huyen L.T. [Research Laboratory for Surface Science, Okayama University, Okayama 700-8530 (Japan); Sakata, Masafumi; Kagayama, Tomoko; Shimizu, Katsuya [Center of Science and Technology under Extreme Conditions, Osaka University, Osaka 560-8531 (Japan)

    2015-07-15

    Highlights: • Aromatic superconductor is one of core research subjects in superconductivity. Superconductivity is observed in certain metal-doped aromatic hydrocarbons. Some serious problems to be solved exist for future advancement of the research. This article shows the present status of aromatic superconductors. - Abstract: ‘Aromatic hydrocarbon’ implies an organic molecule that satisfies the (4n + 2) π-electron rule and consists of benzene rings. Doping solid aromatic hydrocarbons with metals provides the superconductivity. The first discovery of such superconductivity was made for K-doped picene (K{sub x}picene, five benzene rings). Its superconducting transition temperatures (T{sub c}’s) were 7 and 18 K. Recently, we found a new superconducting K{sub x}picene phase with a T{sub c} as high as 14 K, so we now know that K{sub x}picene possesses multiple superconducting phases. Besides K{sub x}picene, we discovered new superconductors such as Rb{sub x}picene and Ca{sub x}picene. A most serious problem is that the shielding fraction is ⩽15% for K{sub x}picene and Rb{sub x}picene, and it is often ∼1% for other superconductors. Such low shielding fractions have made it difficult to determine the crystal structures of superconducting phases. Nevertheless, many research groups have expended a great deal of effort to make high quality hydrocarbon superconductors in the five years since the discovery of hydrocarbon superconductivity. At the present stage, superconductivity is observed in certain metal-doped aromatic hydrocarbons (picene, phenanthrene and dibenzopentacene), but the shielding fraction remains stubbornly low. The highest priority research area is to prepare aromatic superconductors with a high superconducting volume-fraction. Despite these difficulties, aromatic superconductivity is still a core research target and presents interesting and potentially breakthrough challenges, such as the positive pressure dependence of T{sub c} that is clearly

  10. Catalytic domain surface residues mediating catecholamine inhibition in tyrosine hydroxylase.

    Science.gov (United States)

    Briggs, Gabrielle D; Bulley, Jesse; Dickson, Phillip W

    2014-03-01

    Tyrosine hydroxylase (TH) performs the rate-limiting step in catecholamine (CA) synthesis and is a tetramer composed of regulatory, catalytic and tetramerization domains. CAs inhibit TH by binding two sites in the active site; one with high affinity and one with low affinity. Only high affinity CA binding requires the regulatory domain, believed to interact with the catalytic domain in the presence of CA. Without a crystal structure of the regulatory domain, the specific areas involved in this process are largely undefined. It is not clear whether the regulatory domain-catalytic domain interaction is asymmetrical across the tetramer to produce the high and low affinity sites. To investigate this, pure dimeric TH was generated through double substitution of residues at the tetramerization interface and dimerization salt bridge (K170E/L480A). This was shown to be the core regulatory unit of TH for CA inhibition, possessing both high and low affinity CA binding sites, indicating that there is symmetry between dimers of the tetramer. We also examined possible regulatory domain-interacting regions on the catalytic domain that mediate high affinity CA binding. Using site-directed mutagenesis, A297, E362/E365 and S368 were shown to mediate high affinity dopamine inhibition through V(max) reduction and increasing the K(M) for the cofactor.

  11. High carrier frequency of 21-hydroxylase deficiency in Cyprus.

    Science.gov (United States)

    Phedonos, A A P; Shammas, C; Skordis, N; Kyriakides, T C; Neocleous, V; Phylactou, L A

    2013-12-01

    Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is a common autosomal recessive disorder caused by mutations in the CYP21A2 gene. The carrier frequency of CYP21A2 mutations has been estimated to be 1:25 to 1:10 on the basis of newborn screening. The main objective of this study was to determine the carrier frequency in the Cypriot population of mutations in the CYP21A2 gene. Three hundred unrelated subjects (150 males and 150 females) from the general population of Cyprus were screened for mutations in the CYP21A2 gene and its promoter. The CYP21A2 genotype analysis identified six different mutants and revealed a carrier frequency of 9.83% with the mild p.Val281Leu being the most frequent (4.3%), followed by p.Qln318stop (2.5%), p.Pro453Ser (1.33%), p.Val304Met (0.83%), p.Pro482Ser (0.67%) and p.Met283Val (0.17%). The notable high CYP21A2 carrier frequency of the Cypriot population is one of the highest reported so far by genotype analysis. Knowledge of the mutational spectrum of CYP21A2 will enable to optimize mutation detection strategy for genetic diagnosis of 21-OHD not only in Cyprus, but also the greater Mediterranean region.

  12. Prolyl hydroxylase 3 inhibited the tumorigenecity of gastric cancer cells.

    Science.gov (United States)

    Cui, Lei; Qu, Jianguo; Dang, Shengchun; Mao, Zhengfa; Wang, Xuqing; Fan, Xin; Sun, Kang; Zhang, Jianxin

    2014-09-01

    Gastric cancer is one of the most common malignancies and the second leading cause of cancer-related death in the world, and it is very urgent to develop novel therapeutic strategies. Although HIF-1α is the most highly characterized target of prolyl hydroxylase 3 (PHD3), PHD3 has been shown to regulate several signal pathways independent of HIF-1α. Here, we found that the expression of PHD3 was decreased in the clinical gastric cancer samples and reversely correlated with tumor size and tumor stage. Over-expression of PHD3 in the gastric cancer cells significantly inhibited cell growth in vitro and in vivo, while knockdown the expression of PHD3 promoted the tumorigenecity of gastric cancer cells. Mechanistically, it showed that PHD3 downregulated the expression of beta-catenin and inhibited beta-catenin/T-cell factor (TCF) signaling. Taken together, our findings demonstrate that PHD3 inhibits gastric cancer by suppressing the beta-catenin/TCF signaling and PHD3 might be an important therapeutic target in gastric cancer.

  13. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    Science.gov (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  14. Dopamine-beta hydroxylase polymorphism and cocaine addiction

    Directory of Open Access Journals (Sweden)

    Collier David

    2008-01-01

    Full Text Available Abstract Cocaine addiction involves a number of medical, psychological and social problems. Understanding the genetic aetiology of this disorder will be essential for design of effective treatments. Dopamine-beta hydroxylase (DbH catalyzes the conversion of dopamine to norepinephrine and could, therefore, have an influence on both cocaine action and the basal sensitivity of neurotransmitter systems to cocaine. Recently, the -1021C>T polymorphism have been found to strongly correlated with individual variation in plasma DbH activity. To test the influence of this polymorphism on the susceptibility of cocaine addiction, we decided to genotype it in a sample of 689 cocaine addicts and 832 healthy individuals. Genotypic and allelic analyses did not show any evidence of association with cocaine addiction, even after correcting for the effect of population stratification and other possible confounders. Our results do not support a major role of the -1021C>T polymorphism or the gene itself in the development of cocaine addiction but further examination of other variants within this gene will be necessary to completely rule out an effect.

  15. An additional substrate binding site in a bacterial phenylalanine hydroxylase.

    Science.gov (United States)

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Corn, Isaac R; Wagner, Kyle T; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2013-09-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.

  16. Characterization of mutations at the mouse phenylalanine hydroxylase locus

    Energy Technology Data Exchange (ETDEWEB)

    McDonald, J.D.; Charlton, C.K. [Wichita State Univ., KS (United States)

    1997-02-01

    Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. In PAH{sup ENU1}, the phenotype is mild. The Pah{sup enu1} mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. In PAH{sup ENU2} the phenotype is severe. The Pah{sup enu2} mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. In PAH{sup ENU2}, the sequence information was used to devise a direct genotyping system based on the creation of a new Alw26I restriction endonuclease site. 26 refs., 2 figs., 1 tab.

  17. The interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) and 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) in TCDD-induced porphyria

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Cheng Catsby.

    1989-01-01

    Halogenated aryl hydrocarbon(HAH)-induced porphyria is caused by alteration of porphyrin metabolism and results in the accumulation of hepatic and urinary porphyrins. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (75 {mu}/kg) caused significant increases of hepatic porphyrin levels in C57BL/6 male, female and ovariectomized female, and C57BL/10 male mice 3 weeks after treatment. In contrast, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) was inactive at a dose of 750 {mu}mol/kg. Cotreatment with MCDF (750 {mu}mol/kg) and 2,3,7,8-TCDD (75 {mu}g/kg) resulted in partial antagonism of 2,3,7,8-TCDD-induced porphyrin accumulation in female but not in male mice. In female C57BL/6 mice, 2,3,7,8-TCDD-induced porphyria was accompanied by the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities and the inhibition of uroporphyrinogen decarboxylase (UROD) activity. MCDF (750 {mu}mol/kg) did not significantly affect these enzyme activities. In coadministration studies, MCDF partially antagonized 2,3,7,8-TCDD-induced hepatic porphyrin accumulation but did not affect the activities of hepatic AHH, EROD or UROD. These results demonstrate that the induction of the monooxygenase enzyme activities and the inhibition of UROD activity by 2,3,7,8-TCDD and the development of porphyria are not coordinately regulated in C57BL/6 female mice. In cultured chick embryo hepatocytes, 2,3,7,8-TCDD caused a significant increase in porphyrin levels and induced AHH and EROD activities. MCDF and Aroclor 1254 partially antagonized the 2,3,7,8-TCDD induced AHH and EROD activities but not the porphyrin accumulation.

  18. Membrane separation of hydrocarbons

    Science.gov (United States)

    Chang, Y. Alice; Kulkarni, Sudhir S.; Funk, Edward W.

    1986-01-01

    Mixtures of heavy oils and light hydrocarbons may be separated by passing the mixture through a polymeric membrane. The membrane which is utilized to effect the separation comprises a polymer which is capable of maintaining its integrity in the presence of hydrocarbon compounds and which has been modified by being subjected to the action of a sulfonating agent. Sulfonating agents which may be employed will include fuming sulfuric acid, chlorosulfonic acid, sulfur trioxide, etc., the surface or bulk modified polymer will contain a degree of sulfonation ranging from about 15 to about 50%. The separation process is effected at temperatures ranging from about ambient to about 100.degree. C. and pressures ranging from about 50 to about 1000 psig.

  19. Direct hydrocarbon fuel cells

    Science.gov (United States)

    Barnett, Scott A.; Lai, Tammy; Liu, Jiang

    2010-05-04

    The direct electrochemical oxidation of hydrocarbons in solid oxide fuel cells, to generate greater power densities at lower temperatures without carbon deposition. The performance obtained is comparable to that of fuel cells used for hydrogen, and is achieved by using novel anode composites at low operating temperatures. Such solid oxide fuel cells, regardless of fuel source or operation, can be configured advantageously using the structural geometries of this invention.

  20. THERMOCHEMISTRY OF HYDROCARBON RADICALS

    Energy Technology Data Exchange (ETDEWEB)

    Kent M. Ervin, Principal Investigator

    2004-08-17

    Gas phase negative ion chemistry methods are employed to determine enthalpies of formation of hydrocarbon radicals that are important in combustion processes and to investigate the dynamics of ion-molecule reactions. Using guided ion beam tandem mass spectrometry, we measure collisional threshold energies of endoergic proton transfer and hydrogen atom transfer reactions of hydrocarbon molecules with negative reagent ions. The measured reaction threshold energies for proton transfer yield the relative gas phase acidities. In an alternative methodology, competitive collision-induced dissociation of proton-bound ion-molecule complexes provides accurate gas phase acidities relative to a reference acid. Combined with the electron affinity of the R {center_dot} radical, the gas phase acidity yields the RH bond dissociation energy of the corresponding neutral molecule, or equivalently the enthalpy of formation of the R{center_dot} organic radical, using equation: D(R-H) = {Delta}{sub acid}H(RH) + EA(R) - IE(H). The threshold energy for hydrogen abstraction from a hydrocarbon molecule yields its hydrogen atom affinity relative to the reagent anion, providing the RH bond dissociation energy directly. Electronic structure calculations are used to evaluate the possibility of potential energy barriers or dynamical constrictions along the reaction path, and as input for RRKM and phase space theory calculations. In newer experiments, we have measured the product velocity distributions to obtain additional information on the energetics and dynamics of the reactions.

  1. Garlic attenuates chrysotile-mediated pulmonary toxicity in rats by altering the phase I and phase II drug metabolizing enzyme system.

    Science.gov (United States)

    Ameen, Mohamed; Musthapa, M Syed; Abidi, Parveen; Ahmad, Iqbal; Rahman, Qamar

    2003-01-01

    Asbestos and its carcinogenic properties have been extensively documented. Asbestos exposure induces diverse cellular events associated with lung injury. Previously, we have shown that treatment with chrysotile shows significant alteration in phase I and phase II drug metabolizing enzyme system. In this study we have examined some potential mechanisms by which garlic treatment attenuates chrysotile-mediated pulmonary toxicity in rat. Female Wistar rats received an intratracheal instillation of 5 mg chrysotile (0.5 mL saline) as well as intragastric garlic treatment (1% body weight (v/w); 6 days per week). Effect of garlic treatment was evaluated after 1, 15, 30, 90, and 180 days by assaying aryl hydrocarbon hydroxylase (AHH), glutathione (GSH), glutathione S-transferase (GST), and production of thiobarbituric acid reactive substances (TBARS) in rat lung microsome. The results showed that AHH and TBARS formation were significantly reduced at day 90 and day 180 in chrysotile treated garlic cofed rats; GSH recovered 15 days later to the near normal level and GST elevated significantly after treatment of garlic as compared to chrysotile alone treated rat lung microsome. The data obtained shows that inhibition of AHH activity and induction of GST activity could be contributing factor in chrysotile-mediated pulmonary toxicity in garlic cofed rats. However, recovery of GSH and inhibition of TBARS formation by garlic and its constituent(s) showed that garlic may give protection by altering the drug metabolizing enzyme system.

  2. Functional implication of β-carotene hydroxylases in soybean nodulation.

    Science.gov (United States)

    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

    2013-07-01

    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.

  3. Structural features of the regulatory ACT domain of phenylalanine hydroxylase.

    Directory of Open Access Journals (Sweden)

    Carla Carluccio

    Full Text Available Phenylalanine hydroxylase (PAH catalyzes the conversion of L-Phe to L-Tyr. Defects in PAH activity, caused by mutations in the human gene, result in the autosomal recessively inherited disease hyperphenylalaninemia. PAH activity is regulated by multiple factors, including phosphorylation and ligand binding. In particular, PAH displays positive cooperativity for L-Phe, which is proposed to bind the enzyme on an allosteric site in the N-terminal regulatory domain (RD, also classified as an ACT domain. This domain is found in several proteins and is able to bind amino acids. We used molecular dynamics simulations to obtain dynamical and structural insights into the isolated RD of PAH. Here we show that the principal motions involve conformational changes leading from an initial open to a final closed domain structure. The global intrinsic motions of the RD are correlated with exposure to solvent of a hydrophobic surface, which corresponds to the ligand binding-site of the ACT domain. Our results strongly suggest a relationship between the Phe-binding function and the overall dynamic behaviour of the enzyme. This relationship may be affected by structure-disturbing mutations. To elucidate the functional implications of the mutations, we investigated the structural effects on the dynamics of the human RD PAH induced by six missense hyperphenylalaninemia-causing mutations, namely p.G46S, p.F39C, p.F39L, p.I65S, p.I65T and p.I65V. These studies showed that the alterations in RD hydrophobic interactions induced by missense mutations could affect the functionality of the whole enzyme.

  4. Structural features of the regulatory ACT domain of phenylalanine hydroxylase.

    Science.gov (United States)

    Carluccio, Carla; Fraternali, Franca; Salvatore, Francesco; Fornili, Arianna; Zagari, Adriana

    2013-01-01

    Phenylalanine hydroxylase (PAH) catalyzes the conversion of L-Phe to L-Tyr. Defects in PAH activity, caused by mutations in the human gene, result in the autosomal recessively inherited disease hyperphenylalaninemia. PAH activity is regulated by multiple factors, including phosphorylation and ligand binding. In particular, PAH displays positive cooperativity for L-Phe, which is proposed to bind the enzyme on an allosteric site in the N-terminal regulatory domain (RD), also classified as an ACT domain. This domain is found in several proteins and is able to bind amino acids. We used molecular dynamics simulations to obtain dynamical and structural insights into the isolated RD of PAH. Here we show that the principal motions involve conformational changes leading from an initial open to a final closed domain structure. The global intrinsic motions of the RD are correlated with exposure to solvent of a hydrophobic surface, which corresponds to the ligand binding-site of the ACT domain. Our results strongly suggest a relationship between the Phe-binding function and the overall dynamic behaviour of the enzyme. This relationship may be affected by structure-disturbing mutations. To elucidate the functional implications of the mutations, we investigated the structural effects on the dynamics of the human RD PAH induced by six missense hyperphenylalaninemia-causing mutations, namely p.G46S, p.F39C, p.F39L, p.I65S, p.I65T and p.I65V. These studies showed that the alterations in RD hydrophobic interactions induced by missense mutations could affect the functionality of the whole enzyme.

  5. Computational study of missense mutations in phenylalanine hydroxylase.

    Science.gov (United States)

    Réblová, Kamila; Kulhánek, Petr; Fajkusová, Lenka

    2015-04-01

    Hyperphenylalaninemia (HPA) is one of the most common metabolic disorders. HPA, which is transmitted by an autosomal recessive mode of inheritance, is caused by mutations of the phenylalanine hydroxylase gene. Most mutations are missense and lead to reduced protein stability and/or impaired catalytic function. The impact of such mutations varies, ranging from classical phenylketonuria (PKU), mild PKU, to non-PKU HPA phenotypes. Despite the fact that HPA is a monogenic disease, clinical data show that one PKU genotype can be associated with more in vivo phenotypes, which indicates the role of other (still unknown) factors. To better understand the phenotype-genotype relationships, we analyzed computationally the impact of missense mutations in homozygotes stored in the BIOPKU database. A total of 34 selected homozygous genotypes was divided into two main groups according to their phenotypes: (A) genotypes leading to non-PKU HPA or combined phenotype non-PKU HPA/mild PKU and (B) genotypes leading to classical PKU, mild PKU or combined phenotype mild PKU/classical PKU. Combining in silico analysis and molecular dynamics simulations (in total 3 μs) we described the structural impact of the mutations, which allowed us to separate 32 out of 34 mutations between groups A and B. Testing the simulation conditions revealed that the outcome of mutant simulations can be modulated by the ionic strength. We also employed programs SNPs3D, Polyphen-2, and SIFT but based on the predictions performed we were not able to discriminate mutations with mild and severe PKU phenotypes.

  6. Human DJ-1-specific Transcriptional Activation of Tyrosine Hydroxylase Gene*

    Science.gov (United States)

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.

    2010-01-01

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice. PMID:20938049

  7. Functional characterization of salicylate hydroxylase from the fungal endophyte Epichloë festucae.

    Science.gov (United States)

    Ambrose, Karen V; Tian, Zipeng; Wang, Yifei; Smith, Jordan; Zylstra, Gerben; Huang, Bingru; Belanger, Faith C

    2015-06-09

    Epichloë spp. are symbiotic fungal endophytes of many cool season grasses. The presence of the fungal endophytes often confers insect, drought, and disease tolerance to the host grasses. The presence of the fungal endophytes within the host plants does not elicit host defense responses. The molecular basis for this phenomenon is not known. Epichloë festucae, the endophyte of Festuca rubra, expresses a salicylate hydroxylase similar to NahG from the bacterium Pseudomonas putida. Few fungal salicylate hydroxylase enzymes have been reported. The in planta expression of an endophyte salicylate hydroxylase raised the possibility that degradation of plant-produced salicylic acid is a factor in the mechanism of how the endophyte avoids eliciting host plant defenses. Here we report the characterization of the E. festucae salicylate hydroxylase, designated Efe-shyA. Although the fungal enzyme has the expected activity, based on salicylic acid levels in endophyte-free and endophyte-infected plants it is unlikely that expression of the endophyte salicylate hydroxylase is a factor in the lack of a host defense response to the presence of the fungal endophyte.

  8. Development of vitamin D/sub 3/ 25-hydroxylase activity in rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Thierry-Palmer, M.; Cullins, S.; Rashada, S.; Gray, T.K.; Free, A.

    1986-05-01

    The authors have determined the ontogeny of vitamin D/sub 3/ 25-hydroxylase activity in rat liver microsomes. Microsomes from fetuses, neonates, and their mothers were incubated with 44 nM /sup 3/H-vitamin D/sub 3/ in the presence of an NADPH generating system, oxygen, KCl, and MgCl/sub 2/. Lipid extracts of the incubation samples were partially purified by thin-layer chromatography. Tritiated 25-hydroxy vitamin D/sub 3/ (250HD/sub 3/) was analyzed by high-pressure liquid chromatography using 94/6 hexane/isopropanol. Production rate for 250HD/sub 3/ in the mothers ranged from 0.22 to 0.30 pmol/mg protein/hr. Activities in the fetuses and neonates were 2.1, 12.9, 32.0, 35.8, and 71.0% of that of their mothers at -3, 0, 2, 7, and 15 days of age. The cytosolic fraction protected the substrate from degradation, stimulated the vitamin D/sub 3/ 25-hydroxylase reaction in neonates and mothers (1.4 to 1.7 fold increase), and was absolutely required for 25-hydroxylase activity in fetuses. These data suggest that microsomal vitamin D/sub 3/ 25-hydroxylase activity develops slowly and approaches full activity near the weaning stage. A cytosolic factor present as early as -3 days of age stimulates the activity of the microsomal vitamin D/sub 3/ 25-hydroxylase.

  9. Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

    Institute of Scientific and Technical Information of China (English)

    Jian-qing Liang; Li Wang; Jian-cheng He; Xian-dong Hua

    2016-01-01

    Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson’s disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson’s disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson’s disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson’s disease were established and verbascoside (60 mg/kg) was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase mRNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were signiifcantly higher than the Parkinson’s model group. These ifndings suggest that the mechanism by which verbascoside treats Parkinson’s disease is related to the regeneration of tyrosine hy-droxylase-immunoreactive neurons in the substantia nigra.

  10. Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11β-hydroxylase deficiency.

    Science.gov (United States)

    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.

  11. Single turnover kinetics of tryptophan hydroxylase: evidence for a new intermediate in the reaction of the aromatic amino acid hydroxylases.

    Science.gov (United States)

    Pavon, Jorge Alex; Eser, Bekir; Huynh, Michaela T; Fitzpatrick, Paul F

    2010-09-07

    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).6MePH(4).tryptophan, and TrpH.Fe(II).6MePH(4).phenylalanine complexes with O(2) 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(-1) s(-1) irrespective of the presence of tryptophan. Stopped-flow absorbance analyses of the reaction of the TrpH.Fe(II).6MePH(4).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.

  12. Hydrocarbon conversion catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Hoek, A.; Huizinga, T.; Maxwell, I.E.

    1989-08-15

    This patent describes a process for hydrocracking hydrocarbon oils into products of lower average molecular weight and lower average boiling point. It comprises contacting a hydrocarbon oil at a temperature between 250{sup 0}C and 500{sup 0}C and a pressure up to 300 bar in the presence of hydrogen with a catalyst consisting essentially of a Y zeolite modified to have a unit cell size below 24.35A, a water absorption capacity (at 25{sup 0}C and a rho/rho/sub o/ value of 0.2) of at least 8% by weight of the zeolite and a pore volume of at least 0.25 ml/g wherein between 10% and 60% of the total pore volume is made up of pores having a diameter of at least 8 nm; an alumina binder and at least one hydrogenation component selected from the group consisting of a Group VI metal, a Group VIII metal and mixtures thereof.

  13. Influence of various phenolic compounds on phenol hydroxylase activity of a Trichosporon cutaneum strain.

    Science.gov (United States)

    Gerginova, Maria; Manasiev, Jordan; Shivarova, Nedka; Alexieva, Zlatka

    2007-01-01

    The phenol-degrading strain Trichosporon cutaneum R57 utilizes various aromatic and aliphatic compounds as a sole carbon and energy source. The intracellular activities of phenol hydroxylase [EC 1.14.13.7] of a Trichosporon cutaneum R57 strain grown on phenol (0.5 g/l) were measured. Different toxic phenol derivatives (cresols, nitrophenols and hydroxyphenols) were used as substrates in the reaction mixture for determination of the enzyme activity. The data obtained showed that the investigated enzyme was capable to hydroxylate all applied aromatic substrates. The measured activities of phenol hydroxylase varied significantly depending on the aromatic compounds used as substrates. The rate of phenol hydroxylase activity with phenol as a substrate (1.0 U/mg total cell protein) was accepted as 100%.

  14. Tyrosine hydroxylase expression in the midbrain of Parkinson's disease model rats treated with Xifeng Dingchan decoction

    Institute of Scientific and Technical Information of China (English)

    Enli Luo

    2011-01-01

    This study showed that abnormal behavioral changes were greatly improved in rats displaying Parkinson's disease-like symptoms after intragastric administration of Xifeng Dingchan decoction at 15, 7.5, 3.75 g/kg per day. In addition, tyrosine hydroxylase mRNA expression in the substantia nigra of the midbrain was up-regulated, and tyrosine hydroxylase content in the midbrain ventral tegmentum and substantia nigra pars compacta was also increased. The effect of administration of Xifeng Dingchan decoction at 7.5 g/kg per day was similar to that of Madopar at 67.5 mg/kg per day. These results indicate that the therapeutic effect of Xifeng Dingchan decoction on Parkinson's disease is associated with the up-regulated protein and mRNA expression of tyrosine hydroxylase in the midbrain.

  15. Thermophysical Properties of Hydrocarbon Mixtures

    Science.gov (United States)

    SRD 4 NIST Thermophysical Properties of Hydrocarbon Mixtures (PC database for purchase)   Interactive computer program for predicting thermodynamic and transport properties of pure fluids and fluid mixtures containing up to 20 components. The components are selected from a database of 196 components, mostly hydrocarbons.

  16. Hydrocarbon Receptor Pathway in Dogs

    NARCIS (Netherlands)

    Steenbeek, F.G. van; Spee, B.; Penning, L.C.; Kummeling, A.; Gils, I.H.M.; Grinwis, G.C.M.; Leenen, D. van; Holstege, F.C.P.; Vos-Loohuis, M.; Rothuizen, J.; Leegwater, P.A.J.

    2013-01-01

    The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein

  17. Hydrocarbon Receptor Pathway in Dogs

    NARCIS (Netherlands)

    Steenbeek, F.G. van; Spee, B.; Penning, L.C.; Kummeling, A.; Gils, I.H.M.; Grinwis, G.C.M.; Leenen, D. van; Holstege, F.C.P.; Vos-Loohuis, M.; Rothuizen, J.; Leegwater, P.A.J.

    The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting

  18. Chemical profiling of deoxyhypusine hydroxylase inhibitors for antimalarial therapy.

    Science.gov (United States)

    von Koschitzky, Imke; Kaiser, Annette

    2013-11-01

    A first approach to discover new antimalarials has been recently performed in a combined approach with data from GlaxoSmithKline Tres Cantos Antimalarial Set, Novartis-GNF Malaria Box Data set and St. Jude Children's Research Hospital. These data are assembled in the Malaria Box. In a first phenotypic forward chemical genetic approach, 400 chemicals were employed to eradicate the parasite in the erythrocytic stages. The advantage of phenotypic screens for the identification of novel chemotypes is that no a priori assumptions are made concerning a fixed target and that active compounds inherently have cellular bioavailability. In a first screen 40 mostly heterocyclic, highly active compounds (in nmol range of growth inhibition) were identified with EC50 values ≤2 μM against chloroquine-resistant Plasmodium falciparum strains and a therapeutic window ≥10 against two mammalian cell lines. 78 % of the compounds had no violations with the Lipinski Rule of 5 and only 1 % of the compounds showed cytotoxicity when applied at concentrations of 10 μM. This pre-selective step of parasitic eradication will be used further for a test of the Malaria Box with a potential in iron chelating capacity to inhibit deoxyhypusine hydroxylase (DOHH) from P. falciparum and vivax. DOHH, a metalloprotein which consists of ferrous iron and catalyzes the second step of the posttranslational modification at a specific lysine in eukaryotic initiation factor 5A (EIF-5A) to hypusine. Hypusine is a novel, non-proteinogenic amino acid, which is essential in eukaryotes and for parasitic proliferation. DOHH seems to be a "druggable" target, since it has only 26 % amino acid identity to its human orthologue. For a High-throughput Screening (HTS) of DOOH inhibitors, rapid and robust analytical tools are a prerequisite. A proteomic platform for the detection of hypusine metabolites is currently established. Ultra performance Liquid Chromatography enables the detection of hypusine metabolites with

  19. High frequency of cytolytic 21-Hydroxylase specific CD8+ T cells in autoimmune Addison’s disease patients1

    Science.gov (United States)

    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

  20. Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Wit, E.C.M. de; Princen, H.M.G.

    1995-01-01

    In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase' ro

  1. Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

    Science.gov (United States)

    Lee, Chang Woo; Yu, Sang-Cheol; Lee, Joo-Ho; Park, Sun-Ha; Park, Hyun; Oh, Tae-Jin; Lee, Jun Hyuck

    2016-01-01

    Enzymatic alkane hydroxylation reactions are useful for producing pharmaceutical and agricultural chemical intermediates from hydrocarbons. Several cytochrome P450 enzymes catalyze the regio- and stereo-specific hydroxylation of alkanes. We evaluated the substrate binding of a putative CYP alkane hydroxylase (CYP153D17) from the bacterium Sphingomonas sp. PAMC 26605. Substrate affinities to C10–C12 n-alkanes and C10–C14 fatty acids with Kd values varied from 0.42 to 0.59 μM. A longer alkane (C12) bound more strongly than a shorter alkane (C10), while shorter fatty acids (C10, capric acid; C12, lauric acid) bound more strongly than a longer fatty acid (C14, myristic acid). These data displayed a broad substrate specificity of CYP153D17, hence it was named as a putative CYP alkane hydroxylase. Moreover, the crystal structure of CYP153D17 was determined at 3.1 Å resolution. This is the first study to provide structural information for the CYP153D family. Structural analysis showed that a co-purified alkane-like compound bound near the active-site heme group. The alkane-like substrate is in the hydrophobic pocket containing Thr74, Met90, Ala175, Ile240, Leu241, Val244, Leu292, Met295, and Phe393. Comparison with other CYP structures suggested that conformational changes in the β1–β2, α3–α4, and α6–α7 connecting loop are important for incorporating the long hydrophobic alkane-like substrate. These results improve the understanding of the catalytic mechanism of CYP153D17 and provide valuable information for future protein engineering studies. PMID:27941697

  2. A steady-state kinetic analysis of the prolyl-4-hydroxylase mechanism.

    Science.gov (United States)

    Soskel, N T; Kuby, S A

    1981-01-01

    Published kinetic data by Kivirikko, et al. on the prolyl-4-hydroxylase reaction have been re-evaluated using the overall steady-state velocity equation in the forward and reverse directions for an ordered ter ter kinetic mechanism. Qualitatively, the published data for prolyl-4-hydroxylase appear to fit the predicted patterns for this kinetic mechanism. More kinetic data are needed to confirm these results and to quantitate the kinetic parameters but, tentatively, the order of substrate addition would appear to be alpha-ketoglutarate, oxygen, and peptide; and the order of product release would be hydroxylated peptide (or collagen), carbon dioxide, and succinate.

  3. Phenylalanine binding is linked to dimerization of the regulatory domain of phenylalanine hydroxylase.

    Science.gov (United States)

    Zhang, Shengnan; Roberts, Kenneth M; Fitzpatrick, Paul F

    2014-10-28

    Analytical ultracentrifugation has been used to analyze the oligomeric structure of the isolated regulatory domain of phenylalanine hydroxylase. The protein exhibits a monomer-dimer equilibrium with a dissociation constant of ~46 μM; this value is unaffected by the removal of the 24 N-terminal residues or by phosphorylation of Ser16. In contrast, phenylalanine binding (Kd = 8 μM) stabilizes the dimer. These results suggest that dimerization of the regulatory domain of phenylalanine hydroxylase is linked to allosteric activation of the enzyme.

  4. Wheat bran and the induction of intestinal benzo(a)pyrene hydroxylase by dietary benzo(a)pyrene.

    Science.gov (United States)

    Clinton, S K; Visek, W J

    1989-03-01

    The mucosa of the intestine responds to polycyclic aromatic hydrocarbons (PAH) with the rapid induction of benzo(a)pyrene hydroxylase (BPH). Studies were conducted to determine if dietary fiber would reduce exposure of the intestine to dietary benzo(a)pyrene (BP) as indicated by intestinal BPH activity. In all studies, female Sprague-Dawley rats were fed a fiber-free purified diet for 7 d, whereupon they were switched to experimental diets for 48 h. After 48 h their small intestinal mucosa was assayed for BPH activity. Diets for the initial study contained 0, 100, 400, 800, or 1200 mg BP/kg diet, each with and without 10% soft white wheat bran. Enzyme induction with 100 and 400 mg BP/kg diet was partially inhibited by bran, but with higher concentrations of BP there was no protective effect. The inhibition in BP-induced intestinal BPH activity was observed with 10% wheat bran but not with 3.3 or 6.6%. Subsequent studies showed no significant inhibition in BPH induction with cellulose or lignin, whereas all forms of wheat bran (hard red, soft white, or finely ground soft white) caused significant inhibition. In the final study, a diet containing charcoal-broiled beef, a known source of PAH, was compared with diets containing raw beef or soybean protein, each with and without 10% soft white wheat bran. BPH activity remained low with raw beef and soybean protein whether or not fiber was added. However, intestinal BPH activity was raised ninefold by charcoal-broiled beef. The addition of bran reduced BPH activity to 65% of that observed with the fiber-free, charcoal-broiled beef diet.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Cloning and expression of Chromobacterium violaceum phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic amino acid hydroxylases.

    Science.gov (United States)

    Onishi, A; Liotta, L J; Benkovic, S J

    1991-10-05

    The complete amino acid sequence (296 amino acids) of Chromobacterium violaceum phenylalanine hydroxylase (PAH) was determined by nucleotide analysis of a DNA clone isolated using both a synthetic oligonucleotide probe based on the NH2-terminal amino acid sequence and an antibody against this enzyme. The ApaL I fragment (approximately 1.9 kilobase pairs) containing the entire PAH gene was subcloned in pBluescript II and induced by isopropyl-beta-D-thiogalactopyranoside. In order to eliminate fusion proteins the XbaI/ClaI fragment which contained the PAH gene from the Bluescript construct was subcloned into pMAC 5-8 containing the TAC promoter. The recombinant protein reacts with antibody raised to authentic C. violaceum PAH and its NH2-terminal 20-amino acid sequence and COOH-terminal amino acid residue were identical with the wild-type protein. Key physical and chemical characteristics of the recombinant protein, i.e. its copper content and Michaelis-Menten parameters, were the same as wild-type. Comparison of amino acid sequences revealed a highly conserved region between C. violaceum PAH and three different mammalian aromatic amino acid hydroxylases. This conserved area may well be a catalytically important domain of these pterin- and metal-requiring aromatic amino acid hydroxylases. The over-expression of C. violaceum PAH in Escherichia coli will facilitate the analysis of the enzyme mechanism by various spectroscopic methods.

  6. Identification and cloning of a sequence homologue of dopamine β-hydroxylase

    NARCIS (Netherlands)

    Chambers, Kaylene J.; Tonkin, Leath A.; Chang, Edwin; Shelton, Dawne N.; Linskens, Maarten H.; Funk, Walter D.

    1998-01-01

    We have identified and cloned a cDNA encoding a new member of the monooxygenase family of enzymes. This novel enzyme, which we call MOX (monooxygenase X; unknown substrate) is a clear sequence homologue of the enzyme dopamine β-hydroxylase (DBH). MOX maintains many of the structural features of DBH,

  7. Complexity of steroid hydroxylase gent expression in the adrenal cortex A microcosm of regulated transcription.

    Science.gov (United States)

    Waterman, M R; Lund, J; Simpson, E R

    1989-01-01

    Regulation of the expression of steroid hydroxylase genes in the adrenal cortex involves mechanisms required for maintenance of optimal steroidogenesis, tissue specificity, and ontogeny of the steroidogenic pathway. Evaluation of the molecular basis of this complexity promises to unfold new aspects of regulated eukaryotic gene expression.

  8. Tyrosine hydroxylase polymorphism (C-824T) and hypertension: a population-based study

    DEFF Research Database (Denmark)

    Nielsen, Søren J; Jeppesen, Jørgen Lykke; Torp-Pedersen, Christian;

    2010-01-01

    Sympathetic nervous system (SNS) overactivity is present in a large proportion of the hypertensive population and precedes the development of established hypertension. Variations in the proximal promoter of the tyrosine hydroxylase (TH) gene have been shown to influence biochemical and physiologi...

  9. TOPOLOGY OF THE MEMBRANE-BOUND ALKANE HYDROXYLASE OF PSEUDOMONAS-OLEOVORANS

    NARCIS (Netherlands)

    van Beilen, J.B.; PENNINGA, D; Witholt, Bernard

    1992-01-01

    The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P. oleovorans. Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices. The topology of alkane hydroxyla

  10. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J

    2012-02-01

    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 +\\/- 2.6 and 38.8 +\\/- 6.7% (n=16; P<\\/=0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  11. Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

    LENUS (Irish Health Repository)

    Ward, Joseph B J

    2011-02-01

    Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)\\/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

  12. Reduction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1

    NARCIS (Netherlands)

    Sucharitakul, J.; Wongnate, T.; Montersino, S.; Berkel, van W.J.H.; Chaiyen, P.

    2012-01-01

    3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB

  13. Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland.

    Science.gov (United States)

    Barbosa, Roseli; Scialfa, Julieta Helena; Terra, Ilza Mingarini; Cipolla-Neto, José; Simonneaux, Valérie; Afeche, Solange Castro

    2008-02-27

    Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.

  14. TGF- induces Lysyl hydroxylase 2b in human synovial osteoarthritic fibroblasts through ALK5 signaling

    NARCIS (Netherlands)

    Remst, Dennis F. G.; Davidson, Esmeralda N. Blaney; Vitters, Elly L.; Bank, Ruud A.; van den Berg, Wim B.; van der Kraan, Peter M.

    2014-01-01

    Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth fa

  15. Response to Stress in 17 alpha-hydroxylase Deficient Common Carp (Cyprinus carpio L.)

    NARCIS (Netherlands)

    Nematollahi, M.A.; Pelt, van H.; Komen, J.

    2013-01-01

    The aim of this study was to analyze the stress response during 3 hours net confinement stress and recovery period of 22 hours in normal (STD) and in 17 alpha-hydroxylase deficient common carp (E5). Fish were raised for 6 months and sampled at T-0 (control, unstressed), 5 minutes, 20 minutes, 1 hour

  16. The crystal structure of human dopamine  β-hydroxylase at 2.9 Å resolution

    DEFF Research Database (Denmark)

    Vendelboe, Trine Vammen; Harris, Pernille; Zhao, Y.

    2016-01-01

    , Alzheimer’s disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600...

  17. Association analysis of the tyrosine hydroxylase gene polymorphisms with early-onset schizophrenia in Chinese population

    Institute of Scientific and Technical Information of China (English)

    吕钦谕

    2014-01-01

    Objective To investigate the relationship between the tyrosine hydroxylase(TH)gene and early-onset schizophrenia in Chinese Han population.Methods To tag single nucleotide polymorphisms(tag SNPs)rs2070762,rs6356 and rs11042978 in the TH gene were genotyped in 315 early-onset schizophrenics(188 male patients,127 female patients)and 391 controls subjects

  18. Fertility in women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    NARCIS (Netherlands)

    Stikkelbroeck, M.M.L.; Hermus, A.R.M.M.; Braat, D.D.M.; Otten, B.J.

    2003-01-01

    Fertility in women with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency appears to be reduced. The purpose of this review is to summarize the reported evidence about subfertility in women with CAH and to review the causes of reduced fertility. Compared with a non-CAH female pop

  19. Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

    OpenAIRE

    Prabhu, Julia; Schauwecker, Florian; Grammel, Nicolas; Keller, Ullrich; Bernhard, Michael

    2004-01-01

    The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.

  20. Stabilization of Tryptophan Hydroxylase 2 by L-Phenylalanine Induced Dimerization

    DEFF Research Database (Denmark)

    Tidemand, Kasper Damgaard; Christensen, Hans Erik Mølager; Hoeck, Niclas;

    2016-01-01

    Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full length TPH2 is poorly characterized due to low purification qua...

  1. Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases.

    Science.gov (United States)

    Teigen, Knut; McKinney, Jeffrey Alan; Haavik, Jan; Martínez, Aurora

    2007-01-01

    Hydroxylation of the aromatic amino acids phenylalanine, tyrosine and tryptophan is carried out by a family of non-heme iron and tetrahydrobiopterin (BH4) dependent enzymes, i.e. the aromatic amino acid hydroxylases (AAHs). The reactions catalyzed by these enzymes are important for biomedicine and their mutant forms in humans are associated with phenylketonuria (phenylalanine hydroxylase), Parkinson's disease and DOPA-responsive dystonia (tyrosine hydroxylase), and possibly neuropsychiatric and gastrointestinal disorders (tryptophan hydroxylase 1 and 2). We attempt to rationalize current knowledge about substrate and inhibitor specificity based on the three-dimensional structures of the enzymes and their complexes with substrates, cofactors and inhibitors. In addition, further insights on the selectivity and affinity determinants for ligand binding in the AAHs were obtained from molecular interaction field (MIF) analysis. We applied this computational structural approach to a rational analysis of structural differences at the active sites of the enzymes, a strategy that can help in the design of novel selective ligands for each AAH.

  2. Expression analysis of kenaf cinnamate 4-hydroxylase (C4H) ortholog during developmental and stress responses

    Science.gov (United States)

    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...

  3. Elicitor-induced prolyl hydroxylase from French bean (Phaseolus vulgaris). Localization, purification and properties.

    Science.gov (United States)

    Bolwell, G P; Robbins, M P; Dixon, R A

    1985-08-01

    The enzyme prolyl hydroxylase (proline: 2-oxoglutarate dioxygenase, EC 1.14.11.12), induced in suspension-cultured cells of Phaseolus vulgaris L. (French bean) by treatment with an elicitor preparation from the phytopathogenic fungus Colletotrichum lindemuthianum, has been investigated. The enzyme, which catalyses the hydroxylation of poly-L-proline with the stoichiometric decarboxylation of 2-oxoglutarate, has been shown to be localized mainly in smooth endoplasmic reticulum. After solubilization from microsomal membranes, the hydroxylase was purified by ion-exchange chromatography and affinity chromatography on poly-L-proline-Sepharose 4B. The subunit Mr, as assessed by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis, was 65 000, the subunit apparently being recovered as a doublet: the subunits associate under non-denaturing conditions to give at least a tetramer. The bean hydroxylase has kinetic properties and cofactor requirements similar to those previously reported for the enzyme from other plants. Elicitor treatment of suspension-cultured bean cells leads to a rapid induction of prolyl hydroxylase activity concomitant with induction of a protein: arabinosyl-transferase and increased levels of an arabinosylated hydroxyproline-rich protein.

  4. Purification and Properties of p-Hydroxybenzoate Hydroxylases from Rhodococcus Strains.

    NARCIS (Netherlands)

    Jadan, A.P.; Berkel, van W.J.H.; Golovleva, L.A.; Golovlev, E.L.

    2001-01-01

    Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon

  5. Hydrocarbon Leak Detection Sensor Project

    Data.gov (United States)

    National Aeronautics and Space Administration — FTT is proposing the development of a sensor to detect the presence of hydrocarbons in turbopump Inter-Propellant Seals (IPS). The purpose of the IPS is to prevent...

  6. Growth of hydrocarbon utilizing microorganisms

    Digital Repository Service at National Institute of Oceanography (India)

    Bhosle, N.B.; Mavinkurve, S.

    Two isolates from marine mud having broad spectrum hydrocarbon utilizing profile were identified as Arthrobacter simplex and Candida tropicalis.Both the organisms grew exponentially on crude oil. The cell yield of the organisms was influenced...

  7. Induction and characterization of a cytochrome P-450-dependent camphor hydroxylase in tissue cultures of common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.; Croteau, R. (Washington State Univ., Pullman (United States))

    1993-04-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O[sub 2]-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl[sub 2], camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn[sup 2+]-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. 44 refs., 6 figs., 2 tabs.

  8. Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis).

    Science.gov (United States)

    Funk, C.; Croteau, R.

    1993-04-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases.

  9. High frequency of cytolytic 21-hydroxylase-specific CD8+ T cells in autoimmune Addison's disease patients.

    Science.gov (United States)

    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.

  10. Bioassay of polycyclic aromatic hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Van Kirk, E.A.

    1980-08-01

    A positive relationship was found between the photodynamic activity of 24 polycyclic aromatic hydrocarbons versus published results on the mutagenicity, carcinogenicity, and initiation of unscheduled DNA synthesis. Metabolic activation of benzo(a)pyrene resulted in detection of increased mutagenesis in Paramecium tetraurelia as found also in the Ames Salmonella assay. The utility of P. tetraurelia as a biological detector of hazardous polycyclic aromatic hydrocarbons is discussed.

  11. Electrochemical decomposition of chlorinated hydrocarbons

    OpenAIRE

    McGee, Gerard Anthony

    1993-01-01

    This work involves the characterisation of the electrochemical decomposition of chlorinated hydrocarbons. A variety of methods were employed involving the use of catalytic reagents to enhance the rate at which chlorinated organic compounds are reduced. The first reagent used was oxygen which was electrochemically reduced to superoxide in nonaqueous solvents. Superoxide is a reactive intermediate and decomposes chlorinated hydrocarbons. However it was found that since the rate of reaction betw...

  12. Aliphatic hydrocarbons of the fungi.

    Science.gov (United States)

    Weete, J. D.

    1972-01-01

    Review of studies of aliphatic hydrocarbons which have been recently detected in the spores of phytopathogenic fungi, and are found to be structurally very similar to the alkanes of higher plants. It appears that the hydrocarbon components of the few mycelial and yeast forms reported resemble the distribution found in bacteria. The occurence and distribution of these compounds in the fungi is discussed. Suggested functional roles of fungal spore alkanes are presented.

  13. LIQUID HYDROCARBON FUEL CELL DEVELOPMENT.

    Science.gov (United States)

    A compound anode consists of a reforming catalyst bed in direct contact with a palladium-silver fuel cell anode. The objective of this study was to...prove the feasibility of operating a compound anode fuel cell on a liquid hydrocarbon and to define the important parameters that influence cell...performance. Both reformer and fuel cell tests were conducted with various liquid hydrocarbon fuels. Included in this report is a description of the

  14. Monooxygenation of small hydrocarbons catalyzed by bacterial cytochrome p450s.

    Science.gov (United States)

    Shoji, Osami; Watanabe, Yoshihito

    2015-01-01

    Cytochrome P450s (P450s) catalyze the NAD(P)H/O2-dependent monooxygenation of less reactive organic molecules under mild conditions. The catalytic activity of bacterial P450s is very high compared with P450s isolated from animals and plants, and the substrate specificity of bacterial P450s is also very high. Accordingly, their catalytic activities toward nonnative substrates are generally low especially toward small hydrocarbons. However, mutagenesis approaches have been very successful for engineering bacterial P450s for the hydroxylation of small hydrocarbons. On the other hand, "decoy" molecules, whose structures are very similar to natural substrates, can be used to trick the substrate recognition of bacterial P450s, allowing the P450s to catalyze oxidation reactions of nonnative substrates without any substitution of amino acid residues in the presence of decoy molecules. Thus, the hydroxylation of small hydrocarbons such as ethane, propane, butane and benzene can be catalyzed by P450BM3, a long-alkyl-chain hydroxylase, using substrate misrecognition of P450s induced by decoy molecules. Furthermore, a number of H2O2-dependent bacterial P450s can catalyze the peroxygenation of a variety of nonnative substrates through a simple substrate-misrecognition trick, in which catalytic activities and enantioselectivity are dependent on the structure of decoy molecules.

  15. HYDROCARBONS RESERVES IN VENEZUELA

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez Cruz, D.J.

    2007-07-01

    Venezuela is an important player in the energy world, because of its hydrocarbons reserves. The process for calculating oil and associated gas reserves is described bearing in mind that 90% of the gas reserves of Venezuela are associated to oil. Likewise, an analysis is made of the oil reserves figures from 1975 to 2003. Reference is also made to inconsistencies found by international experts and the explanations offered in this respect by the Ministry of Energy and Petroleum (MENPET) and Petroleos de Venezuela (PDVSA) regarding the changes that took place in the 1980s. In turn, Hubbert's Law is explained to determine peak production of conventional oil that a reservoir or field will reach, as well as its relationship with remaining reserves. Emphasis is placed on the interest of the United Nations on this topic. The reserves of associated gas are presented along with their relationship with the different crude oils that are produced and with injected gas, as well as with respect to the possible changes that would take place in the latter if oil reserves are revised. Some recommendations are submitted so that the MENPET starts preparing the pertinent policies ruling reserves. (auth)

  16. Evaluation of hydrocarbon potential

    Energy Technology Data Exchange (ETDEWEB)

    Cashman, P.H.; Trexler, J.H. Jr. [Univ. of Nevada, Reno, NV (United States)

    1992-09-30

    Task 8 is responsible for assessing the hydrocarbon potential of the Yucca Mountain vincinity. Our main focus is source rock stratigraphy in the NTS area in southern Nevada. (In addition, Trexler continues to work on a parallel study of source rock stratigraphy in the oil-producing region of east central Nevada, but this work is not funded by Task 8.) As a supplement to the stratigraphic studies, we are studying the geometry and kinematics of deformation at NTS, particularly as these pertain to reconstructing Paleozoic stratigraphy and to predicting the nature of the Late Paleozoic rocks under Yucca Mountain. Our stratigraphic studies continue to support the interpretation that rocks mapped as the {open_quotes}Eleana Formation{close_quotes} are in fact parts of two different Mississippian units. We have made significant progress in determining the basin histories of both units. These place important constraints on regional paleogeographic and tectonic reconstructions. In addition to continued work on the Eleana, we plan to look at the overlying Tippipah Limestone. Preliminary TOC and maturation data indicate that this may be another potential source rock.

  17. From "Ugh" to "Ahh": The Power of Sketchbooks

    Science.gov (United States)

    Carlson, Pam

    2010-01-01

    "Art history? Ugh!" is often the response of high-school visual arts students when faced with an art-history assignment. With the exception of very few students who might have an affection for the historical aspects of art, getting art history into the hearts and minds of high-school students can be a challenge. In this article, the author shares…

  18. Mutagenesis of a Specificity-Determining Residue in Tyrosine Hydroxylase Establishes that the Enzyme is a Robust Phenylalanine Hydroxylase but a Fragile Tyrosine Hydroxylase†

    Science.gov (United States)

    Daubner, S. Colette; Avila, Audrey; Bailey, Johnathan O.; Barrera, Dimitrios; Bermudez, Jaclyn Y.; Giles, David H.; Khan, Crystal A.; Shaheen, Noel; Thompson, Janie Womac; Vasquez, Jessica; Oxley, Susan P.; Fitzpatrick, Paul F.

    2013-01-01

    The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites, but PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme (Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652–9661). Alanine scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 104) decreases in the Vmax and Vmax/Ktyr values for tyrosine hydroxylation, but only small decreases or even increases in the Vmax and Vmax/Kphe values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the Vmax value with tyrosine as substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur. PMID:23368961

  19. Membrane-bound hydroxylases in elicitor-treated bean cells. Rapid induction of the synthesis of prolyl hydroxylase and a putative cytochrome P-450.

    Science.gov (United States)

    Bolwell, G P; Dixon, R A

    1986-08-15

    Treatment of cell-suspension cultures of bean (Phaseolus vulgaris cv. Canadian Wonder) with an elicitor preparation heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid changes in the activities of two microsomal oxygenases, cinnamic acid 4-hydroxylase, involved in accumulation of wall-bound phenolics and phytoalexins, and proline 2-oxoglutarate dioxygenase (prolyl hydroxylase) involved in the post-translational modification of hydroxyproline-rich glycoproteins. An anti-(cytochrome P-450) monoclonal antibody, originally raised against rat cytochrome P-450 isoform c, has been shown to bind to bean microsomes and recognise in Western blots an Mr-48,000 polypeptide, which comigrates with a haeme-containing protein on SDS/polyacrylamide gel analysis and which has been tentatively identified as a cytochrome P-450 capable of the hydroxylation of cinnamic acid. A preparation of proline 2-oxoglutarate dioxygenase purified to homogeneity was used to immunise rabbits for the production of antiserum. An elicitor-induced polypeptide of Mr 65,000 was identified as prolyl hydroxylase while an antigenically related polypeptide of Mr 60,000 was also immunoprecipitated but not induced by elicitor treatment. Use of the two antibodies has demonstrated rapid transient increases in the synthesis of the Mr 48,000 and Mr 65,000 oxygenases in vivo and for mRNAs as measured in in vitro translations, particularly for the putative cytochrome P-450. These increases slightly precede corresponding changes in the synthesis of the soluble enzyme phenylalanine ammonia-lyase, in common with which these oxygenases probably share a mechanism of gene activation underlying the increased activities seen in response to elicitor treatment.

  20. Polycyclic Aromatic Hydrocarbons

    Science.gov (United States)

    Salama, Farid

    2010-01-01

    Carbonaceous materials play an important role in space. Polycyclic Aromatic Hydrocarbons (PAHs) are a ubiquitous component of the carbonaceous materials. PAHs are the best-known candidates to account for the IR emission bands. They are also thought to be among the carriers of the diffuse interstellar absorption bands (DIBs). PAH ionization states reflect the ionization balance of the medium while PAH size, composition, and structure reflect the energetic and chemical history of the medium. A major challenge is to reproduce in the laboratory the physical conditions that exist in the emission and absorption interstellar zones. The harsh physical conditions of the ISM -low temperature, collisionless, strong UV radiation fields- are simulated in the laboratory by associating a molecular beam with an ionizing discharge to generate a cold plasma expansion. PAH ions and radicals are formed from the neutral precursors in an isolated environment at low temperature and probed with high-sensitivity cavity ringdown spectroscopy in the NUV-NIR range. Carbon nanoparticles are also formed during the short residence time of the precursors in the plasma and are characterized with time-offlight mass spectrometry. These experiments provide unique information on the spectra of large carbonaceous molecules and ions in the gas phase that can now be directly compared to interstellar and circumstellar observations (IR emission bands, DIBs, extinction curve). These findings also hold great potential for understanding the formation process of interstellar carbonaceous grains. We will review recent progress in the experimental and theoretical studies of PAHs, compare the laboratory data with astronomical observations and discuss the global implications.

  1. Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism.

    Science.gov (United States)

    Zampolli, Jessica; Collina, Elena; Lasagni, Marina; Di Gennaro, Patrizia

    2014-01-01

    Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.

  2. Characterization of the medium- and long-chain n-alkanes degrading Pseudomonas aeruginosa strain SJTD-1 and its alkane hydroxylase genes.

    Science.gov (United States)

    Liu, Huan; Xu, Jing; Liang, Rubing; Liu, Jianhua

    2014-01-01

    A gram-negative aliphatic hydrocarbon-degrading bacterium SJTD-1 isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa by comparative analyses of the 16S rRNA sequence, phenotype, and physiological features. SJTD-1 could efficiently mineralize medium- and long-chain n-alkanes (C12-C30) as its sole carbon source within seven days, showing the most optimal growth on n-hexadecane, followed by n-octadecane, and n-eicosane. In 36 h, 500 mg/L of tetradecane, hexadecane, and octadecane were transformed completely; and 2 g/L n-hexadecane was degraded to undetectable levels within 72 h. Two putative alkane-degrading genes (gene 3623 and gene 4712) were characterized and our results indicated that their gene products were rate-limiting enzymes involved in the synergetic catabolism of C12-C16 alkanes. On the basis of bioinformatics and transcriptional analysis, two P450 monooxygenases, along with a putative AlmA-like oxygenase, were examined. Genetically defective mutants lacking the characteristic alkane hydroxylase failed to degrade n-octadecane, thereby suggesting a different catalytic mechanism for the microbial transformation of alkanes with chain lengths over C18.

  3. Indirect assays for deoxyhypusine hydroxylase using dual-label ratio changes and oxidative release of radioactivity

    Energy Technology Data Exchange (ETDEWEB)

    Abbruzzese, A.; Park, M.H.; Folk, J.E.

    1986-05-01

    Two procedures for rapid assay of deoxyhypusine hydroxylase activity are described. One of these assays measures changes in the /sup 3/H:/sup 14/C ratio of dual-labeled protein that results from the release of tritium from a specific position in the side chain of the /sup 3/H, /sup 14/C-labeled constituent amino acid deoxyhypusine upon its conversion to (/sup 3/H, /sup 14/C)hypusine. The other assay relies upon release of radioactivity from product protein by periodate oxidation of the radiolabeled side chain of component hypusine. The good correspondence of each of these assays with the ion exchange chromatographic method which measures hypusine and deoxyhypusine in acid hydrolysates of protein indicates that each provides a valid means of determining deoxyhypusine hydroxylase activity.

  4. The crystal structure of tryptophan hydroxylase with bound amino acid substrate

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Petersen, Charlotte Rode; Christensen, Hans Erik Mølager

    2008-01-01

    124−Asp139 and Ile367−Thr369 close around the active site. Similar structural changes are seen in the catalytic domain of phenylalanine hydroxylase (PAH) upon binding of substrate analogues norleucine and thienylalanine to the PAH·BH4 complex. In fact, the chicken TPH1·Trp·imidazole structure......Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O2, and tetrahydrobiopterin (BH4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis...... of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). We have determined the 1.9 Å resolution crystal structure of the catalytic domain (Δ1−100/Δ415−445) of chicken TPH isoform 1 (TPH1) in complex with the tryptophan substrate and an iron-bound imidazole. This is the first structure of any aromatic amino...

  5. Biotransformation of benzene and toluene to catechols by phenol hydroxylase from Arthrobacter sp. W1.

    Science.gov (United States)

    Ma, Fang; Shi, Sheng-Nan; Sun, Tie-Heng; Li, Ang; Zhou, Ji-Ti; Qu, Yuan-Yuan

    2013-06-01

    Phenol hydroxylase gene engineered microorganism (PHIND) was used to synthesize catechols from benzene and toluene by successive hydroxylation reaction. HPLC-MS and (1)H NMR analysis proved that the products of biotransformation were the corresponding catechols via the intermediate production of phenols. It was indicated that the main products of toluene oxidation were o-cresol and p-cresol. 3-Methylcatechol was the predominant product for m-cresol biotransformation. Formation rate of catechol (25 μM/min/g cell dry weight) was 1.43-fold higher than that of methylcatechols. It was suggested that phenol hydroxylase could be successfully used to transform both benzene and toluene to catechols by successive hydroxylation.

  6. Enrichment of light hydrocarbon mixture

    Science.gov (United States)

    Yang; Dali; Devlin, David; Barbero, Robert S.; Carrera, Martin E.; Colling, Craig W.

    2010-08-10

    Light hydrocarbon enrichment is accomplished using a vertically oriented distillation column having a plurality of vertically oriented, nonselective micro/mesoporous hollow fibers. Vapor having, for example, both propylene and propane is sent upward through the distillation column in between the hollow fibers. Vapor exits neat the top of the column and is condensed to form a liquid phase that is directed back downward through the lumen of the hollow fibers. As vapor continues to ascend and liquid continues to countercurrently descend, the liquid at the bottom of the column becomes enriched in a higher boiling point, light hydrocarbon (propane, for example) and the vapor at the top becomes enriched in a lower boiling point light hydrocarbon (propylene, for example). The hollow fiber becomes wetted with liquid during the process.

  7. Functional analysis of Antirrhinum kelloggii flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes; critical role in flower color and evolution in the genus Antirrhinum.

    Science.gov (United States)

    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.

  8. Development of plasma 21-deoxycortisol radioimmunoassay and application to the diagnosis of patients with 21-hydroxylase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Milewicz, A.; Vecsei, P.; Korth-Schuetz, S.; Haack, D.; Lichtwald, K.; Lewicka, S. (Heidelberg Univ. (Germany, F.R.)); Roesler, A. (Hadassah Medical School, Jerusalem (Israel)); Mittelstaedt, G.V. (Heidelberg Univ. (Germany, F.R.). Dept of Nephrology, Medical Faculty)

    1984-08-01

    Specific 21-deoxycortisol (21-DF) antiserum was raised in rabbits using a 21-DF-3,20-oxime-bovine serum albumin complex. Plasma radioimmunoassay of 21-DF was developed and used together with a radioimmunoassay of 17-hydroxyprogesterone (17-OH-P) for diagnosis of patients with 21-hydroxylase deficiency of congenital and postpubertal forms. The response of plasma 21-DF and 17-OH-P to iv.v. ACTH (25 IU) was studied in 15 adult controls and compared to 8 women with the late onset form of 21-hydroxylase deficiency and 23 women with idiopathic hirsutism. Normal 21-DF values for women were 6.9 +- 3.6 ng/dl and for men 9.71 +- 2.73 ng/dl. Newborn children (age: 3-10 days) had a value of 8.3 +- 4.8 ng/dl. During the menstrual cycle the 21-DF values did not change. The baseline and post-stimulated concentrations of hormone were similar in controls and women with hirsutism but significantly higher in women with the late onset form of 21-hydroxylase deficiency. In the congenital form of 21-hydroxylase deficiency, 21-DF values (baseline) were high. In general, the 21-DF and 17-OH-P values have shown parallel changes. However, one case of 21-hydroxylase deficiency with elevated 21-DF but normal 17-OH-P was observed. The use of 21-DF for the diagnosis of 21-hydroxylase deficiency is suggested.

  9. Mineralocorticoid replacement during infancy for salt wasting congenital adrenal hyperplasia due to 21-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Larissa G. Gomes

    2013-01-01

    Full Text Available OBJECTIVE: The protocols for glucocorticoid replacement in children with salt wasting 21-hydroxylase deficiency are well established; however, the current recommendation for mineralocorticoid replacement is general and suggests individualized dose adjustments. This study aims to retrospectively review the 9-∝-fludrocortisone dose regimen in salt wasting 21-hydroxylase deficient children who have been adequately treated during infancy. METHODS: Twenty-three salt wasting 21-hydroxylase deficient patients with good anthropometric and hormonal control were followed in our center since diagnosis. The assessments of cortisone acetate and 9-∝-fludrocortisone doses, anthropometric parameters, and biochemical and hormonal levels were rigorously evaluated in pre-determined intervals from diagnosis to two years of age. RESULTS: The 9-∝-fludrocortisone doses decreased over time during the first and second years of life; the median fludrocortisone doses were 200 µg at 0-6 months, 150 µg at 7-18 months and 125 µg at 19-24 months. The cortisone acetate dose per square meter was stable during follow-up (median = 16.8 mg/m²/day. The serum sodium, potassium and plasma rennin activity levels during treatment were normal, except in the first month of life, when periodic 9-∝-fludrocortisone dose adjustments were made. CONCLUSIONS: The mineralocorticoid needs of salt wasting 21-hydroxylase deficient patients are greater during early infancy and progressively decrease during the first two years of life, which confirms that a partial aldosterone resistance exists during this time. Our study proposes a safety regiment for mineralocorticoid replacement during this critical developmental period.

  10. Effects of coumarate 3-hydroxylase down-regulation on lignin structure

    Science.gov (United States)

    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...

  11. Functional characterization of salicylate hydroxylase from the fungal endophyte Epichloë festucae

    OpenAIRE

    Ambrose, Karen V.; Zipeng Tian; Yifei Wang; Jordan Smith; Gerben Zylstra; Bingru Huang; Belanger, Faith C.

    2015-01-01

    Epichloë spp. are symbiotic fungal endophytes of many cool season grasses. The presence of the fungal endophytes often confers insect, drought, and disease tolerance to the host grasses. The presence of the fungal endophytes within the host plants does not elicit host defense responses. The molecular basis for this phenomenon is not known. Epichloë festucae, the endophyte of Festuca rubra, expresses a salicylate hydroxylase similar to NahG from the bacterium Pseudomonas putida. Few fungal sal...

  12. Recurrence of the R408W Mutation in the Phenylalanine Hydroxylase Locus in Europeans

    OpenAIRE

    Eisensmith, Randy C.; Goltsov, Alexei A.; O'Neill, Charles; Tyfield, Linda A.; Schwartz, Eugene I.; Kuzmin, Alexei I.; Baranovskaya, Svetlana S.; Tsukerman, Gennady L.; Treacy, Eileen; Scriver, Charles R.; Güttler, Flemming; Guldberg, Per; Eiken, Hans G; Apold, Jaran; Svensson, Elisabeth

    1995-01-01

    The relative frequency of the common phenylalanine hydroxylase (PAH) mutation R408W and its associations with polymorphic RFLP, VNTR, and short-tandem-repeat (STR) sites in the PAH gene were examined in many European populations and one representative North American population of defined European descent. This mutation was found to cluster in two regions: in northwest Europe among Irish and Scottish peoples, and in eastern Europe, including the Commonwealth of Independent States. This allele ...

  13. Serial studies of serum dopamine-B-hydroxylase and urinary vanillylmandelic and homovanillic acids in neuroblastoma.

    Science.gov (United States)

    Brewster, M A; Berry, D H

    1979-01-01

    Recent reports have suggested that elevations of serum dopamine-B-hydroxylase (DBH) activity may correlate with diagnosis in neuroblastoma patients excreting vanillylmandelic acid (VMA). We have serially studied serum DBH and urinary homovanillic acid (HVA) and VMA excretion during the disease course of five patients with neuroblastoma. DBH activities did not indicate clinical course, therapies, or prognosis. Dilution studies revealed an age-related alteration in DBH effectors and they suggest that these may be different in neuroblastoma.

  14. 24-Hydroxylase in Cancer: Impact on Vitamin D-based Anticancer Therapeutics

    Science.gov (United States)

    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

  15. Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

    Science.gov (United States)

    Nowicki, Magda; Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2015-11-01

    Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

  16. Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5.

    Science.gov (United States)

    Liu, Chenli; Wang, Wanpeng; Wu, Yehui; Zhou, Zhongwen; Lai, Qiliang; Shao, Zongze

    2011-05-01

    Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.

  17. Structural Insights into Diversity and n-Alkane Biodegradation Mechanisms of Alkane Hydroxylases

    Directory of Open Access Journals (Sweden)

    Yurui eJi

    2013-03-01

    Full Text Available Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini depending on the type of enzymes. In this review, we summarize the different types of alkane hydroxylases, their degrading steps and compare typical enzymes from various classes with regard to their three dimensional structures, in order to provide insights into how the enzymes mediate their different roles in the degradation of n-alkanes and what determines their different substrate ranges. Through the above analyses, the degrading mechanisms of enzymes can be elucidated and molecular biological methods can be utilized to expand their catalytic roles in the petrochemical industry or in bioremediation of oil-contaminated environments.

  18. CYP17A1 intron mutation causing cryptic splicing in 17α-hydroxylase deficiency.

    Directory of Open Access Journals (Sweden)

    Daw-Yang Hwang

    Full Text Available 17α-Hydroxylase/17, 20-lyase deficiency (17OHD is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90% of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination.

  19. The Activities of Lysyl Hydroxylase 3 (LH3) Regulate the Amount and Oligomerization Status of Adiponectin

    Science.gov (United States)

    Ruotsalainen, Heli; Wang, Yu; Karppinen, Marjo; Bergmann, Ulrich; Kvist, Ari-Pekka; Pospiech, Helmut; Herzig, Karl-Heinz; Myllylä, Raili

    2012-01-01

    Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the lysyl hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDL-cholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance. PMID:23209641

  20. Who is a carrier? Detection of unsuspected mutations in 21-hydroxylase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Witchel, S.S.; Lee, P.A.; Trucco, M. [Univ. of Pittsburgh School of Medicine, Pittsburgh, PA (United States)

    1996-01-02

    Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. During our routine genotyping of affected individuals and their relatives using allele-specific oligonucleotide hybridization and single-strand conformational polymorphism analysis, we identified two families each segregating three mutations. In both families, a mutation known to be associated with 21-hydroxylase deficiency was identified in healthy individuals but was not detected in the propositus. The propositus in family 1 was shown to be a homozygous carrier for G at nucleotide 655, which alters the splice acceptor site at exon 3. The propositus in family 2 carried the same splicing mutation on the maternal allele and a gene deletion/conversion on the paternal allele. In both families, other clinically unaffected relatives carried the Q318X mutation in exon 8. If molecular diagnostic studies had been limited to the mutation carried by the propositi, relatives would have been misinformed regarding their status as carriers or mildly affected individuals. The findings in these two families emphasize the high frequency of alleles causing 21-hydroxylase deficiency in the population. 29 refs., 3 figs., 2 tabs.

  1. Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses.

    Science.gov (United States)

    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.

  2. Tyrosine Hydroxylase as a Target for Deltamethrin in the Nigrostriatal Dopaminergic Pathway

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To study the effects of deltamethrin on tyrosine hydroxylase in nigrostriatum of male rats. Methods Sprague-Dawley rats were daily treated with deltamethrin at 6.25 or 12.5 mg/kg body weight by gavage for 10 days. Then HPLC-fluorescence detection was used to analyze the contents of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homoranillic acid (HVA) in substantial nigra and striatum. The activities of tyrosine hydroxylase (TH) were also detected by HPLC-fluorescence detection. TH mRNA or TH protein levels were measured by RT-PCR and immunohistochemistry method. Results The content of DA in striatum was significantly decreased by the treatments, suggesting an inhibition of DA synthesis by deltamethrin. The contents of DA metabolites DOPAC and HVA increased, indicating increased dopamine turnover. Furthermore, deltamethrin significantly decreased the activity, as well as the mRNA and protein levels of TH.Conclusions These findings reveal a novel aspect of deltamethrin neurotoxicity and suggest tyrosine hydroxylase as a molecular target of deltamethin on dopamine metabolism in the nigrostriatal pathway.

  3. Stringency of the 2-His-1-Asp active-site motif in prolyl 4-hydroxylase.

    Directory of Open Access Journals (Sweden)

    Kelly L Gorres

    Full Text Available 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 alpha-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.

  4. Periplasmically-exported lupanine hydroxylase undergoes transition from soluble to functional inclusion bodies in Escherichia coli.

    Science.gov (United States)

    Stampolidis, Pavlos; Kaderbhai, Naheed N; Kaderbhai, Mustak A

    2009-04-01

    Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca(2+)-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors. Increased growth temperatures ranging from 18 to 30 degrees C gradually raised the enzyme production to a peak together with its concomitant aggregation as red solid particles, readily activatable in a fully functional form by mild chaotropic treatment. Here, we demonstrate that the exported lupanine hydroxylase undergoes a cascade transition from a soluble to "non-classical" inclusion body form when build-up in the periplasm exceeded a basal threshold concentration. These periplasmic aggregates were distinct from the non-secreted, signal-sequenceless counterpart that occurred as misfolded, non-functional concatamers in the form of classical inclusion bodies. We discuss our findings in the light of current models of how aggregation of lupanine hydroxylase arises in the periplasmic space.

  5. Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase.

    Science.gov (United States)

    Li, Jun; Ilangovan, Udayar; Daubner, S Colette; Hinck, Andrew P; Fitzpatrick, Paul F

    2011-01-15

    The hydroxylation of phenylalanine to tyrosine by the liver enzyme phenylalanine hydroxylase is regulated by the level of phenylalanine. Whether there is a distinct allosteric binding site for phenylalanine outside of the active site has been unclear. The enzyme contains an N-terminal regulatory domain that extends through Thr117. The regulatory domain of rat phenylalanine hydroxylase was expressed in Escherichia coli. The purified protein behaves as a dimer on a gel filtration column. In the presence of phenylalanine, the protein elutes earlier from the column, consistent with a conformational change in the presence of the amino acid. No change in elution is seen in the presence of the non-activating amino acid proline. ¹H-¹⁵N HSQC NMR spectra were obtained of the ¹⁵N-labeled protein alone and in the presence of phenylalanine or proline. A subset of the peaks in the spectrum exhibits chemical shift perturbation in the presence of phenylalanine, consistent with binding of phenylalanine at a specific site. No change in the NMR spectrum is seen in the presence of proline. These results establish that the regulatory domain of phenylalanine hydroxylase can bind phenylalanine, consistent with the presence of an allosteric site for the amino acid.

  6. The analysis of the phenylalanine hydroxylase gene mutations by sequencing and ARMS techniques in Turkish patients

    Directory of Open Access Journals (Sweden)

    Umit Luleyap

    2016-12-01

    Full Text Available Purpose: Phenylketonuria is an autosomal recessive deficiency of the hepatic enzyme phenylalanine hydroxylase. With this study, detection of the most frequent phenylalanine hydroxylase gene mutations in Turkish population is aimed. Material and Methods: 23 unrelated phenylketonuria patients (46 alleles who are living in Cukurova region, Turkey were investigated.First, all exons were screened by using DHPLC method then the direct sequencing technique and ARMS methods were used to analyze mutation suspected samples. Results: IVS10-11g and #61614;a splicing mutation in 27 alleles (58.7%, R261Q mutation in 7 alleles (15.2% and E178G, P281L, R243X, R243Q, R408W, Y386C mutations, all in a frequency of 2/46 (4.3% is found. Conclusion: The arginine amino acid, accounting for 68.4% of changes in exon sites 7 and 12, where R243X, R243Q, R261Q and R408W mutations occur, is thought to be important for amino acid changes in phenylalanine hydroxylase gene among phenylketonuria patients in Cukurova region. Single-base mutations like IVS10-11g and #61614;a and P281L could be detected with an accuracy of 100% by the use of specifically designed primers by authors according to ARMS technique and it is relatively cheaper and requesting less technical equipment. [Cukurova Med J 2016; 41(4.000: 702-708

  7. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog.

    Science.gov (United States)

    van de Loo, F J; Broun, P; Turner, S; Somerville, C

    1995-01-01

    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 and with approximately 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. Images Fig. 3 Fig. 4 Fig. 5 PMID:7624314

  8. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

    Energy Technology Data Exchange (ETDEWEB)

    Van De Loo, F.J. [Univ. of Kentucky, Lexington, KY (United States); Broun, P.; Turner, S.; Somerville, C. [Carnegie Institution of Washington, Stanford, CA (United States)

    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 and 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.

  9. High Pressure Preignition Chemistry of Hydrocarbons and Hydrocarbon Mixtures

    Science.gov (United States)

    2007-11-02

    and hydrocarbon blends in our various combustion systems, with emphasis on the effects of elevated pressure using our pressurized flow reactor ( PFR ...facility. Detailed experimental data were generated from the PFR for use in associated kinetic modeling work. We continued to develop and extend both

  10. Total Petroleum Hydrocarbons (TPH): ToxFAQs

    Science.gov (United States)

    ... a state: This map displays locations where Total Petroleum Hydrocarbons (TPH) is known to be present. On ... I get more information? ToxFAQs TM for Total Petroleum Hydrocarbons (TPH) ( Hidrocarburos Totales de Petróleo (TPH) ) August ...

  11. Assessment of the toxic potential of PCB congener 81 (3,4,4[prime],5-tetrachlorobiphenyl) to fish in relation to other non-ortho-substituted PCB congeners

    Energy Technology Data Exchange (ETDEWEB)

    Harris, G.E.; Kiparissis, Y.; Metcalfe, C.D. (Trent Univ., Peterborough, Ontario (Canada). Environmental and Resource Studies Program)

    1994-09-01

    PCB congener 81 (3,4,4[prime],5-tetrachlorobiphenyl) has been detected in fish tissues from various sites in North America. The embryotoxicity of this compound to medaka (Oryzias latipes) and the induction of hepatic aryl hydrocarbon hydroxylase (AHH) in rainbow trout (Oncorhynchus mykiss) were determined to assess the toxic potency of this compound relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and two other non-ortho-substituted PCB compounds, congener 77 (3,3[prime],4,4[prime]-tetrachlorobiphenyl) and congener 126 (3,3[prime],4,4[prime],5-pentachlorobiphenyl). The TCDD toxic equivalency factors (TEFs) estimated for congener 81 from two end points in the medaka embryotoxicity assay were 0.0014 (from mortality data) and 0.006 (from swim bladder inflation data). The TEF estimated for congener 81 from data on AHH induction in rainbow trout was 0.004. All TEFs were greater than those estimated for congener 77 but were less than the TEFs estimated for congener 126. On the basis of these toxicity data, it is suggested that this congener may contribute significantly to the toxic burden of planar halogenated aromatic hydrocarbon in fish.

  12. 33 CFR 157.166 - Hydrocarbon emissions.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Hydrocarbon emissions. 157.166... Crude Oil Washing (COW) System on Tank Vessels Cow Operations § 157.166 Hydrocarbon emissions. If the... ballasted in that port the hydrocarbon vapors in each tank are contained by a means under § 157.132....

  13. Compositions and methods for hydrocarbon functionalization

    Energy Technology Data Exchange (ETDEWEB)

    Gunnoe, Thomas Brent; Fortman, George; Boaz, Nicholas C.; Groves, John T.

    2017-03-28

    Embodiments of the present disclosure provide for methods of hydrocarbon functionalization, methods and systems for converting a hydrocarbon into a compound including at least one group ((e.g., hydroxyl group) (e.g., methane to methanol)), functionalized hydrocarbons, and the like.

  14. Effective viscosity of confined hydrocarbons

    DEFF Research Database (Denmark)

    Sivebæk, Ion Marius; Samoilov, V.N.; Persson, B.N.J.

    2012-01-01

    We present molecular dynamics friction calculations for confined hydrocarbon films with molecular lengths from 20 to 1400 carbon atoms. We find that the logarithm of the effective viscosity ηeff for nanometer-thin films depends linearly on the logarithm of the shear rate: log ηeff=C-nlog γ̇, where...

  15. Fire-safe hydrocarbon fuels

    Energy Technology Data Exchange (ETDEWEB)

    Fodor, G.E.; Weatherford, W.D. Jr.; Wright, B.R.

    1979-11-06

    A stabilized, fire-safe, aqueous hydrocarbon fuel emulsion prepared by mixing: a diesel fuel; an emulsifier (consisting of oleyl diethanolamide, diethanolamine, and diethanolamine soap of oleic acid) which has been treated with about 0 to 7 1/2 of oleic acid. A modified version of this fuel also contains 0 to 0.5% of an antimisting agent, and water.

  16. Hydrophobic encapsulation of hydrocarbon gases.

    Science.gov (United States)

    Leontiev, Alexander V; Saleh, Anas W; Rudkevich, Dmitry M

    2007-04-26

    [reaction: see text] Encapsulation data for hydrophobic hydrocarbon gases within a water-soluble hemicarcerand in aqueous solution are reported. It is concluded that hydrophobic interactions serve as the primary driving force for the encapsulation, which can be used for the design of gas-separating polymers with intrinsic inner cavities.

  17. Correlation between expression of 1α-hydroxylase and hypocalcaemia in rats with severe pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Ping Zhou; Li Chang; Xiao-Hong Zhang; You-Dai Chen; Xuan-Lin Feng; Lei Deng; Jian-Dong Wang

    2015-01-01

    Objective:To investigate the essential biochemical indices like 1α-hydroxylase and hypocalcaemia in the rats with severe acute pancreatitis and explore the correlation between them.Methods:A total of 120 SPF grade Wistar male rats which were in similar physiological status were selected and randomly divided into two groups: sham group (SO group) and severe acute pancreatitis group (SAP group). Then they were divided into 1 h, 3 h, 6 h, and 12 h subgroups according to the killing time. The severe acute pancreatitis model was established by retrograde injection of 5% sodium taurocholate. Serum calcium, serum creatinine, serum urea nitrogen and serum amylase were measured at different time. Serum 1, 25 dihydroxy vitamin D3 level was determined by enzyme linked immunosorbentassay. The expression of 1α-hydroxylase protein in the kidney tissue was determined with Western blotting and immunohistochemistry to observe its location. The pathologic features of the kidney tissue section was observed under light microscope and submicroscopic structure of the proximal convoluted tubule epithelial cell was observed under transmission electron microscope. Results:Compared with the SO group, rats in the SAP group showed continuous pathological injury as time went by. There was significant increase in serum creatinine, serum urea nitrogen and serum amylase in SAP group compared with the SO group 1, 3,6,12 hours after the operation (P<0.05). There was significant decrease in serum calcium and 1, 25 dihydroxy vitamin D3 3,6,12 hours after the operation (P<0.05). It also showed that the expression of the 1α-hydroxylase protein in kidney tissues was upregulated at 1 h, 3 h and decreased at 6 h,12 h compared with the SO group. The serum calcium, 1, 25 dihydroxy vitamin D3 and the expression of the 1α-hydroxylase protein in kidney tissues of the SAP group showed sustaining decrease. Western blotting showed positive correlation between the 1α-hydroxylase expression and serum calcium

  18. Biodegradation of petroleum hydrocarbons in hypersaline environments

    Directory of Open Access Journals (Sweden)

    Luiz Fernando Martins

    2012-09-01

    Full Text Available Literature on hydrocarbon degradation in extreme hypersaline media presents studies that point to a negative effect of salinity increase on hydrocarbonoclastic activity, while several others report an opposite tendency. Based on information available in the literature, we present a discussion on the reasons that justify these contrary results. Despite the fact that microbial ability to metabolize hydrocarbons is found in extreme hypersaline media, indeed some factors are critical for the occurrence of hydrocarbon degradation in such environments. How these factors affect hydrocarbon degradation and their implications for the assessment of hydrocarbon biodegradation in hypersaline environments are presented in this review.

  19. Syngas Upgrading to Hydrocarbon Fuels Technology Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Talmadge, M.; Biddy, M.; Dutta, A.; Jones, S.; Meyer, A.

    2013-03-01

    This technology pathway case investigates the upgrading of woody biomass derived synthesis gas (syngas) to hydrocarbon biofuels. While this specific discussion focuses on the conversion of syngas via a methanol intermediate to hydrocarbon blendstocks, there are a number of alternative conversion routes for production of hydrocarbons through a wide array of intermediates from syngas. Future work will also consider the variations to this pathway to determine the most economically viable and lowest risk conversion route. Technical barriers and key research needs have been identified that should be pursued for the syngas-to-hydrocarbon pathway to be competitive with petroleum-derived gasoline-, diesel- and jet-range hydrocarbon blendstocks.

  20. Biodegradation of petroleum hydrocarbons in hypersaline environments

    Science.gov (United States)

    Martins, Luiz Fernando; Peixoto, Raquel Silva

    2012-01-01

    Literature on hydrocarbon degradation in extreme hypersaline media presents studies that point to a negative effect of salinity increase on hydrocarbonoclastic activity, while several others report an opposite tendency. Based on information available in the literature, we present a discussion on the reasons that justify these contrary results. Despite the fact that microbial ability to metabolize hydrocarbons is found in extreme hypersaline media, indeed some factors are critical for the occurrence of hydrocarbon degradation in such environments. How these factors affect hydrocarbon degradation and their implications for the assessment of hydrocarbon biodegradation in hypersaline environments are presented in this review. PMID:24031900

  1. Aqueous reactions of chlorine dioxide with hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Rav-Acha, C.; Choshen, E.

    1987-11-01

    In contrast to mechanisms proposed earlier in the literature, according to which chlorine dioxide (ClO/sub 2/) reacts with various hydrocarbons in aqueous media by abstracting allylic or benzylic hydrogens, it is shown that ClO/sub 2/ reacts with olefins through initial electron transfer. Hydrocarbons that can undergo facile oxidation, such as polycyclic aromatic hydrocarbons (PAH) and some olefins, react with ClO/sub 2/ quite rapidly, while saturated aliphatic hydrocarbons, some aromatic hydrocarbons, and olefins substituted with electron-withdrawing groups remain unreactive. This was substantiated by comparing the reactivities toward ClO/sub 2/ of a variety of hydrocarbons, including aliphatic and aromatic hydrocarbons, saturated and unsaturated acids, PAH, or cyclic and acyclic olefins. The results were supported by a detailed kinetic and product study of the reaction between ClO/sub 2/ and some model compounds.

  2. Transcriptome Analysis Reveals Key Flavonoid 3'-Hydroxylase and Flavonoid 3',5'-Hydroxylase Genes in Affecting the Ratio of Dihydroxylated to Trihydroxylated Catechins in Camellia sinensis.

    Directory of Open Access Journals (Sweden)

    Kang Wei

    Full Text Available The ratio of dihydroxylated to trihydroxylated catechins (RDTC is an important indicator of tea quality and biochemical marker for the study of genetic diversity. It is reported to be under genetic control but the underlying mechanism is not well understood. Flavonoid 3'-hydroxylase (F3'H and flavonoid 3',5'-hydroxylase (F3'5'H are key enzymes involved in the formation of dihydroxylated and trihydroxylated catechins. The transcriptome and HPLC analysis of tea samples from Longjing43 and Zhonghuang2 under control and shading treatment were performed to assess the F3'H and F3'5'H genes that might affect RDTC. A total of 74.7 million reads of mRNA seq (2×101bp data were generated. After de novo assembly, 109,909 unigenes were obtained, and 39,982 of them were annotated using 7 public databases. Four key F3'H and F3'5'H genes (including CsF3'5'H1, CsF3'H1, CsF3'H2 and CsF3'H3 were identified to be closely correlated with RDTC. Shading treatment had little effect on RDTC, which was attributed to the stable expression of these key F3'H and F3'5'H genes. The correlation of the coexpression of four key genes and RDTC was further confirmed among 13 tea varieties by real time PCR and HPLC analysis. The coexpression of three F3'H genes and a F3'5'H gene may play a key role in affecting RDTC in Camellia sinensis. The current results may establish valuable foundation for further research about the mechanism controlling catechin composition in tea.

  3. Differential hepatocellular zonation pattern of cholesterol 7alpha-hydroxylase (Cyp7a1) and sterol 12alpha-hydroxylase (Cyp8b1) in the mouse.

    Science.gov (United States)

    Wang, Jin; Olin, Maria; Rozell, Björn; Björkhem, Ingemar; Einarsson, Curt; Eggertsen, Gösta; Gåfvels, Mats

    2007-03-01

    The synthesis of primary bile acids is confined to the hepatocytes. This study aimed to evaluate the expression pattern within the liver architecture of the rate-limiting enzyme of the neutral pathway, cholesterol 7alpha-hydroxylase (Cyp7a1), and sterol 12alpha-hydroxylase (Cyp8b1), the enzyme necessary for the synthesis of cholic acid. Specific Cyp8b1 and Cyp7a1 peptide antiserums were used for immunohistochemical staining of livers from wild type and Cyp8b1 null mice, the latter instead expressing beta-galactosidase (beta-Gal) as a replacement reporter gene. Cyp8b1 was mainly expressed in the hepatocytes in a zonal pattern surrounding the central vein while the areas surrounding the portal zones showed much lower levels. The zonation was maintained in cholic acid-depleted mice using beta-Gal as a reporter protein. Cyp7a1 expression in wild type mice also showed a zonal distribution pattern, although less distinct, with a maximal expression within a 1-2 cell thick layer of hepatocytes surrounding the central vein. In Cyp8b1 null mice, a more intense staining was obtained, in accordance with the higher expression level of Cyp7a1, although the overall expression pattern was maintained. Our results in mice indicate possible differences in the regulation of the cellular zonation of Cyp7a1 and Cyp8b1. Also, cholic acid affects the set-point of Cyp7a1 expression but not its zonal distribution.

  4. Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and saccharomyces cerevisiae.

    Science.gov (United States)

    Haudenschild, C; Schalk, M; Karp, F; Croteau, R

    2000-07-01

    The oxygenation pattern of the essential oil monoterpenes of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In spearmint (M. spicata), C6-allylic hydroxylation leads to (-)-trans-carveol and thence (-)-carvone, whereas in peppermint (M. x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol and ultimately (-)-menthol. cDNAs encoding the C6-hydroxylase and C3-hydroxylase from spearmint and peppermint, respectively, were isolated by a combination of reverse genetic and homology-based cloning methods (S. Lupien, F. Karp, M. Wildung, and R. Croteau, Arch. Biochem. Biophys. 368, 181-192, 1999). Although both hydroxylase genes were confirmed by functional expression using the baculovirus-Spodoptera system, too little protein was available by this approach to permit detailed study of the structure-function relationships of these catalysts, especially the substrate binding determinants that underlie the regiochemistry and stereochemistry of the reactions. Therefore, heterologous overexpression systems based on Escherichia coli and Saccharomyces cerevisiae were developed to produce several N-terminally modified versions of the recombinant hydroxylases. Ancillary methods for the solubilization, purification, and reconstitution (with recombinant spearmint cytochrome P450 reductase) of the limonene hydroxylases were also devised, with which substrate binding behavior and precise regiochemistry and stereochemistry of product formation were determined. Copyright 2000 Academic Press.

  5. Hypoxia-inducible Factor Prolyl 4-Hydroxylase Inhibition A TARGET FOR NEUROPROTECTION IN THE CENTRAL NERVOUS SYSTEM*

    Science.gov (United States)

    Siddiq, Ambreena; Ayoub, Issam A.; Chavez, Juan C.; Aminova, Leila; Shah, Sapan; LaManna, Joseph C.; Patton, Stephanie M.; Connor, James R.; Cherny, Robert A.; Volitakis, Irene; Bush, Ashley I.; Langsetmo, Ingrid; Seeley, Todd; Gunzler, Volkmar; Ratan, Rajiv R.

    2008-01-01

    Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1α and HIF-2α. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1α and up-regulate HIF-dependent target genes (e.g. enolase, p21waf1/cip1, vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress. PMID:16227210

  6. Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response.

    Science.gov (United States)

    Kuiper, Caroline; Dachs, Gabi U; Currie, Margaret J; Vissers, Margreet C M

    2014-04-01

    Hypoxia-inducible factor (HIF)-1 drives the transcription of hundreds of genes to support cell survival under conditions of microenvironmental and metabolic stress. HIF-1 is downregulated by iron-containing 2-oxoglutarate-dependent enzymes that require ascorbate as a cofactor. The HIF hydroxylases control both protein stability and the formation of an active transcription complex and, consequently, ascorbate could affect HIF-1α stabilization and/or gene expression, but the relative effect of ascorbate on these separate processes has not been well characterized. In this study we examined the effects of known intracellular ascorbate concentrations on both processes in response to various means of hydroxylase inhibition, including CoCl2, NiCl2, desferrioxamine, dimethyloxalylglycine, and hypoxia. Ascorbate inhibited HIF-1 activity most dramatically with all mechanisms of iron competition. In addition, HIF-1-dependent gene expression was effectively prevented by ascorbate and was inhibited even under conditions that allowed HIF-1α protein stabilization. This suggests that (1) ascorbate acts primarily to stabilize and reduce the iron atom in the hydroxylase active site and (2) the asparagine hydroxylase controlling HIF-1 transcriptional activity is particularly susceptible to fluctuations in intracellular ascorbate. These findings suggest that ascorbate plays a significant role in supporting HIF-hydroxylase function and that it could thereby modulate the cell survival response.

  7. Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger.

    Science.gov (United States)

    Matsuda, J; Okabe, S; Hashimoto, T; Yamada, Y

    1991-05-25

    Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. niger.

  8. Microbial production of gaseous hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Fukuda, Hideo

    1987-10-20

    Microbial production of ethylene, isobutane and a saturated gaseous hydrocarbon mixture was described. Microbial ethylene production was studied with Penicillium digitatum IFO 9372 and a novel pathway of the ethylene biosynthesis through alpha-ketoglutarate was proposed. Rhodotorula minuta IFO 1102 was selected for the microbial production of isobutane and the interesting actions of L-leucine and L-phenylalanine for the isobutane production were found. It was finally presented about the microbial production of a saturated gaseous hydrocarbon mixture with Rhizopus japonicus IFO 4758 was described. A gas mixture was produced through a chemical reaction of SH compounds and some cellular component such as squalene under aerobic conditions. (4 figs, 7 tabs, 41 refs)

  9. Hydrocarbon Rocket Technology Impact Forecasting

    Science.gov (United States)

    Stuber, Eric; Prasadh, Nishant; Edwards, Stephen; Mavris, Dimitri N.

    2012-01-01

    Ever since the Apollo program ended, the development of launch propulsion systems in the US has fallen drastically, with only two new booster engine developments, the SSME and the RS-68, occurring in the past few decades.1 In recent years, however, there has been an increased interest in pursuing more effective launch propulsion technologies in the U.S., exemplified by the NASA Office of the Chief Technologist s inclusion of Launch Propulsion Systems as the first technological area in the Space Technology Roadmaps2. One area of particular interest to both government agencies and commercial entities has been the development of hydrocarbon engines; NASA and the Air Force Research Lab3 have expressed interest in the use of hydrocarbon fuels for their respective SLS Booster and Reusable Booster System concepts, and two major commercially-developed launch vehicles SpaceX s Falcon 9 and Orbital Sciences Antares feature engines that use RP-1 kerosene fuel. Compared to engines powered by liquid hydrogen, hydrocarbon-fueled engines have a greater propellant density (usually resulting in a lighter overall engine), produce greater propulsive force, possess easier fuel handling and loading, and for reusable vehicle concepts can provide a shorter turnaround time between launches. These benefits suggest that a hydrocarbon-fueled launch vehicle would allow for a cheap and frequent means of access to space.1 However, the time and money required for the development of a new engine still presents a major challenge. Long and costly design, development, testing and evaluation (DDT&E) programs underscore the importance of identifying critical technologies and prioritizing investment efforts. Trade studies must be performed on engine concepts examining the affordability, operability, and reliability of each concept, and quantifying the impacts of proposed technologies. These studies can be performed through use of the Technology Impact Forecasting (TIF) method. The Technology Impact

  10. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334 ameliorates murine colitis

    Directory of Open Access Journals (Sweden)

    Gupta R

    2014-01-01

    Full Text Available Ram Gupta,1 Anita R Chaudhary,2 Binita N Shah,1 Avinash V Jadhav,3 Shitalkumar P Zambad,1 Ramesh Chandra Gupta,4 Shailesh Deshpande,4 Vijay Chauthaiwale,4 Chaitanya Dutt4 1Department of Pharmacology, 2Cellular and Molecular Biology, 3Preclinical Safety Evaluation, 4Discovery, Torrent Research Centre, Torrent Pharmaceuticals Ltd, Gandhinagar, Gujarat, India Background and aim: Mucosal healing in inflammatory bowel disease (IBD can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods: The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn's disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results: TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion: Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor

  11. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334) ameliorates murine colitis

    Science.gov (United States)

    Gupta, Ram; Chaudhary, Anita R; Shah, Binita N; Jadhav, Avinash V; Zambad, Shitalkumar P; Gupta, Ramesh Chandra; Deshpande, Shailesh; Chauthaiwale, Vijay; Dutt, Chaitanya

    2014-01-01

    Background and aim Mucosal healing in inflammatory bowel disease (IBD) can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF) has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn’s disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor ameliorates IBD in disease models. These findings highlight the potential of TRC160334 for its clinical application in the treatment of IBD. PMID:24493931

  12. Crystal structure of the ectoine hydroxylase, a snapshot of the active site.

    Science.gov (United States)

    Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H J

    2014-10-24

    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.

  13. Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Fitzpatrick, Susan F.; Fábián, Zsolt; Schaible, Bettina; Lenihan, Colin R.; Schwarzl, Thomas [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Rodriguez, Javier [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Zheng, Xingnan; Li, Zongwei [Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC (United States); Tambuwala, Murtaza M. [School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, BT52 1SA, Northern Ireland (United Kingdom); Higgins, Desmond G.; O' Meara, Yvonne [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Slattery, Craig [School of Biomolecular and Biomedical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Manresa, Mario C. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Fraisl, Peter; Bruning, Ulrike [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Baes, Myriam [Laboratory for Cell Metabolism, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven (Belgium); Carmeliet, Peter; Doherty, Glen [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Kriegsheim, Alex von [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Cummins, Eoin P. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); and others

    2016-06-03

    Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1{sup −/−} hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease. -- Highlights: •Genetic ablation of PHD1 upregulates NF-kappaB (NF-κB) in hepatocytes. •Activation of NF-κB leads to differential DNA-binding of p50/p65 and results in differential regulation of apoptotic genes. •We identified proline 191 in the beta subunit of the I-kappaB kinase as a target for PHD1-mediated hydroxylation. •Blockade of prolyl-4-hydroxylases has been found cytoprotective in liver cells.

  14. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    Science.gov (United States)

    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.

  15. Deep desulfurization of hydrocarbon fuels

    Science.gov (United States)

    Song, Chunshan [State College, PA; Ma, Xiaoliang [State College, PA; Sprague, Michael J [Calgary, CA; Subramani, Velu [State College, PA

    2012-04-17

    The invention relates to processes for reducing the sulfur content in hydrocarbon fuels such as gasoline, diesel fuel and jet fuel. The invention provides a method and materials for producing ultra low sulfur content transportation fuels for motor vehicles as well as for applications such as fuel cells. The materials and method of the invention may be used at ambient or elevated temperatures and at ambient or elevated pressures without the need for hydrogen.

  16. Hydrocarbon prospectivity in Western Greece

    Energy Technology Data Exchange (ETDEWEB)

    Maravelis, Angelos; Makrodimitras, George; Zelilidis, Avraam [Patras Univ. (Greece). Lab. of Sedimentology

    2012-06-15

    The geology of Western Greece is dominated by the most external zones of the Hellenide fold-and-thrust belt, namely the Pre-Apulian (or Paxoi) and Ionian zones. With Western Greece and Albania having undergone, in broad terms, similar geological histories, also the hydrocarbon potentials of both areas may be compared. Likewise, the hydrocarbon potential of Italy's Apulian Platform, adjoining in the westerly offshore, may serve as an analogue. Three basin types within Western Greece that deserve hydrocarbon exploration have been examined and are grouped, correlated to major tectonic features, namely foreland (Ionian thrusts' foreland basin), piggy-back (Ionian thrusts' back-arc basin) and strike-slip basins. Additionally, strike-slip basins are further subdivided into the basin north of the Borsh-Khardhiqit strike-slip fault and the Preveza basin, north of Cephalonia transfer fault. Their filling histories suggest the occurrence of Mesozoic carbonate plays and Oligocene/Miocene sandstone plays both for oil and gas.

  17. Abnormal pressure in hydrocarbon environments

    Science.gov (United States)

    Law, B.E.; Spencer, C.W.

    1998-01-01

    Abnormal pressures, pressures above or below hydrostatic pressures, occur on all continents in a wide range of geological conditions. According to a survey of published literature on abnormal pressures, compaction disequilibrium and hydrocarbon generation are the two most commonly cited causes of abnormally high pressure in petroleum provinces. In young (Tertiary) deltaic sequences, compaction disequilibrium is the dominant cause of abnormal pressure. In older (pre-Tertiary) lithified rocks, hydrocarbon generation, aquathermal expansion, and tectonics are most often cited as the causes of abnormal pressure. The association of abnormal pressures with hydrocarbon accumulations is statistically significant. Within abnormally pressured reservoirs, empirical evidence indicates that the bulk of economically recoverable oil and gas occurs in reservoirs with pressure gradients less than 0.75 psi/ft (17.4 kPa/m) and there is very little production potential from reservoirs that exceed 0.85 psi/ft (19.6 kPa/m). Abnormally pressured rocks are also commonly associated with unconventional gas accumulations where the pressuring phase is gas of either a thermal or microbial origin. In underpressured, thermally mature rocks, the affected reservoirs have most often experienced a significant cooling history and probably evolved from an originally overpressured system.

  18. Self-hydroxylation of the splicing factor lysyl hydroxylase, JMJD6

    DEFF Research Database (Denmark)

    Mantri, M.; Webby, C.J.; Loik, N.D.

    2012-01-01

    The lysyl 5S-hydroxylase, JMJD6 acts on proteins involved in RNA splicing. We find that in the absence of substrate JMJD6 catalyses turnover of 2OG to succinate. H-NMR analyses demonstrate that consumption of 2OG is coupled to succinate formation. MS analyses reveal that JMJD6 undergoes self......-hydroxylation in the presence of Fe(ii) and 2OG resulting in production of 5S-hydroxylysine residues. JMJD6 in human cells is also found to be hydroxylated. Self-hydroxylation of JMJD6 may play a regulatory role in modulating the hydroxylation status of proteins involved in RNA splicing. This journal is...

  19. The crystal structure of tryptophan hydroxylase with bound amino acid substrate

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Petersen, Charlotte Rode; Christensen, Hans Erik Mølager

    2008-01-01

    acid hydroxylase with bound natural amino acid substrate. The iron coordination can be described as distorted trigonal bipyramidal coordination with His273, His278, and Glu318 (partially bidentate) and one imidazole as ligands. The tryptophan stacks against Pro269 with a distance of 3.9 Å between...... of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). We have determined the 1.9 Å resolution crystal structure of the catalytic domain (Δ1−100/Δ415−445) of chicken TPH isoform 1 (TPH1) in complex with the tryptophan substrate and an iron-bound imidazole. This is the first structure of any aromatic amino...

  20. 17-α-Hydroxylase deficiency: An unusual case with primary amenorrhea and hypertension

    Directory of Open Access Journals (Sweden)

    Sunil Kumar Kota

    2011-01-01

    Full Text Available A 14-year-old girl presented with acute onset quadriparesis and newly detected hypertension. Parental consanguinity, delayed puberty with normal stature form the additional information. Hypokalemia with metabolic alkalosis, low cortisol, high ACTH and FSH pointed to the possibility of CAH with 17α hydroxylase deficiency. 46XX karyotype and high progesterone supported this. Normalization of hypokalemia and hypertension with glucocorticoid treatment confirmed the diagnosis. In summary, the possibility of 17 OHD should be suspected in patients with hypokalemic myopathy, Hypertension and hypogonadism so that appropriate therapy can be implemented.

  1. Brain development and cognitive, psychosocial, and psychiatric functioning in classical 21-hydroxylase deficiency.

    Science.gov (United States)

    Meyer-Bahlburg, Heino F L

    2011-01-01

    The disturbance of the hypothalamic-pituitary- adrenal axis characteristic of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is likely to affect brain development, yet neuroanatomic work is only beginning. Fetal hyperandrogenemia in 46, XX 21-OHD leads to masculinized brain organization and, consequently, at later stages of development, to masculinized gender-related behavior and cognitive function, including, although relatively uncommonly, gender identity. Genital masculinization as well as its surgical treatment has implications for social stigmatization and sexual functioning. CAH-associated electrolyte crises in infancy and later may result in severe cognitive impairment. Psychiatric disorders are somewhat increased, especially in patients with severe degrees of CAH.

  2. Distribution pattern of tyrosine hydroxylase in the brain and pituitary of the lungfish Protopterus annectens.

    Science.gov (United States)

    Bruzzone, F; d'Amora, M; Angelini, C; Vallarino, M

    2009-04-01

    The distribution of tyrosine hydroxylase (TH) was investigated in the brain and pituitary of the African lungfish Protopterus annectens by using immunohistochemistry. TH was expressed in the olfactory bulbs, pallium and subpallium, hypothalamus, tegmentum, reticular formation, nucleus of the solitary tract, and pituitary. In some cells of the hypothalamus and pituitary, TH is colocalized with vasoactive intestinal peptide. In the intermediate and neuronal lobes of the pituitary, TH is expressed in glial fibrillary acidic protein-contained cells. These data suggest that, in lungfish, catecholamine may play a role in olfaction information processing, regulation of visceral activities, and pituitary secretion.

  3. Molecular analysis of contiguous exons of the phenylalanine hydroxylase gene: identification of a new PKU mutation.

    OpenAIRE

    Dianzani, I; Camaschella, C.; Saglio, G; Ferrero, G B; Ramus, S; Ponzone, A; Cotton, R G

    1993-01-01

    A modified application of the chemical cleavage of mismatch (CCM) method has been used to screen three contiguous exons (exons 9, 10, and 11) of the phenylalanine hydroxylase gene in 17 Italian PKU patients. A new nonsense heterozygous C-->G transversion within exon 11 (S359X) was identified in a single patient. Only one of the four mutations previously reported in this DNA region in Caucasians was found. This lesion, IVS X-546, was detected in five of the 34 PKU alleles examined. Our results...

  4. Biochemical and genetic characterization of three molybdenum cofactor hydroxylases in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Hoff, Tine; Frandsen, Gitte Inselmann; Rocher, Anne

    1998-01-01

    Aldehyde oxidases and xanthine dehydrogenases/oxidases belong to the molybdenum cofactor dependent hydroxylase class of enzymes. Zymograms show that Arabidopsis thaliana has at least three different aldehyde oxidases and one xanthine oxidase. Three different cDNA clones encoding putative aldehyde...... oxidases (AtAO1, 2, 3) were isolated. An aldehyde oxidase is the last step in abscisic acid (ABA) biosynthesis. AtAO1 is mainly expressed in seeds and roots which might reflect that it is involved in ABA biosynthesis....

  5. Hydrocarbon Reserves: Abundance or Scarcity

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2005-07-01

    IFP and the OAPEC jointly organize a regular international seminar dealing with world oil-related problems appearing in the news. For the first time, this seminar has been opened to oil and gas company specialists, service companies, research centers and independents. This year's theme concerns oil and gas reserves: are they abundant or are we headed towards the shortages announced by some experts? This theme is especially topical in that: oil and gas currently meet two thirds of world energy needs and almost completely dominate the transport sector; the reserves declared by the OAPEC countries account for nearly half of world reserves; the price of a barrel of oil went through the roof in 2004; world energy demand is growing fast and alternative sources of energy are far from ready to take over from oil and gas in the next few decades. Since the reserves correspond to the volume it is technically and economically viable to produce, the seminar has, of course, dealt with the technical and economic questions that arise in connection with exploration and production, but it has also considered changes in the geopolitical context. Presentations by the leading companies of the OAPEC countries and by the IFP group were completed by presentation from the International Energy Agency (IEA), the United States Geological Survey (USGS), the IHS Energy Group, Total and Gaz de France. This document gathers the transparencies of the following presentations: Hydrocarbon reserves in OAPEC members countries: current and future (M. Al-Lababidi); Non OAPEC liquid reserves and production forecasts (Y. Mathieu); World oil and gas resources and production outlook (K. Chew); Global investments in the upstream (F. Birol); Total's policy in the oil and gas sector (C. de Margerie); Gaz de France's policy in the oil and gas sector (J. Abiteboul); NOC/IOC's opportunities in OPEC countries (I. Sandrea); Relationships between companies, countries and investors: How they may

  6. Cinnamic acid 4-hydroxylase of sorghum [Sorghum biocolor (L.) Moench] gene SbC4H1 restricts lignin synthesis in Arabidopsis

    Science.gov (United States)

    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...

  7. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF

    National Research Council Canada - National Science Library

    Koivunen, Peppi; Hirsilä, Maija; Remes, Anne M; Hassinen, Ilmo E; Kivirikko, Kari I; Myllyharju, Johanna

    ... (HIF-P4Hs) and one HIF asparaginyl hydroxylase (FIH). We have studied possible links between metabolic pathways and HIF hydroxylases by analyzing the abilities of citric acid cycle intermediates to inhibit purified human HIF-P4Hs and FIH...

  8. Regulation of phenylalanine hydroxylase: conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry.

    Science.gov (United States)

    Li, Jun; Fitzpatrick, Paul F

    2013-07-15

    The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.

  9. Correlation between hydrocarbon distribution and water-hydrocarbon ratio in Fischer-Tropsch synthesis

    Institute of Scientific and Technical Information of China (English)

    Xiaofeng Zhou; Qingling Chen; Yuewu Tao; Huixin Weng

    2011-01-01

    In order to shorten the evaluation cycle of cobalt catalyst before the optimized catalyst is fixed on,a mathematical method is proposed to calculate weight percentage of C5+ hydrocarbons.Based on the carbide polymerization mechanism and the main hydrocarbons being linear alkanes and α-olefins,the correlation between hydrocarbon distribution and the molecular mass ratio of water to hydrocarbons is discussed.The result shows the ratio was within the range of 1.125-1.286 and the lower the ratio,the more gaseous hydrocarbons were obtained.Moreover,a linear equation between the weight percentage of C5+ hydrocarbons and the weight ratio of C5+ hydrocarbons to the total water is established.These results are validated by corresponding experiments.The weight percentage of C5+ hydrocarbons could be immediately calculated by this linear equation without detailed gas chromatography (GC) analysis of them.

  10. HYDROCARBON AND SULFUR SENSORS FOR SOFC SYSTEMS

    Energy Technology Data Exchange (ETDEWEB)

    A.M. Azad; Chris Holt; Todd Lesousky; Scott Swartz

    2003-11-01

    The following report summarizes work conducted during the Phase I program Hydrocarbon and Sulfur Sensors for SOFC Systems under contract No. DE-FC26-02NT41576. For the SOFC application, sensors are required to monitor hydrocarbons and sulfur in order to increase the operation life of SOFC components. This report discusses the development of two such sensors, one based on thick film approach for sulfur monitoring and the second galvanic based for hydrocarbon monitoring.

  11. Sustained high blood pressure reduction with etamicastat, a peripheral selective dopamine β-hydroxylase inhibitor.

    Science.gov (United States)

    Igreja, Bruno; Wright, Lyndon C; Soares-da-Silva, Patricio

    2016-03-01

    The aim of the present study was to evaluate the influence of chronic inhibition of dopamine ß-hydroxylase by etamicastat on the development of hypertension in the spontaneously hypertensive rat (SHR) and the sustainability of effects on the systolic and diastolic blood pressure in the SHR and the normotensive Wistar-Kyoto rat (WKY). WKY and SHR received etamicastat (10 mg/kg/d) from 5 weeks of age for 35 weeks in drinking water, and cardiovascular assessments were performed on a weekly basis. Etamicastat reduced systolic and diastolic blood pressure when SHRs reached the age of 16 weeks with mean decreases of 37 and 32 mm Hg, respectively, for the subsequent for 24 weeks of treatment, but did not prevent the increase in blood pressure (BP) aged between 5 and 11 week. The BP lowering effect of etamicastat in SHR was reversible on discontinuation and quickly resumed after reinstatement of therapy and was not accompanied by changes in heart rate. Etamicastat affected neither BP nor heart rate in WKY during 36 weeks of treatment. Etamicastat reduced urinary excretion of norepinephrine to a similar extent in WKY and SHR, accompanied by significant increases in urinary dopamine in SHR. Chronic administration of etamicastat did not adversely affected development of animals. Chronic dopamine ß-hydroxylase inhibition with etamicastat effectively decreases BP, although does not prevent the development of hypertension in the SHR.

  12. 17-Alpha Hydroxylase Deficiency: A Rare Case of Primary Amenorrhea and Hypertension

    Directory of Open Access Journals (Sweden)

    Yasin Şimşek

    2014-12-01

    Full Text Available 17-alpha hydroxylase deficiency (17OHD is an uncommon cause of congenital adrenal hyperplasia (CAH resulting from mutation in the CYP17 gene. It is an autosomal recessive disease leading to the deficiency of enzyme activity which results in impaired synthesis of cortisol, androgen and sex steroids. A 35-year-old woman was admitted to the hospital with hypokalemia, weakness, and fatigue. Medical history revealed two attempts of in vitro fertilization (IVF without achieving pregnancy. Laboratory evaluation showed hypokalemia, hypocortisolemia, hypergonadotropic hypogonadism and increased levels of 11-deoxycorticosterone. Her karyotype was 46XX. The patient had no history of menarche. She had Tanner stage 2 breast development. Genotyping showed homozygous mutation located in exon 7 which abolishes both 17-alpha hydroxylase and 17,20 lyase activities of the CYP17A1 protein. After starting dexamethasone 0.5 mg/day, her potassium level was normalized, and blood pressure improved. In conclusion, a comprehensive evaluation should be performed before using assisted reproductive techniques, such as in vitro fertilization in hypertensive women with sexual infantilism, primary amenorrhea, and hypokalemia.

  13. Estrogen-2-hydroxylase in the brain of the male African catfish, Clarias gariepinus

    Energy Technology Data Exchange (ETDEWEB)

    Timmers, R.J.; Granneman, J.C.; Lambert, J.G.; van Oordt, P.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; 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.

  14. Mutation in the CYP21B gene (Ile-172. -->. Asn) causes steroid 21-hydroxylase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Amor, M.; Parker, K.L.; Globerman, H.; New, M.I.; White, P.C.

    1988-03-01

    Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia. It results from a deficiency in a specific cytochrome P450, P450c21 (P450XXIA). The gene encoding this protein (CYP21B) and a closely linked pseudogene (CYP21A) are located in the HLA complex on chromosome 6p. Many mutant alleles are associated with deletions of CYP21B; the authors report the cloning and characterization of a nondeleted mutant CYP21B gene. This mutant gene is expressed on transfection into mouse Y1 adrenal cells, producing mRNA levels similar to those seen after transfection of the normal CYP21B gene. In codon 172 of the mutant gene, the normal codon ATC, encoding isoleucine, has been changed to AAC, encoding asparagine. This mutation is normally present in the CYP21A pseudogene, so that it may have been transferred to the mutant CYP21B gene by gene conversion. Hybridization of oligonucleotide probes corresponding to this and two other mutations normally present in CYP21A demonstrated that 4 out of 20 patients carried the codon 172 mutation; in one of these patients, the mutation was present as part of a larger gene conversion involving at least exons 3-6. Gene conversion may be a frequent cause of 21-hydroxylase deficiency.

  15. Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

    Science.gov (United States)

    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.

  16. Tyrosine hydroxylase positive nerves and mast cells in the porcine gallbladder

    Directory of Open Access Journals (Sweden)

    I. Stefanov

    2017-03-01

    Full Text Available The aim of this study was to detect the localisation of tyrosine hydroxylase (TH positive nerve fibres (THN and distribution of tyrosine hydroxylase positive mast cells (THMC in the wall of porcine gallbladder. THN were observed as single fibres, nerve fibres forming perivascular plexuses and nerve fibres grouped within the nerve fascicles. In the gallbladder`s fundus, body and neck, the TH+ fibres formed mucosal, muscular and serosal nonganglionated nerve plexuses. Toluidine blue (TB staining was used to confirm that the TH positive cells were mast cells. The number of THMC in the propria of gallbladder`s fundus, body and neck was significantly higher than in the muscular and serosal layers in both genders. The number of mast cells in the musculature was higher than in the serosa. The density and location of the THMC were similar to the TB positive (with gamma meta-chromasia mast cells in both males and females, and statistically significant difference was not established. In conclusion, original data concerning the existence and localisation of catecholaminergic nerves and THMC distribution in the porcine gallbladder’s wall are presented. The results could con-tribute to the body of knowledge of functional communication between autonomic nerves and mast cells in the gallbladder.

  17. Tertiary alcohol preferred: Hydroxylation of trans-3-methyl-L-proline with proline hydroxylases

    Directory of Open Access Journals (Sweden)

    Christian Klein

    2011-12-01

    Full Text Available The enzymatic synthesis of tertiary alcohols by the stereospecific oxidation of tertiary alkyl centers is a most-straightforward but challenging approach, since these positions are sterically hindered. In contrast to P450-monooxygenases, there is little known about the potential of non-heme iron(II oxygenases to catalyze such reactions. We have studied the hydroxylation of trans-3-methyl-L-proline with the α-ketoglutarate (α-KG dependent oxygenases, cis-3-proline hydroxylase type II and cis-4-proline hydroxylase (cis-P3H_II and cis-P4H. With cis-P3H_II, the tertiary alcohol product (3R-3-hydroxy-3-methyl-L-proline was obtained exclusively but in reduced yield (~7% compared to the native substrate L-proline. For cis-P4H, a complete shift in regioselectivity from C-4 to C-3 was observed so that the same product as with cis-P3H_II was obtained. Moreover, the yields were at least as good as in control reactions with L-proline (~110% relative yield. This result demonstrates a remarkable potential of non-heme iron(II oxygenases to oxidize substrates selectively at sterically hindered positions.

  18. Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations.

    Science.gov (United States)

    Song, Minmin; Kim, Amy C; Gorzalski, Andrew J; MacLean, Marina; Young, Sharon; Ginzel, Matthew D; Blomquist, Gary J; Tittiger, Claus

    2013-04-01

    Ips pini bark beetles use myrcene hydroxylases to produce the aggregation pheromone component, ipsdienol, from myrcene. The enantiomeric ratio of pheromonal ipsdienol is an important prezygotic mating isolation mechanism of I. pini and differs among geographically distinct populations. We explored the substrate and product ranges of myrcene hydroxylases (CYP9T2 and CYP9T3) from reproductively-isolated western and eastern I. pini. The two cytochromes P450 share 94% amino acid identity. CYP9T2 mRNA levels were not induced in adults exposed to myrcene-saturated atmosphere. Functional assays of recombinant enzymes showed both hydroxylated myrcene, (+)- and (-)-α-pinene, 3-carene, and R-(+)-limonene, but not α-phellandrene, (-)-β-pinene, γ-terpinene, or terpinolene, with evidence that CYP9T2 strongly preferred myrcene over other substrates. They differed in the enantiomeric ratios of ipsdienol produced from myrcene, and in the products resulting from different α-pinene enantiomers. These data provide new information regarding bark beetle pheromone evolution and factors affecting cytochrome P450 structure-function relationships.

  19. Scallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-α.

    Science.gov (United States)

    Zhou, Zhi; Wang, Lingling; Wang, Mengqiang; Zhang, Huan; Wu, Tiantian; Qiu, Limei; Song, Linsheng

    2011-12-01

    Phenylalanine hydroxylase (PAH) is an important metabolic enzyme of aromatic amino acids, which is responsible for the irreversible oxidation of phenylalanine to tyrosine. In the present study, the full-length cDNA encoding PAH from Chlamys farreri (designated CfPAH) was cloned by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The open reading frame of CfPAH encoded a polypeptide of 460 amino acids, and its sequence shared 64.4-74.2% similarity with those of PAHs from other animals. There were an N-terminal regulatory ACT domain and a C-terminal catalytic Biopterin_H domain in the deduced CfPAH protein. The mRNA transcripts of CfPAH could be detected in all the tested tissues, including adductor muscle, mantle, gill, gonad, haemocytes and hepatopancreas. And its expression level in haemocytes was increased significantly during 3-48 h after bacteria Vibrio anguillarum challenge with the highest level (9.1-fold, P phenylalanine to tyrosine within 1 min (nmol min(-1) mg(-1) protein) in vitro. These results indicated collectively that CfPAH, as a homologue of phenylalanine hydroxylase in scallop C. farreri, could be induced by cytokine and involved in the immunomodulation of scallops by supplying the starting material tyrosine for the synthesis of melanin and catecholamines.

  20. Cloning, functional expression, and characterization of a chalcone 3-hydroxylase from Cosmos sulphureus.

    Science.gov (United States)

    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.

  1. Cosuppression of limonene-3-hydroxylase in peppermint promotes accumulation of limonene in the essential oil.

    Science.gov (United States)

    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.

  2. Cloning and Functional Characterization of the Maize (Zea mays L. Carotenoid Epsilon Hydroxylase Gene.

    Directory of Open Access Journals (Sweden)

    Shu Chang

    Full Text Available 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.

  3. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    Energy Technology Data Exchange (ETDEWEB)

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

    2006-01-01

    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  4. 40 CFR 90.316 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 90... Equipment Provisions § 90.316 Hydrocarbon analyzer calibration. (a) Calibrate the FID and HFID hydrocarbon... thereafter, adjust the FID and HFID hydrocarbon analyzer for optimum hydrocarbon response as specified...

  5. 40 CFR 86.121-90 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 18 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 86... Complete Heavy-Duty Vehicles; Test Procedures § 86.121-90 Hydrocarbon analyzer calibration. The hydrocarbon... FID and HFID hydrocarbon analyzers shall be adjusted for optimum hydrocarbon response....

  6. 40 CFR 91.316 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 91....316 Hydrocarbon analyzer calibration. (a) Calibrate the FID and HFID hydrocarbon analyzer as described... thereafter, adjust the FID and HFID hydrocarbon analyzer for optimum hydrocarbon response as specified...

  7. 40 CFR 89.319 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 89... Equipment Provisions § 89.319 Hydrocarbon analyzer calibration. (a) The FID hydrocarbon analyzer shall... and at least annually thereafter, adjust the FID hydrocarbon analyzer for optimum hydrocarbon...

  8. Allele-specific marker development and selection efficiencies for both flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes in soybean subgenus soja.

    Science.gov (United States)

    Guo, Yong; Qiu, Li-Juan

    2013-06-01

    Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3'-hydroxylase (F3'H; EC1.14.3.21) and flavonoid 3',5'-hydroxylase (F3'5'H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3'H and the W1 locus co-segregated with a gene encoding F3'5'H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3'H and F3'5'H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3'H and one of three alleles for GmF3'5'H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3'H and F3'5'H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3'H allele explained 92.2 % of the variation in tawny and two gmf3'h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3'5'H alleles and one gmF3'5'h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3'h-b and gmf3'5'h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.

  9. Isolation and characterization of the human tyrosine hydroxylase gene: identification of 5' alternative splice sites responsible for multiple mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    O' Malley, K.L.; Anhalt, M.J.; Martin, B.M.; Kelsoe, J.R.; Winfield, S.L.; Ginns, E.I.

    1987-11-03

    A full-length genomic clone for human tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.2) has been isolated. A human brain genomic library constructed in EMBL3 was screened by using a rat cDNA for tyrosine hydroxylase as a probe. Out of one million recombinant phage, one clone was identified that hybridized to both 5' and 3' rat cDNA probes. Restriction endonuclease mapping, Southern blotting, and sequence analysis revealed that, like its rodent counterpart, the human gene is single copy, contains 13 primary exons, and spans approximately 8 kilobases (kb). In contrast to the rat gene, human tyrosine hydroxylase undergoes alternative RNA processing within intron 1, generating at least three distinct mRNAs. A comparison of the human tyrosine hydroxylase and phenylalanine hydroxylase genes indicates that although both probably evolved from a common ancestral gene, major changes in the size of introns have occurred since their divergence.

  10. Detection of hydrocarbons in irradiated foods

    Energy Technology Data Exchange (ETDEWEB)

    Miyahara, Makoto; Maitani, Tamio [National Inst. of Health Sciences, Tokyo (Japan); Saito, Akiko; Kamimura, Tomomi; Nagasawa, Taeko [Kitasato Univ., Sagamihara, Kanagawa (Japan). School of Allied Health Sciences; Kobayashi, Yasuo; Ito, Hitoshi [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Establishment

    2003-06-01

    The hydrocarbon method for the detection of irradiated foods is now recognized as the international technique. This method is based on radiolysis of fatty acids in food to give hydrocarbons. In order to expand this technique's application, ten foods (butter, cheese, chicken, pork, beef, tuna, dry shrimp, avocado, papaya, and mango) were irradiated in the range from 0.5 to 10 kGy and the hydrocarbons in them were detected. Recoveries of the hydrocarbons from most foods were acceptable (38-128%). Some hydrocarbons were found in non-irradiated foods, particularly, in butter, cheese, tuna, and shrimp. Seven irradiated foods, butter, cheese, chicken, beef, pork, tuna, dry shrimp, and avocado were detectable at their practical doses by measuring the appropriate marker hydrocarbons. In most case, marker hydrocarbon will be 1,7-hexadecadiene. However, the marker hydrocarbons produced only in irradiated foods varied from food to food; therefore, it is necessary to check a specific irradiated food for marker hydrocarbons. On the other hand, two irradiated foods (papaya and mango which were irradiated at their practical doses) were difficult to distinguish from non-irradiated foods using this method. (author)

  11. Applied bioremediation of petroleum hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Hinchee, R.E.; Kittel, J.A. [eds.] [Battelle Memorial Inst., Columbus, OH (United States); Reisinger, H.J. [ed.] [Integrated Science and Technology, Inc., Marietta, GA (United States)

    1995-12-31

    This volume is part of a ten volume set of papers derived from the Third International In Situ and On-Site Bioreclamation Symposium which was held in San Diego, California, in April 1995. The purpose of the conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on bioremediation. The papers in this volume focus on petroleum hydrocarbon bioremediation, with an emphasis on pilot-scale and field-scale applications. Individual papers have been processed separately for inclusion in the appropriate data bases.

  12. 40 CFR 52.1877 - Control strategy: Photochemical oxidants (hydrocarbons).

    Science.gov (United States)

    2010-07-01

    ... oxidants (hydrocarbons). 52.1877 Section 52.1877 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....1877 Control strategy: Photochemical oxidants (hydrocarbons). (a) The requirements of Subpart G of this... national standard for photochemical oxidants (hydrocarbons) in the Metropolitan Cincinnati...

  13. Hydrocarbon degradation potential and plant growth-promoting activity of culturable endophytic bacteria of Lotus corniculatus and Oenothera biennis from a long-term polluted site.

    Science.gov (United States)

    Pawlik, Małgorzata; Cania, Barbara; Thijs, Sofie; Vangronsveld, Jaco; Piotrowska-Seget, Zofia

    2017-07-06

    Many endophytic bacteria exert beneficial effects on their host, but still little is known about the bacteria associated with plants growing in areas heavily polluted by hydrocarbons. The aim of the study was characterization of culturable hydrocarbon-degrading endophytic bacteria associated with Lotus corniculatus L. and Oenothera biennis L. collected in long-term petroleum hydrocarbon-polluted site using culture-dependent and molecular approaches. A total of 26 hydrocarbon-degrading endophytes from these plants were isolated. Phylogenetic analyses classified the isolates into the phyla Proteobacteria and Actinobacteria. The majority of strains belonged to the genera Rhizobium, Pseudomonas, Stenotrophomonas, and Rhodococcus. More than 90% of the isolates could grow on medium with diesel oil, approximately 20% could use n-hexadecane as a sole carbon and energy source. PCR analysis revealed that 40% of the isolates possessed the P450 gene encoding for cytochrome P450-type alkane hydroxylase (CYP153). In in vitro tests, all endophytic strains demonstrated a wide range of plant growth-promoting traits such as production of indole-3-acetic acid, hydrogen cyanide, siderophores, and phosphate solubilization. More than 40% of the bacteria carried the gene encoding for the 1-aminocyclopropane-1-carboxylic acid deaminase (acdS). Our study shows that the diversity of endophytic bacterial communities in tested plants was different. The results revealed also that the investigated plants were colonized by endophytic bacteria possessing plant growth-promoting features and a clear potential to degrade hydrocarbons. The properties of isolated endophytes indicate that they have the high potential to improve phytoremediation of petroleum hydrocarbon-polluted soils.

  14. Hydrocarbon conversion process and catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Hoek, A.; Huizinga, T.; Maxwell, I.E.

    1989-08-15

    This patent describes a process for hydrocracking hydrocarbon oils into products of lower average molecular weight and lower average boiling point. It comprises contacting hydrocarbon oil at a temperature between 250{sup 0}C and 500{sup 0}C and a pressure up to 300 bar in the presence of hydrogen with a catalyst consisting essentially of a Y zeolite modified to have a unit cell size below 24.40 A, a water adsorption capacity (at 25{sup 0}C and a rho/rho/sub o/ value of 0.2) of between 10% and 15% by weight of the zeolite and a pore volume of at least 0.25 ml/g wherein between 10% and 60% of the total pore volume is made up of pores having a diameter of at least 8 nm; am amorphous cracking component, a binder and at least one hydrogenation component selected from the group consisting of a Group VI metal, a Group VIII metal and mixtures thereof.

  15. Hydrocarbon degradation by antarctic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Cavanagh, J.A.E.; Nichols, P.D.; McMeekin, T.A.; Franzmann, P.D. [Univ. of Tasmania (Australia)] [and others

    1996-12-31

    Bacterial cultures obtained from sediment samples collected during a trial oil spill experiment conducted at Airport beach, Eastern Antarctica were selectively enriched for n-alkane-degrading and phenanthrenedegrading bacteria. Samples were collected from a control site and sites treated with different hydrocarbon mixtures - Special Antarctic blend (SAB), BP-Visco and orange roughy oils. One set of replicate sites was also treated with water from Organic Lake which had previously been shown to contain hydrocarbon-degrading bacteria. No viable bacteria were obtained from samples collected from sites treated with orange roughy oil. Extensive degradation of n-alkanes by enrichment cultures obtained from sites treated with SAB and BP-Visco occurred at both 25{degrees}C and 10{degrees}C. Extensive degradation of phenanthrene also occurred in enrichment cultures from these sites grown at 25{degrees}C. Concurrent increases of polar lipid in these cultures were also observed. The presence of 1,4-naphthaquinone and 1-naphthol during the growth of the cultures on phenanthrene is unusual and warrants further investigation of the mechanism of phenanthrene-degradation by these Antarctic bacteria.

  16. Cool Sooting Flames of Hydrocarbons

    Institute of Scientific and Technical Information of China (English)

    Z.A. MANSUROV

    2001-01-01

    This paper presents the study of polycyclic aromatic hydrocarbons (PAH) and paramagnetism of soot particles sampled from cool sooting flames of methane and propane in a separately-heated two-sectional reactor under atmospheric pressure at the reactor temperatures of 670-1170 K. The temperature profiles of the flames were studied. The sampling was carried out with a quartz sampler and the samples were frozen with liquid nitrogen. A number of polyaromatic hydrocarbons such as pyrene, fluoranthene, coronene, anthanthrene, 1,12-benzperylene,were identified by spectroscopic methods in the extract of soot. The processes of soot formation at methaneoxygen mixture combustion in the electric field with applied potential changed from 0 to 2,2 kV at different polarity of electrodes have been investigated. It has been stated that at the electrical field application, an increase in soot particle sizes and soot yield occurs; besides, at the application of the field, speeding up the positively charged particles, the interplanar distance decreases. On the basis of investigation of soot particles paramagnetism, it was shown that initially soot particles have high carcinogetic activity and pollute the environment owing to a rapid decrease of the number of these radical centers. The reduction of the radical concentration is connected with radical recombination on soot.

  17. Phenotypic variation in a Chinese family with 46,XY and 46,XX 17α-hydroxylase deficiency.

    Science.gov (United States)

    Bee, Yong Mong; Manju, Chandran; Papari-Zareei, Mahboubeh; Auchus, Richard J

    2012-04-01

    17α-hydroxylase deficiency is a rare autosomal recessive disorder characterized by sexual infantilism, amenorrhea, hypertension and hypokalemia, which is caused by mutations in the CYP17A1 gene. To date, more than 50 mutations in this gene have been described. The clinical features and biochemical data of a pair of 46,XY and 46,XX Chinese siblings with 17α-hydroxylase deficiency from Singapore were studied. Direct DNA sequence analysis of the CYP17A1 gene was performed. There was significant phenotypic variation between the siblings. The proband (46,XY) presented classically with sexual infantilism, amenorrhea and hypertension. The younger sibling (46,XX) also presented with amenorrhea, but she had breast development and absence of hypokalemic hypertension. The same compound heterozygous mutations in CYP17A1 gene were identified in both patients. A missense mutation (P409R) was detected in exon 7, and a 9-bp deletion (D487-S488-F489del) was detected in exon 8. We confirmed the diagnosis of 17α-hydroxylase deficiency in these two patients. Both P409R and D487-S488-F489del have been described previously and are widely propagated in the Chinese population in East and Southeast Asia. We propose that the phenotypic expression of affected individuals with 17α-hydroxylase deficiency is karyotype-dependent, with individuals having the 46,XX karyotype having less pronounced clinical symptoms.

  18. CRYSTAL-STRUCTURE OF THE REDUCED FORM OF P-HYDROXYBENZOATE HYDROXYLASE REFINED AT 2.3-A RESOLUTION

    NARCIS (Netherlands)

    SCHREUDER, HA; VANDERLAAN, JM; SWARTE, MBA; KALK, KH; HOL, WGJ; DRENTH, J

    1992-01-01

    The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzym

  19. 3-Ketosteroid 9 alpha-hydroxylase enzymes : Rieske non-heme monooxygenases essential for bacterial steroid degradation

    NARCIS (Netherlands)

    Petrusma, Mirjan; van der Geize, Robert; Dijkhuizen, Lubbert

    2014-01-01

    Various micro-organisms are able to use sterols/steroids as carbon- and energy sources for growth. 3-Ketosteroid 9 alpha-hydroxylase (KSH), a two component Rieske non-heme monooxygenase comprised of the oxygenase KshA and the reductase KshB, is a key-enzyme in bacterial steroid degradation. It initi

  20. Monomer formation and functioning of p hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide-water mixtures

    NARCIS (Netherlands)

    Kudryashova, E.V.; Visser, A.J.W.G.; Berkel, van W.J.H.

    2008-01-01

    It has previously been postulated that the dimeric form of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH) is important for catalysis. Here it is demonstrated that the monomeric form of PHBH is active. In a water/AOT/isooctane reverse micellar system, the function of the monomeric and dimeric

  1. CRYSTAL-STRUCTURE OF THE REDUCED FORM OF P-HYDROXYBENZOATE HYDROXYLASE REFINED AT 2.3-A RESOLUTION

    NARCIS (Netherlands)

    SCHREUDER, HA; VANDERLAAN, JM; SWARTE, MBA; KALK, KH; HOL, WGJ; DRENTH, J

    1992-01-01

    The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized

  2. Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms.

    Directory of Open Access Journals (Sweden)

    Nils Widderich

    Full Text Available 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

  3. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

    Directory of Open Access Journals (Sweden)

    Lin Yuheng

    2012-04-01

    Full Text Available Abstract Background Caffeic acid (3,4-dihydroxycinnamic acid is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. Results We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H, is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. Conclusion We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis

  4. Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms.

    Science.gov (United States)

    Widderich, Nils; Höppner, Astrid; 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

  5. Conversion of organic solids to hydrocarbons

    Science.gov (United States)

    Greenbaum, Elias

    1995-01-01

    A method of converting organic solids to liquid and gaseous hydrocarbons includes impregnating an organic solid with photosensitizing ions and exposing the impregnated solid to light in a non-oxidizing atmosphere for a time sufficient to photocatalytically reduce the solid to at least one of a liquid and a gaseous hydrocarbon.

  6. Versatility of hydrocarbon production in cyanobacteria.

    Science.gov (United States)

    Xie, Min; Wang, Weihua; Zhang, Weiwen; Chen, Lei; Lu, Xuefeng

    2017-02-01

    Cyanobacteria are photosynthetic microorganisms using solar energy, H2O, and CO2 as the primary inputs. Compared to plants and eukaryotic microalgae, cyanobacteria are easier to be genetically engineered and possess higher growth rate. Extensive genomic information and well-established genetic platform make cyanobacteria good candidates to build efficient biosynthetic pathways for biofuels and chemicals by genetic engineering. Hydrocarbons are a family of compounds consisting entirely of hydrogen and carbon. Structural diversity of the hydrocarbon family is enabled by variation in chain length, degree of saturation, and rearrangements of the carbon skeleton. The diversified hydrocarbons can be used as valuable chemicals in the field of food, fuels, pharmaceuticals, nutrition, and cosmetics. Hydrocarbon biosynthesis is ubiquitous in bacteria, yeasts, fungi, plants, and insects. A wide variety of pathways for the hydrocarbon biosynthesis have been identified in recent years. Cyanobacteria may be superior chassis for hydrocabon production in a photosynthetic manner. A diversity of hydrocarbons including ethylene, alkanes, alkenes, and terpenes can be produced by cyanobacteria. Metabolic engineering and synthetic biology strategies can be employed to improve hydrocarbon production in cyanobacteria. This review mainly summarizes versatility and perspectives of hydrocarbon production in cyanobacteria.

  7. Primary biodegradation of petroleum hydrocarbons in seawater

    Energy Technology Data Exchange (ETDEWEB)

    Comber, M.I.H.; Den Haan, K.H.; Djemel, N.; Eadsforth, C.V.; King, D.; Paumen, M.L.; Parkerton, T.; Dmytrasz, B.

    2012-12-15

    This report describes primary biodegradation experiments performed to determine the persistence of higher molecular weight petroleum hydrocarbons in seawater. Results from the biodegradation experiments show that the majority of tested petroleum hydrocarbons have half-lives in seawater less than 60 days.

  8. 南极低温降解菌Shewanella sp. NJ49羟化酶分离纯化及最适产酶条件研究%Separation and purification of hydroxylase from Antarctic psychrophilic bacteriumShewanella sp. NJ49 and optimum conditions for its enzyme production

    Institute of Scientific and Technical Information of China (English)

    刘芳明; 王以斌; 曲长凤; 梁强; 郑洲; 缪锦来; 肖天

    2016-01-01

    Hydroxylase is a key enzyme with respect to bacteria during hydrocarbon degradation. In this paper, we investigate the Antarctic psychrophilic bacteriumShewanella sp. NJ49. To obtain a single component of hydroxy-lase, we separated and purified hydroxylase from NJ49 in a series of steps, i.e., ammonium sulfate precipitation, ultrafiltration concentration, and column chromatography with Sephadex G-75 as the column media. In addition, we studied the relationships between the activity of NJ49 hydroxylase and environmental factors such as temperature, pH, surfactant, and salinity. We found that purified hydroxylase comprised subunitsα,β, andγ, with estimated molecular masses of 56, 45, and 36 kD, respectively. The optimum conditions for producing NJ49 hydroxylase were a temperature of 15℃, a pH of 7.2, and a salinity of 55‰. Different types of surfactants had different effects on enzyme activity. Although individual amphoteric, cationic, and nonionic surfactants did not inhibit enzyme activity, a mixture of anionic and nonionic surfactants, as well as a mixture of cationic and nonionic surfactants, improved the activity of the hydroxylase.%羟化酶是细菌降解烷烃的关键酶,本文以南极低温降解菌Shewanella sp. NJ49为研究对象,通过硫酸铵沉淀、超滤浓缩及葡聚糖凝胶Sephadex G-75柱层析方法,分离纯化NJ49的羟化酶,获得了单一羟化酶组分,研究了温度、pH、表面活性剂和盐度等环境因素对 NJ49羟化酶酶活活性影响。结果表明,羟化酶组分由α、β、γ三个亚基组成,分子质量分别为56,45和36 kD。NJ49最适产酶条件为:温度15℃、pH7.2、盐度55;不同类型表面活性剂对酶的活性影响效应不一,两性离子表面活性剂、阳离子表面活性剂、非离子表面活性剂对酶活没有作用,阴离子表面活性剂+非离子表面活性剂混和剂和阳离子表面活性剂+非离子表面活性剂混和剂能提高羟化酶的活性。

  9. Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Berod, A.; Biguet, N.F.; Dumas, S.; Bloch, B.; Mallet, J.

    1987-03-01

    cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as control probe. Using the tyrosine hydroxylase probe, the authors ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of /sup 35/S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.

  10. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

    Science.gov (United States)

    Chowdhury, Rasheduzzaman; Leung, Ivanhoe K. H.; Tian, Ya-Min; Abboud, Martine I.; Ge, Wei; Domene, Carmen; Cantrelle, François-Xavier; Landrieu, Isabelle; Hardy, Adam P.; Pugh, Christopher W.; Ratcliffe, Peter J.; Claridge, Timothy D. W.; Schofield, Christopher J.

    2016-08-01

    The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

  11. Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase.

    Science.gov (United States)

    Whittington, D A; Rosenzweig, A C; Frederick, C A; Lippard, S J

    2001-03-27

    To investigate the role of protein cavities in facilitating movement of the substrates, methane and dioxygen, in the soluble methane monooxygenase hydroxylase (MMOH), we determined the X-ray structures of MMOH from Methylococcus capsulatus (Bath) cocrystallized with dibromomethane or iodoethane, or by using crystals pressurized with xenon gas. The halogenated alkanes bind in two cavities within the alpha-subunit that extend from one surface of the protein to the buried dinuclear iron active site. Two additional binding sites were located in the beta-subunit. Pressurization of two crystal forms of MMOH with xenon resulted in the identification of six binding sites located exclusively in the alpha-subunit. These results indicate that hydrophobic species bind preferentially in preexisting cavities in MMOH and support the hypothesis that such cavities may play a functional role in sequestering and enhancing the availability of the physiological substrates for reaction at the active site.

  12. Dark rearing maintains tyrosine hydroxylase expression in retinal amacrine cells following optic nerve transection

    Institute of Scientific and Technical Information of China (English)

    Wei Wan; Zhenghai Liu; Xiaosheng Wang; Xuegang Luo

    2012-01-01

    The present study examined changes in retinal tyrosine hydroxylase (TH) expression in rats having undergone optic nerve transection and housed under a normal day/night cycle or in the dark. The aim was to investigate the effects of amacrine cells on axonal regeneration in retinal ganglion cells and on the synapses that transmit visual signals. The results revealed that retinal TH expression gradually decreased following optic nerve transection in rats housed under a normal day/night cycle, reaching a minimum at 5 days. In contrast, retinal TH expression decreased to a minimum at 1 day following optic nerve transection in dark reared rats, gradually increasing afterward and reaching a normal level at 5-7 days. The number of TH-positive synaptic particles correlated with the TH levels, indicating that dark rearing can help maintain TH expression during the synaptic degeneration stage (5-7 days after optic nerve injury) in retinal amacrine cells.

  13. Reproductive outcomes of female patients with congenital adrenal hyperplasia due to 21-hydroxylase defi ciency

    Directory of Open Access Journals (Sweden)

    Mouna Feki Mnif

    2013-01-01

    Full Text Available Fertility in women with congenital adrenal hyperplasia (CAH due to 21-hydroxylase deficiency (21-OHD appears to be reduced, especially in women with the classic salt-wasting type. Several factors have been suggested to contribute to this subfertility such as androgen excess, adrenal progesterone hypersecretion, consequences of genital reconstructive surgery, secondary polycystic ovaries syndrome, and psychosexual factors. In contrast to this subfertility, pregnancies are commonly normal and uneventful. Adequate glucocorticoid therapy and improvement of surgical and psychological management could contribute to optimize fertility in CAH female patients, even among women with the classic variant. This review provides current information regarding the reproductive outcomes of women with CAH due to 21-OHD and the fertility and pregnancy issues in this population.

  14. Human tyrosine hydroxylase (TH) genomic fragment (pHGTH4) identifies a PstI polymorphism

    Energy Technology Data Exchange (ETDEWEB)

    Kelsoe, J.R.; Stubblefield, B.K.; Ginns, E.I. (National Institute of Mental Health, Bethesda, MD (USA))

    1988-08-11

    pHGTH4 is a 2.3 kb Bam HI genomic fragment of tyrosine hydroxylase isolated from a lambda EMBL3 Sau3A partial digest human genomic library prepared from lymphoblasts. The fragment was subcloned into the Bam HI site of pBLUESCRIPT. The absence of a polymorphic Pst I site results in the 1.3 kb fragment, A1; whereas its presence results in a 0.7 kb and a 0.6 kb fragment, A2, as shown in the figure. It was located at 11p15 by in situ hybridization. Mendelian inheritance was demonstrated in a 23 member family with 12 children. Pst I was not polymorphic (all homoallelic for A2) in a panel of 6 subjects from Amish pedigree 110 (IMR 884), in which two other chromosome 11 RFLP's have been reported to be linked to manic-depressive illness.

  15. Overexpression, crystallization and preliminary X-ray crystallographic analysis of the ectoine hydroxylase from Sphingopyxis alaskensis.

    Science.gov (United States)

    Hoeppner, Astrid; Widderich, Nils; Bremer, Erhard; Smits, Sander H J

    2014-04-01

    The ectoine hydroxylase (EctD) is a member of the non-haem-containing iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily. Its mononuclear iron centre is a prerequisite for the activity of this enzyme and promotes the O2-dependent oxidative decarboxylation of 2-oxoglutarate, which is coupled to a two-electron oxidation of the substrate ectoine to yield 5-hydroxyectoine. An expression and purification protocol for the EctD enzyme from Sphingopyxis alaskensis was developed and the protein was crystallized using the sitting-drop vapour-diffusion method. This resulted in two different crystal forms, representing the apo and iron-bound forms of the enzyme.

  16. Chronic stress effects on working memory: association with prefrontal cortical tyrosine hydroxylase.

    Science.gov (United States)

    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.

  17. Recent Advances in Developing Inhibitors for Hypoxia-Inducible Factor Prolyl Hydroxylases and Their Therapeutic Implications

    Directory of Open Access Journals (Sweden)

    So Yeon Kim

    2015-11-01

    Full Text Available Hypoxia-inducible factor (HIF prolyl hydroxylases (PHDs are members of the 2-oxoglutarate dependent non-heme iron dioxygenases. Due to their physiological roles in regulation of HIF-1α stability, many efforts have been focused on searching for selective PHD inhibitors to control HIF-1α levels for therapeutic applications. In this review, we first describe the structure of PHD2 as a molecular basis for structure-based drug design (SBDD and various experimental methods developed for measuring PHD activity. We further discuss the current status of the development of PHD inhibitors enabled by combining SBDD approaches with high-throughput screening. Finally, we highlight the clinical implications of small molecule PHD inhibitors.

  18. An Exploratory Evaluation of Tyrosine Hydroxylase Inhibition in Planaria as a Model for Parkinsonism

    Directory of Open Access Journals (Sweden)

    David Prokai

    2013-11-01

    Full Text Available Planaria are the simplest organisms with bilateral symmetry and a central nervous system (CNS with cephalization; therefore, they could be useful as model organisms to investigate mechanistic aspects of parkinsonism and to screen potential therapeutic agents. Taking advantage of the organism’s anti-tropism towards light, we measured a significantly reduced locomotor velocity in planaria after exposure to 3-iodo-L-tyrosine, an inhibitor of tyrosine hydroxylase that is an enzyme catalyzing the first and rate-limiting step in the biosynthesis of catecholamines. A simple semi-automatic assay using videotaped experiments and subsequent evaluation by tracking software was also implemented to increase throughput. The dopaminergic regulation of locomotor velocity was confirmed by bromocriptine, a drug whose mechanisms of action to treat Parkinson’s disease is believed to be through the stimulation of nerves that control movement.

  19. Sequence variation at the phenylalanine hydroxylase gene in the British Isles

    Energy Technology Data Exchange (ETDEWEB)

    Tyfield, L.A. [Southmead Hospital, Bristol (United Kingdom)]|[Univ. of Bristol (United Kingdom); Stephenson, A. [Southmead Hospital, Bristol (United Kingdom); Cockburn, F. [Royal Hospital for Sick Children, Glasgow (United Kingdom)] [and others

    1997-02-01

    Using mutation and haplotype analysis, we have examined the phenylalanine hydroxylase gene in the phenylketonuria populations of four geographical areas of the British Isles: the west of Scotland, southern Wales, and southwestern and southeastern England. The enormous genetic diversity of this locus within the British Isles is demonstrated in the large number of different mutations characterized and in the variety of genetic backgrounds on which individual mutations are found. Allele frequencies of the more common mutations exhibited significant nonrandom distribution in a north/south differentiation. Differences between the west of Scotland and southwestern England may be related to different events in the recent and past histories of their respective populations. Similarities between southern Wales and southeastern England are likely to reflect the heterogeneity that is seen in and around two large capital cities. Finally, comparison with more recently colonized areas of the world corroborates the genealogical origin by range expansion of several mutations. 38 refs., 2 tabs.

  20. Towards the identification of the allosteric Phe-binding site in phenylalanine hydroxylase.

    Science.gov (United States)

    Carluccio, Carla; Fraternali, Franca; Salvatore, Francesco; Fornili, Arianna; Zagari, Adriana

    2016-01-01

    The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe-PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant.

  1. To cheat or not to cheat: Tryptophan hydroxylase 2 SNP variants contribute to dishonest behavior

    Directory of Open Access Journals (Sweden)

    Qiang eShen

    2016-05-01

    Full Text Available Although lying (bear false witness is explicitly prohibited in the Decalogue and a focus of interest in philosophy and theology, more recently the behavioral and neural mechanisms of deception are gaining increasing attention from diverse fields especially economics, psychology and neuroscience. Despite the considerable role of heredity in explaining individual differences in deceptive behavior, few studies have investigated which specific genes contribute to the heterogeneity of lying behavior across individuals. Also, little is known concerning which specific neurotransmitter pathways underlie deception. Towards addressing these two key questions, we implemented a neurogenetic strategy and modeled deception by an incentivized die-under-cup task in a laboratory setting. The results of this exploratory study provide provisional evidence that SNP variants across the tryptophan hydroxylase 2 (TPH2 gene, that encodes the rate-limiting enzyme in the biosynthesis of brain serotonin, contribute to individual differences in deceptive behavior.

  2. Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase.

    Science.gov (United States)

    Bursy, Jan; Pierik, Antonio J; Pica, Nathalie; Bremer, Erhard

    2007-10-26

    By using natural abundance (13)C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine- and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctD-related proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ectABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).

  3. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency - management in adults.

    Science.gov (United States)

    Ambroziak, Urszula; Bednarczuk, Tomasz; Ginalska-Malinowska, Maria; Małunowicz, Ewa Maria; Grzechocińska, Barbara; Kamiński, Paweł; Bablok, Leszek; Przedlacki, Jerzy; Bar-Andziak, Ewa

    2010-01-01

    Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive hereditary diseases. The impairment of cortisol synthesis leads to excessive stimulation of the adrenal glands by adrenocorticotropic hormone (ACTH), adrenal hyperplasia, and excessive androgen synthesis. The syndrome is characterised by a considerable correlation between the genotype and the phenotype with the type of CYP21A2 gene mutation affecting the severity of 21-hydroxylase deficiency. The clinical manifestations of CAH in adults result from adrenocortical and adrenomedullary insufficiency, hyperandrogenism, and the adverse effects of glucocorticosteroids used for the treatment of the condition. Non-classic CAH may sometimes be asymptomatic. In patients with classic CAH obesity, hyperinsulinaemia, insulin resistance, and hyperleptinaemia are more often seen than in the general population. These abnormalities promote the development of metabolic syndrome and its sequelae, including endothelial dysfunction, and cardiovascular disease. Long-term glucocorticosteroid treatment is also a known risk factor for osteoporosis. Patients with CAH require constant monitoring of biochemical parameters (17a-hydroxyprogesterone [17-OHP] and androstenedione), clinical parameters (body mass, waist circumference, blood pressure, glucose, and lipids), and bone mineral density by densitometry. The principal goal of treatment in adults with CAH is to improve quality of life, ensure that they remain fertile, reduce the manifestations of hyperandrogenisation in females, and minimise the adverse effects of glucocorticosteroid treatment. Patients with classic CAH require treatment with glucocorticosteroids and, in cases of salt wasting, also with a mineralocorticosteroid. Radical measures, such as bilateral adrenalectomy, are very rarely needed. Asymptomatic patients with non-classic CAH require monitoring: treatment is not always necessary. Medical care for patients

  4. [Congenital adrenal hyperplasia due to 21-hydroxylase deficiency--management in adults].

    Science.gov (United States)

    Ambroziak, Urszula; Bednarczuk, Tomasz; Ginalska-Malinowska, Maria; Małunowicz, Ewa Maria; Grzechocińska, Barbara; Kamiński, Paweł; Bablok, Leszek; Przedlacki, Jerzy; Bar-Andziak, Ewa

    2010-01-01

    Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive hereditary diseases. The impairment of cortisol synthesis leads to excessive stimulation of the adrenal glands by adrenocorticotropic hormone (ACTH), adrenal hyperplasia, and excessive androgen synthesis. The syndrome is characterised by a considerable correlation between the genotype and the phenotype with the type of CYP21A2 gene mutation affecting the severity of 21-hydroxylase deficiency. The clinical manifestations of CAH in adults result from adrenocortical and adrenomedullary insufficiency, hyperandrogenism, and the adverse effects of glucocorticosteroids used for the treatment of the condition. Non-classic CAH may sometimes be asymptomatic. In patients with classic CAH obesity, hyperinsulinaemia, insulin resistance, and hyperleptinaemia are more often seen than in the general population. These abnormalities promote the development of metabolic syndrome and its sequelae, including endothelial dysfunction, and cardiovascular disease. Long-term glucocorticosteroid treatment is also a known risk factor for osteoporosis. Patients with CAH require constant monitoring of biochemical parameters (17a-hydroxyprogesterone and androstenedione), clinical parameters (body mass, waist circumference, blood pressure, glucose, and lipids), and bone mineral density by densitometry. The principal goal of treatment in adults with CAH is to improve quality of life, ensure that they remain fertile, reduce the manifestations of hyperandrogenisation in females, and minimise the adverse effects of glucocorticosteroid treatment. Patients with classic CAH require treatment with glucocorticosteroids and, in cases of salt wasting, also with a mineralocorticosteroid. Radical measures, such as bilateral adrenalectomy, are very rarely needed. Asymptomatic patients with non-classic CAH require monitoring: treatment is not always necessary. Medical care for patients with CAH

  5. Method for producing diene hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Tsaylingol' d, A.L.; Abayev, G.N.; Mikhaylov, R.K.; Stepanov, G.A.; Troitskiy, A.P.

    1980-04-28

    A method is claimed for producing diene hydrocarbons by oxidational dehydration of paraffin or olefin hydrocarbons in a fluidized bed of a concentrate with circulation of the latter between the zones of the reaction of regeneration with the help of circulation stand pipes. To increase the efectiveness of the process, it is proposed to circulate the concentrate between the zones of reaction and regeneration, sequentially disposed in a common apparatus with a difference in the concentration of the concentrate in the circulation stand pipes disposed in the same apparatus and the zone of the reaction equal to 20-700 kg/m/sup 3/. For example, the process of oxidational dehydration of butane through the proposed system is conducted in an apparatus with a diameter of 1,000 mm, a circulation stand pipe diameter of 500 mm, a linear gas speed in the reaction zone of 0.6 m/s, and in the circulation stand pipe of 0.15 m/s. The concentration of the concentrate in the dehydration zone is 640 kg/m/sup 3/ and in the stand pipe, 970 kg/m/sup 3/. The volumetric ratio of the n-C/sub 4/H/sub 10/:air, air:vapor vapor in the form of a condensate is 1:7.2:4.5:5.5. The output of the butadiene is: in the passed butane, 32.9% and in the broken down butane, 52.5%. The butane conversion is 62.6%. The losses of the concentrate with the contact gas and with the regeneration gases is 1/3 as much for the supplied butane, than in a known method. The method makes it possible to reduce the air expenditure by 60%, to reduce the concentrate losses by 2-3 times and to simplify the industrial system.

  6. A modified microbial adhesion to hydrocarbons assay to account for the presence of hydrocarbon droplets.

    Science.gov (United States)

    Zoueki, Caroline Warne; Tufenkji, Nathalie; Ghoshal, Subhasis

    2010-04-15

    The microbial adhesion to hydrocarbons (MATH) assay has been used widely to characterize microbial cell hydrophobicity and/or the extent of cell adhesion to hydrophobic liquids. The classical MATH assay involves spectrophotometric absorbance measurements of the initial and final cell concentrations in an aqueous cell suspension that has been contacted with a hydrocarbon liquid. In this study, microscopic examination of the aqueous cell suspension after contact with hexadecane or a hexadecane/toluene mixture revealed the presence of hydrocarbon droplets. The hydrocarbon droplets contributed to the absorbance values during spectrophotometric measurements and caused erroneous estimates of cell concentrations and extents of microbial adhesion. A modified MATH assay that avoids such artefacts is proposed here. In this modified assay, microscopic examination of the aqueous suspension and direct cell counts provides cell concentrations that are free of interference from hydrocarbon droplets. The presence of hydrocarbon droplets was noted in MATH assays performed with three bacterial strains, and two different hydrocarbons, at ionic strengths of 0.2 mM and 20 mM and pH 6. In these experiments, the formation of quasi-stable hydrocarbon droplets cannot be attributed to the presence of biosurfactants, or stabilization by biocolloids. The presence of surface potential at the hydrocarbon-water interface that was characterized by electrophoretic mobility of up to -1 and -2 microm cm/Vs, likely caused the formation of the quasi-stable hydrocarbon droplets that provided erroneous results using the classical MATH assay.

  7. Hydrocarbon degradation abilities of psychrotolerant Bacillus strains

    Directory of Open Access Journals (Sweden)

    Fulya Kolsal

    2017-06-01

    Full Text Available Biodegradation requires identification of hydrocarbon degrading microbes and the investigation of psychrotolerant hydrocarbon degrading microbes is essential for successful biodegradation in cold seawater. In the present study, a total of 597 Bacillus isolates were screened to select psychrotolerant strains and 134 isolates were established as psychrotolerant on the basis of their ability to grow at 7 °C. Hydrocarbon degradation capacities of these 134 psychrotolerant isolate were initially investigated on agar medium containing different hydrocarbons (naphthalene, n-hexadecane, mineral oil and 47 positive isolates were grown in broth medium containing hydrocarbons at 20 °C under static culture. Bacterial growth was estimated in terms of viable cell count (cfu ml–1. Isolates showing the best growth in static culture were further grown in presence of crude oil under shaking culture and viable cell count was observed between 8.3 × 105–7.4 × 108 cfu ml–1. In the final step, polycyclic aromatic hydrocarbon (PAH (chrysene and naphthalene degradation yield of two most potent isolates was determined by GC-MS along with the measurement of pH, biomass and emulsification activities. Results showed that isolates Ege B.6.2i and Ege B.1.4Ka have shown 60% and 36% chrysene degradation yield, respectively, while 33% and 55% naphthalene degradation yield, respectively, with emulsification activities ranges between 33–50%. These isolates can be used to remove hydrocarbon contamination from different environments, particularly in cold regions.

  8. Waste Plastic Converting into Hydrocarbon Fuel Materials

    Energy Technology Data Exchange (ETDEWEB)

    Sarker, Moinuddin; Mamunor Rashid, Mohammad; Molla, Mohammad

    2010-09-15

    The increased demand and high prices for energy sources are driving efforts to convert organic compounds into useful hydrocarbon fuels. Although much of this work has focused on biomass, there are strong benefits to deriving fuels from waste plastic material. Natural State Research Inc. (NSR) has invented a simple and economically viable process to decompose the hydrocarbon polymers of waste plastic into the shorter chain hydrocarbon of liquid fuel (patent pending). The method and principle of the production / process will be discussed. Initial tests with several widely used polymers indicate a high potential for commercialization.

  9. Using supercritical fluids to refine hydrocarbons

    Science.gov (United States)

    Yarbro, Stephen Lee

    2015-06-09

    A system and method for reactively refining hydrocarbons, such as heavy oils with API gravities of less than 20 degrees and bitumen-like hydrocarbons with viscosities greater than 1000 cp at standard temperature and pressure, using a selected fluid at supercritical conditions. A reaction portion of the system and method delivers lightweight, volatile hydrocarbons to an associated contacting unit which operates in mixed subcritical/supercritical or supercritical modes. Using thermal diffusion, multiphase contact, or a momentum generating pressure gradient, the contacting unit separates the reaction products into portions that are viable for use or sale without further conventional refining and hydro-processing techniques.

  10. Emulsification of hydrocarbons by subsurface bacteria

    Science.gov (United States)

    Francy, D.S.; Thomas, J.M.; Raymond, R.L.; Ward, C.H.

    1991-01-01

    Biosurfactants have potential for use in enhancement of in situ biorestoration by increasing the bioavailability of contaminants. Microorganisms isolated from biostimulated, contaminated and uncontaminated zones at the site of an aviation fuel spill and hydrocarbon-degrading microorganisms isolated from sites contaminated with unleaded gasoline were examined for their abilities to emulsify petroleum hydrocarbons. Emulsifying ability was quantified by a method involving agitation and visual inspection. Biostimulated-zone microbes and hydrocarbon-degrading microorganisms were the best emulsifiers as compared to contaminated and uncontaminated zone microbes. Biostimulation (nutrient and oxygen addition) may have been the dominant factor which selected for and encouraged growth of emulsifiers; exposure to hydrocarbon was also important. Biostimulated microorganisms were better emulsifiers of aviation fuel (the contaminant hydrocarbon) than of heavier hydrocarbon to which they were not previously exposed. By measuring surface tension changes of culture broths, 11 out of 41 emulsifiers tested were identified as possible biosurfactant producers and two isolates produced large surface tension reductions indicating the high probability of biosurfactant production.Biosurfactants have potential for use in enhancement of in situ biorestoration by increasing the bioavailability of contaminants. Microorganisms isolated from biostimulated, contaminated and uncontaminated zones at the site of an aviation fuel spill and hydrocarbon-degrading microorganisms isolated from sites contaminated with unleaded gasoline were examined for their abilities to emulsify petroleum hydrocarbons. Emulsifying ability was quantified by a method involving agitation and visual inspection. Biostimulated-zone microbes and hydrocarbon-degrading microorganisms were the best emulsifiers as compared to contaminated and uncontaminated zone microbes. Biostimulation (nutrient and oxygen addition) may have been

  11. Controlled oxidation of aliphatic CH bonds in metallo-monooxygenases: mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons.

    Science.gov (United States)

    Chen, Yao-Sheng; Luo, Wen-I; Yang, Chung-Ling; Tu, Yi-Jung; Chang, Chun-Wei; Chiang, Chih-Hsiang; Chang, Chi-Yao; Chan, Sunney I; Yu, Steve S-F

    2014-05-01

    The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions.

  12. In situ detection of alkB2 gene involved in Alcanivorax borkumensis SK2(T) hydrocarbon biodegradation.

    Science.gov (United States)

    Matturro, Bruna; Frascadore, Emanuela; Cappello, Simone; Genovese, Mariella; Rossetti, Simona

    2016-09-15

    This study aimed to develop a new assay based on the whole cell hybridization in order to monitor alkane hydroxylase genes (alkB system) of the marine bacterium Alcanivorax borkumensis SK2(T) commonly reported as the predominant microorganism responsible for the biodegradation of n-alkanes which are the major fraction of petroleum hydrocarbons. The assay based on the whole cell hybridization targeting alkB2 gene was successfully developed and calibrated on a pure culture of Alcanivorax borkumensis SK2(T) with a detection efficiency up to 80%. The approach was further successfully validated on hydrocarbon-contaminated seawater and provided cells abundance (6.74E+04alkB2-carryingcellsmL(-1)) higher of about one order of magnitude than those obtained by qPCR (4.96E+03alkB2genecopiesmL(-1)). This study highlights the validity of the assay for the detection at single cell level of key-functional genes involved in the biodegradation of n-alkanes.

  13. 40 CFR 86.317-79 - Hydrocarbon analyzer specifications.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 18 2010-07-01 2010-07-01 false Hydrocarbon analyzer specifications....317-79 Hydrocarbon analyzer specifications. (a) Hydrocarbon measurements are to be made with a heated... measures hydrocarbon emissions on a dry basis is permitted for gasoline-fueled testing; Provided,...

  14. 40 CFR 92.119 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 92... Hydrocarbon analyzer calibration. The HFID hydrocarbon analyzer shall receive the following initial and... into service and at least annually thereafter, the HFID hydrocarbon analyzer shall be adjusted...

  15. 40 CFR 86.1321-94 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 19 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 86... Procedures § 86.1321-94 Hydrocarbon analyzer calibration. The FID hydrocarbon analyzer shall receive the... into service and at least annually thereafter, the FID hydrocarbon analyzer shall be adjusted...

  16. 21 CFR 178.3530 - Isoparaffinic petroleum hydrocarbons, synthetic.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Isoparaffinic petroleum hydrocarbons, synthetic... hydrocarbons, synthetic. Isoparaffinic petroleum hydrocarbons, synthetic, may be safely used in the production... isoparaffinic petroleum hydrocarbons, produced by synthesis from petroleum gases consist of a mixture of...

  17. 21 CFR 172.882 - Synthetic isoparaffinic petroleum hydrocarbons.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Synthetic isoparaffinic petroleum hydrocarbons. 172... hydrocarbons. Synthetic isoparaffinic petroleum hydrocarbons may be safely used in food, in accordance with the... liquid hydrocarbons meeting the following specifications: Boiling point 93-260 °C as determined by...

  18. Absence of exercise-induced leptin suppression associated with insufficient epinephrine reserve in patients with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    NARCIS (Netherlands)

    Riepe, F.G.; Krone, N.; Kruger, S.N.; Sweep, C.G.J.; Lenders, J.W.M.; Dotsch, J.; Monig, H.; Sippell, W.G.; Partsch, C.J.

    2006-01-01

    OBJECTIVE: Patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency suffer from glucocorticoid and mineralocorticoid deficiency. They have insufficient epinephrine reserves and increased basal leptin levels and are often insulin resistant. In healthy subjects, an inhibitor

  19. Informative document halogenated hydrocarbon-containing waste

    NARCIS (Netherlands)

    Verhagen H

    1992-01-01

    This "Informative document halogenated hydrocarbon-containing waste" forms part of a series of "Informative documents waste materials". These documents are conducted by RIVM on the instructions of the Directorate General for the Environment, Waste Materials Directorate, in behal

  20. Quantitative Hydrocarbon Energies from the PMO Method.

    Science.gov (United States)

    Cooper, Charles F.

    1979-01-01

    Details a procedure for accurately calculating the quantum mechanical energies of hydrocarbons using the perturbational molecular orbital (PMO) method, which does not require the use of a computer. (BT)

  1. Compost bioremediation of hydrocarbon-contaminated soil ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

    May 16, 2008 ... Total petroleum hydrocarbons (TPH) was reduced by 17% in the .... Identification of bacterial isolates was done by biochemical tests. Atagana 1517 ..... control the prolonged thermophilic period in two-phase olive oil mill.

  2. Infrared Spectra of Polycyclic Aromatic Hydrocarbons (PAHs)

    Science.gov (United States)

    Bauschlicher, Charles W., Jr.; Bakes, E. L. O.

    2000-01-01

    We have computed the synthetic infrared spectra of some polycyclic aromatic hydrocarbons containing up to 54 carbon atoms. The species studied include ovalene, circumcoronene, dicoronylene, and hexabenzocoronene. We report spectra for anions, neutrals, cations, and multiply charged cations.

  3. Molecular characterization of autochthonous hydrocarbon utilizing ...

    African Journals Online (AJOL)

    Hydrocarbon utilizing bacteria in water, soil and sediment samples collected from ... of the genomic DNA extracted from each bacterial isolate was amplified with ... that16S rRNA-gene-based techniques be used when studying the bacterial ...

  4. Gravity observations for hydrocarbon reservoir monitoring

    NARCIS (Netherlands)

    Glegola, M.A.

    2013-01-01

    In this thesis the added value of gravity observations for hydrocarbon reservoir monitoring and characterization is investigated. Reservoir processes and reservoir types most suitable for gravimetric monitoring are identified. Major noise sources affecting time-lapse gravimetry are analyzed. The

  5. Determination of some polycyclic aromatic hydrocarbons (PAHs ...

    African Journals Online (AJOL)

    cinthia

    2013-08-01

    Aug 1, 2013 ... In this study, polycyclic aromatic hydrocarbons (PAHs) associated with airborne particulate ... compounds from the heavily industrialized Vaal Triangle region. ... benzene ring as cluster, linear or angular (Maliszewska-.

  6. Biodegradation of polycyclic hydrocarbons by Phanerochaete chrysosporium

    Science.gov (United States)

    The ability of the white rot fungus Phanerochaete chrysosporium to degrade polycyclic aromatic hydrocarbons (PAHs) that are present in anthracene oil (a distillation product obtained from coal tar) was demonstrated. Analysis by capillary gas chromatography and high-performance li...

  7. Volatilisation of aromatic hydrocarbons from soil

    DEFF Research Database (Denmark)

    Lindhardt, B.; Christensen, T.H.

    1996-01-01

    The non-steady-state fluxes of aromatic hydrocarbons were measured in the laboratory from the surface of soils contaminated with coal tar Four soil samples from a former gasworks site were used for the experiments. The fluxes were quantified for 11 selected compounds, 4 mono- and 7 polycyclic...... aromatic hydrocarbons, for a period of up to 8 or 16 days. The concentrations of the selected compounds in the soils were between 0.2 and 3,100 mu g/g. The study included the experimental determination of the distribution coefficient of the aromatic hydrocarbons between the sorbed phase and the water under...... saturated conditions. The determined distribution coefficients showed that the aromatic hydrocarbons were more strongly sorbed to the total organic carbon including the coal tar pitch - by a factor of 8 to 25 - than expected for natural organic matter. The fluxes were also estimated using an analytical...

  8. Biodegradation of polycyclic hydrocarbons by Phanerochaete chrysosporium

    Science.gov (United States)

    The ability of the white rot fungus Phanerochaete chrysosporium to degrade polycyclic aromatic hydrocarbons (PAHs) that are present in anthracene oil (a distillation product obtained from coal tar) was demonstrated. Analysis by capillary gas chromatography and high-performance li...

  9. Macroporous polymer foams by hydrocarbon templating

    OpenAIRE

    Shastri, Venkatram Prasad; Martin, Ivan; Langer, Robert

    2000-01-01

    Porous polymeric media (polymer foams) are utilized in a wide range of applications, such as thermal and mechanical insulators, solid supports for catalysis, and medical devices. A process for the production of polymer foams has been developed. This process, which is applicable to a wide range of polymers, uses a hydrocarbon particulate phase as a template for the precipitation of the polymer phase and subsequent pore formation. The use of a hydrocarbon template allows for enhanced control ov...

  10. Zeolitic catalytic conversion of alochols to hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Narula, Chaitanya K.; Davison, Brian H.; Keller, Martin

    2017-01-03

    A method for converting an alcohol to a hydrocarbon, the method comprising contacting said alcohol with a metal-loaded zeolite catalyst at a temperature of at least 100.degree. C. and up to 550.degree. C., wherein said alcohol can be produced by a fermentation process, said metal is a positively-charged metal ion, and said metal-loaded zeolite catalyst is catalytically active for converting said alcohol to said hydrocarbon.

  11. Pyrolysis of hydrocarbons from lignite semicoking tar

    Energy Technology Data Exchange (ETDEWEB)

    Platonov, V.V.; Ryl' tsova, S.V.; Proskuryakov, V.A.; Rozental, D.A.; Polovetskaya, O.S.

    2000-07-01

    Pyrolysis of hydrocarbons from lignite semicoking tar in the range 750-900{degree}C at a contact time within 0.5-6.0 s was studied. The yields of pyrocarbons, pyrolysis gas, and liquid products and the group and component compositions of the liquid and gaseous products were determined. The optimal pyrolysis parameters from the viewpoint of obtaining the maximal yield of particular 'secondary' hydrocarbons were recommended.

  12. Nitrocarburising in ammonia-hydrocarbon gas mixtures

    DEFF Research Database (Denmark)

    Pedersen, Hanne; Christiansen, Thomas; Somers, Marcel A. J.

    2010-01-01

    The present work investigates the possibility of nitrocarburising in ammonia-acetylene-hydrogen and ammoniapropene- hydrogen gas mixtures, where unsaturated hydrocarbon gas is the carbon source during nitrocarburising. Consequently, nitrocarburising is carried out in a reducing atmosphere...... microscopy and X-ray diffraction analysis. It is shown that the use of unsaturated hydrocarbon gas in nitrocarburising processes is a viable alternative to traditional nitrocarburising methods....

  13. Nitrocarburizing in ammonia-hydrocarbon gas mixtures

    DEFF Research Database (Denmark)

    Pedersen, Hanne; Christiansen, Thomas; Somers, Marcel A. J.

    2011-01-01

    The present work investigates the possibility of nitrocarburising in ammonia-acetylene-hydrogen and ammonia-propene-hydrogen gas mixtures, where unsaturated hydrocarbon gas is the carbon source during nitrocarburising. Consequently, nitrocarburising is carried out in a reducing atmosphere...... microscopy and X-ray diffraction analysis. It is shown that the use of unsaturated hydrocarbon gas in nitrocarburising processes is a viable alternative to traditional nitrocarburising methods....

  14. Formation of hydrocarbons by bacteria and algae

    Energy Technology Data Exchange (ETDEWEB)

    Tornabene, T.G.

    1980-12-01

    A literature review has been performed summarizing studies on hydrocarbon synthesis by microorganisms. Certain algal and bacterial species produce hydrocarbons in large quantities, 70 to 80% of dry cell mass, when in a controlled environment. The nutritional requirements of these organisms are simple: CO/sub 2/ and mineral salts. The studies were initiated to determine whether or not microorganisms played a role in petroleum formation. 90 references. (DMC)

  15. Electrokinetic Remediation of Petroleum Hydrocarbons Spiked Soils

    OpenAIRE

    , M. Bilgin; , G. Akıncı

    2011-01-01

    In the presented study, remediation studies were conducted to determine the effectiveness of electrokinetic method on the treatment of natural soil contaminated with petroleum hydrocarbons, in laboratory scale reactors. Electokinetic remediation of agricultural soil with an initial TPHs (Total Petroleum Hydrocarbons) concentration of 10000 ppm was investigated under 20 V or 40 V direct current by using NaOH and acetic acid as electrolyte solution, treatment efficiencies were observed accordin...

  16. The future of oil and hydrocarbon man

    CERN Document Server

    Campbell, Colin

    1999-01-01

    Man appeared on the planet about four million years ago, and by 1850 numbered about one billion Ten came Hydrocarbon man. World population has since increased six-fold. After the oil price shocks of the 1970s, people asked "when will production peak?". It is not easy to answer this question because of the very poor database. Reserves and the many different hydrocarbon categories are poorly defined, reporting practices are ambiguous, revisions are not backdated...

  17. Application of real-time PCR, DGGE fingerprinting, and culture-based method to evaluate the effectiveness of intrinsic bioremediation on the control of petroleum-hydrocarbon plume.

    Science.gov (United States)

    Kao, Chih-Ming; Chen, Colin S; Tsa, Fu-Yu; Yang, Kai-Hsing; Chien, Chih-Ching; Liang, Shih-Hsiung; Yang, Chin-an; Chen, Ssu Ching

    2010-06-15

    Real-time polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and the culture-based method were applied in the intrinsic bioremediation study at a petroleum-hydrocarbon contaminated site. The genes of phenol hydroxylase (PHE), ring-hydroxylating toluene monooxygenase (RMO), naphthalene dioxygenase (NAH), toluene monooxygenase (TOL), toluene dioxygenase (TOD), and biphenyl dioxygenase (BPH4) were quantified by real-time PCR. Results show that PHE gene was detected in groundwater contaminated with benzene, toluene, ethylbenzene, xylene isomers (BTEX) and methyl tert-butyl ether (MTBE), and this indicates that intrinsic bioremediation occurred at this contaminated site. Results from DGGE analyses reveal that the petroleum-hydrocarbon plume caused the variation in microbial communities. In this study, MTBE degraders including Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01, and Enterobacter sp. NKNU02 were isolated from the contaminated groundwater using the cultured-based method. Results from MTBE biodegradation experiment show that the isolated bacteria were affected by propane. This indicates that propane may influence the metabolic pathway of MTBE by these bacteria. Knowledge and comprehension obtained from this study will be helpful in evaluating the occurrence and effectiveness of intrinsic bioremediation on the remediation of petroleum-hydrocarbon contaminated groundwater.

  18. Hypoxia-inducible factor prolyl 4-hydroxylases regulating erythropoiesis, and hypoxia-inducible lysyl oxidase regulating skeletal muscle development during embryogenesis

    OpenAIRE

    Laitala, A. (Anu)

    2014-01-01

    Abstract Erythropoiesis is the process of red blood cell production. The main regulator is the erythropoietin (EPO) hormone, which is strongly upregulated in low oxygen concentration (hypoxia) in cells via the hypoxia-inducible transcription factor HIF. The stability of HIF is regulated in an oxygen-dependent manner by three HIF prolyl 4-hydroxylases, all of which are known to participate in the regulation of erythropoiesis. A role in erythropoiesis of a fourth prolyl 4-hydroxylase, P4H-TM...

  19. Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

    Science.gov (United States)

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

  20. Regulation of phenylalanine hydroxylase: conformational changes upon phenylalanine binding detected by hydrogen/deuterium exchange and mass spectrometry.

    Science.gov (United States)

    Li, Jun; Dangott, Lawrence J; Fitzpatrick, Paul F

    2010-04-20

    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.

  1. Hydrocarbon degradation by Antarctic coastal bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Cavanagh, J.E. [University of Tasmania, Hobart (Australia). Antarctic Cooperative Research Centre; CSIRO Div of Marine Research, Hobart (Australia); University of Tasmania, Hobart (Australia). Dept. of Agricultural Science; Nichols, P.D. [University of Tasmania, Hobart (Australia). Antarctic Cooperative Research Centre; CSIRO Div. of Marine Research, Hobart (Australia); Franzmann, P.D. [CSIRO Land and Water, Wembley (Australia); McMeekin, T.A. [University of Tasmania, Hobart (Australia). Antarctic Cooperative Research Centre

    1999-07-01

    Bacterial cultures obtained through selective enrichment of beach sand collected 60 days and one year after treatment of sites in a pilot oil spill trial conducted at Airport Beach, Vestfold Hills, East Antarctica, were examined for the ability to degrade n-alkanes and phenanthrene. The effects of different hydrocarbon mixtures (Special Antarctic Blend [SAB] and BP-Visco), (fish oil [orange roughy]) and inoculation of replicate sites with water from Organic Lake, (previously shown to contain hydrocarbon-degrading bacteria) on the indigenous microbial population, were examined. Of the cultures obtained, those from sites treated with SAB and BP-Visco degraded n-alkanes most consistently and typically to the greatest extent. Two mixed cultures obtained from samples collected at 60 days and two isolates obtained from these cultures extensively degraded phenanthrene. 1-Hydroxy-naphthoic acid formed the major phenanthrene metabolite. Lower levels of salicyclic acid, 1-naphthol, 1,4-naphthaquinone and phenanthrene 9-10 dihydrodiol were detected in extracts of phenanthrene grown cultures. This study shows that under laboratory conditions indigenous Antarctica bacteria can degrade n-alkanes and the more recalcitrant polycyclic aromatic hydrocarbon, phenanthrene. The enrichment of hydrocarbon degrading microorganisms in Antarctic ecosystems exposed to hydrocarbons, is relevant for the long term fate of hydrocarbon spills in this environment. (author)

  2. Deuterated polycyclic aromatic hydrocarbons: Revisited

    CERN Document Server

    Doney, Kirstin D; Mori, Tamami; Onaka, Takashi; Tielens, A G G M

    2016-01-01

    The amount of deuterium locked up in polycyclic aromatic hydrocarbons (PAHs) has to date been an uncertain value. We present a near-infrared (NIR) spectroscopic survey of HII regions in the Milky Way, Large Magellanic Cloud (LMC), and Small Magellanic Cloud (SMC) obtained with AKARI, which aims to search for features indicative of deuterated PAHs (PAD or Dn-PAH) to better constrain the D/H ratio of PAHs. Fifty-three HII regions were observed in the NIR (2.5-5 {\\mu}m), using the Infrared Camera (IRC) on board the AKARI satellite. Through comparison of the observed spectra with a theoretical model of deuterated PAH vibrational modes, the aromatic and (a)symmetric aliphatic C-D stretch modes were identified. We see emission features between 4.4-4.8 {\\mu}m, which could be unambiguously attributed to deuterated PAHs in only six of the observed sources, all of which are located in the Milky Way. In all cases, the aromatic C-D stretching feature is weaker than the aliphatic C-D stretching feature, and, in the case o...

  3. Birds and polycyclic aromatic hydrocarbons

    Science.gov (United States)

    Albers, P.H.

    2006-01-01

    Polycyclic aromatic hydrocarbons (PAH) are present throughout the global environment and are produced naturally and by activities of humans. Effects of PAH on birds have been determined by studies employing egg injection, egg immersion, egg shell application, single and multiple oral doses, subcutaneous injection, and chemical analysis of field-collected eggs and tissue. The four-to six-ring aromatic compounds are the most toxic to embryos, young birds, and adult birds. For embryos, effects include death, developmental abnormalities, and a variety of cellular and biochemical responses. For adult and young birds, effects include reduced egg production and hatching, increased clutch or brood abandonment, reduced growth, increased organweights, and a variety of biochemical responses. Trophic level accumulation is unlikely. Environmental exposure to PAH in areas of high human population or habitats affected by recent petroleum spills might be sufficient to adversely affect reproduction. Evidence of long-term effects of elevated concentrations of environmental PAH on bird populations is very limited and the mechanisms of effect are unclear.

  4. Microbial biodegradation of polyaromatic hydrocarbons.

    Science.gov (United States)

    Peng, Ri-He; Xiong, Ai-Sheng; Xue, Yong; Fu, Xiao-Yan; Gao, Feng; Zhao, Wei; Tian, Yong-Sheng; Yao, Quan-Hong

    2008-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are widespread in various ecosystems and are pollutants of great concern due to their potential toxicity, mutagenicity and carcinogenicity. Because of their hydrophobic nature, most PAHs bind to particulates in soil and sediments, rendering them less available for biological uptake. Microbial degradation represents the major mechanism responsible for the ecological recovery of PAH-contaminated sites. The goal of this review is to provide an outline of the current knowledge of microbial PAH catabolism. In the past decade, the genetic regulation of the pathway involved in naphthalene degradation by different gram-negative and gram-positive bacteria was studied in great detail. Based on both genomic and proteomic data, a deeper understanding of some high-molecular-weight PAH degradation pathways in bacteria was provided. The ability of nonligninolytic and ligninolytic fungi to transform or metabolize PAH pollutants has received considerable attention, and the biochemical principles underlying the degradation of PAHs were examined. In addition, this review summarizes the information known about the biochemical processes that determine the fate of the individual components of PAH mixtures in polluted ecosystems. A deeper understanding of the microorganism-mediated mechanisms of catalysis of PAHs will facilitate the development of new methods to enhance the bioremediation of PAH-contaminated sites.

  5. Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear.

    Science.gov (United States)

    Gumucio, J P; Flood, M D; Bedi, A; Kramer, H F; Russell, A J; Mendias, C L

    2017-01-01

    Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred.Cite this article: J. P. Gumucio, M. D. Flood, A. Bedi, H. F. Kramer, A. J

  6. 25-Hydroxyvitamin D3 1-Alpha-Hydroxylase-Dependent Stimulation of Renal Klotho Expression by Spironolactone

    Directory of Open Access Journals (Sweden)

    Ioana Alesutan

    2013-11-01

    Full Text Available Background: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1 by FGF23. Conversely, 1,25(OH2D3 stimulates, by activating the vitamin D3 receptor (Vdr, the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. Methods: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293 or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR or of mineralocorticoid receptor (NR3C2. Results: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours and mice (8 hours or 5 days. Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours. Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. Conclusion: Besides blocking the effects of

  7. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, Frances H.

    2012-02-27

    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  8. Mutation R96W in cytochrome P450c17 gene causes combined 17{alpha}-hydroxylase/17-20-lyase deficiency in two french canadian patients

    Energy Technology Data Exchange (ETDEWEB)

    LaFlamme, N.; Leblanc, J.F.; Mailloux, J. [Laval Univ., Quebec (Canada)

    1996-01-01

    Congenital adrenal hyperplasia (CAH) is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to CAH caused by 21{alpha}-hydroxylase and 11{beta}-hydroxylase deficiencies, which impairs steroid formation in the adrenal exclusively, 17{alpha}-hydroxylase/17,20-lyase deficiency impairs steroid biosynthesis in the adrenals and gonads. The sequence of CYP17 gene was determined by direct sequencing of asymmetric PCR products in two French-Canadian 46,XY pseudohermaphrodite siblings suffering from combined 17{alpha}-hydroxylase/17,20-lyase deficiency. The two patients are homozygous for the novel missense mutation R96W caused by a C to T transition converting codon Arg{sup 96} (CGG) into a Trp (TGG) in exon 1. Both parents are heterozygous for this missense mutation. We assessed the effect of the R96W mutation on 17{alpha}-hydroxylase/17,20-lyase activity by analysis of mutant enzyme, generated by site-directed mutagenesis, expressed in COS-1 cells. The presence of R96W substitution almost completely abolished the activity of the mutant protein. The present findings provide a molecular explanation for the signs and symptoms of combined 17 {alpha}-hydroxylase/17,20-lyase deficiency in these two patients and provide useful information on the structure-activity relationships of the P450c17 enzyme. 31 refs., 4 figs., 1 tab.

  9. Pharmacogenetics of glucocorticoid replacement could optimize the treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Ricardo P. P. Moreira

    2011-01-01

    Full Text Available INTRODUCTION: 21-hydroxylase deficiency is an autosomal recessive disorder that causes glucocorticoid deficiency and increased androgen production. Treatment is based on glucocorticoid replacement; however, interindividual variability in the glucocorticoid dose required to achieve adequate hormonal control has been observed. OBJECTIVE: The present study aimed to evaluate the association between polymorphic variants involved inglucocorticoid action and/or metabolism and the mean daily glucocorticoid dose in 21-hydroxylase deficiency patients. METHODS: We evaluated 53 patients with classical forms of 21-hydroxylase deficiency who were receiving cortisone acetate. All patients were between four and six years of age and had normal androgen levels. RESULTS: The P450 oxidoreductase A503V, HSD11B1 rs12086634, and CYP3A7*1C variants were found in 19%, 11.3% and 3.8% of the patients, respectively. The mean ± SD glucocorticoid dose in patients with the CYP3A7*1C and wild-type alleles was 13.9 ± 0.8 and 19.5 ± 3.2 mg/m²/d, respectively. We did not identify an association between the P450 oxidoreductase or HSD11B1 allelic variants and the mean glucocorticoid dose. CONCLUSION: Patients carrying the CYP3A7*1C variant required a significantly lower mean glucocorticoid dose. Indeed, the CYP3A7*1C allele accounted for 20% of the variability in the cortisone acetate dose. The analysis of genes involved in glucocorticoid metabolism may be useful in the optimization of treatment of 21-hydroxylase deficiency.

  10. Increased cholesterol 7α-hydroxylase expression and size of the bile acid pool in the lactating rat

    Science.gov (United States)

    Wooton-Kee, Clavia Ruth; Cohen, David E.; Vore, Mary

    2008-01-01

    Maximal bile acid secretory rates and expression of bile acid transporters in liver and ileum are increased in lactation, possibly to facilitate increased enterohepatic recirculation of bile acids. We determined changes in the size and composition of the bile acid pool and key enzymes of the bile acid synthetic pathway [cholesterol 7α-hydroxylase (Cyp7a1), sterol 27-hydroxylase (Cyp27a1), and sterol 12α-hydroxylase (Cyp8b1)] in lactating rats relative to female virgin controls. The bile acid pool increased 1.9 to 2.5-fold [postpartum (PP) days 10, 14, and 19–23], compared with controls. A 1.5-fold increase in cholic acids and a 14 to 20% decrease in muricholic acids in lactation significantly increased the hydrophobicity index. In contrast, the hepatic concentration of bile acids and small heterodimer partner mRNA were unchanged in lactation. A 2.8-fold increase in Cyp7a1 mRNA expression at 16 h (10 h of light) demonstrated a shift in the diurnal rhythm at day 10 PP; Cyp7a1 protein expression and cholesterol 7α-hydroxylase activity were significantly increased at this time and remained elevated at day 14 PP but decreased to control levels by day 21 PP. There was an overall decrease in Cyp27a1 mRNA expression and a 20% decrease in Cyp27a1 protein expression, but there was no change in Cyp8b1 mRNA or protein expression at day 10 PP. The increase in Cyp7a1 expression PP provides a mechanism for the increase in the bile acid pool. PMID:18292185

  11. Restricted expression of Neuroglobin in the mouse retina and co-localization with Melanopsin and Tyrosine Hydroxylase

    Energy Technology Data Exchange (ETDEWEB)

    Hundahl, C.A., E-mail: c.hundahl@gmail.com [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark); Centre of Excellence for Translational Medicine, University of Tartu, Tartu (Estonia); Department of Physiology, University of Tartu, Tartu (Estonia); Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen (Denmark); Fahrenkrug, J. [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark); Luuk, H. [Centre of Excellence for Translational Medicine, University of Tartu, Tartu (Estonia); Department of Physiology, University of Tartu, Tartu (Estonia); Hay-Schmidt, A. [Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen (Denmark); Hannibal, J. [Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen (Denmark)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Restricted Neuroglobin expression in the mouse retina. Black-Right-Pointing-Pointer Antibody validation using Neuroglobin-null mice. Black-Right-Pointing-Pointer Co-expression of Neuroglobin with Melanopsin and tyrosine hydroxylase. Black-Right-Pointing-Pointer No effect of Neuroglobin deficiency on neuronal survival. -- Abstract: Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.

  12. Lignification in transgenics deficient in 4-coumarate 3-hydroxylase (C3H)or the associated hydroxycinnamoyl transferase (HCT)

    Science.gov (United States)

    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...

  13. Improvement of semen quality in an infertile man with 21-hydroxylase deficiency, suppressed serum gonadotropins and testicular adrenal rest tumours

    DEFF Research Database (Denmark)

    Mouritsen, Annette; Juul, Anders; Jørgensen, Niels

    2010-01-01

    Here, we report improvement of semen quality in a 30-year-old man with congenital adrenal hyperplasia (CAH) because of 21-hydroxylase deficiency, bilateral testicular adrenal rest tumours (TART) and a 1.5-year infertility history. His adrenal substitution therapy was changed from hydrocortisone 1...... for the presence of TART and disturbed reproductive hormones levels, leading to impaired semen quality. Optimizing the medical treatment may at least in some cases improve fecundity....

  14. Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations

    Science.gov (United States)

    Menabò, Soara; Polat, Seher; Baldazzi, Lilia; Kulle, Alexandra E; Holterhus, Paul-Martin; Grötzinger, Joachim; Fanelli, Flaminia; Balsamo, Antonio; Riepe, Felix G

    2014-01-01

    Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling. PMID:24022297

  15. Evidence for a high-spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase.

    Science.gov (United States)

    Panay, Aram Joel; Lee, Michael; Krebs, Carsten; Bollinger, J Martin; Fitzpatrick, Paul F

    2011-03-22

    Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-(57)Fe(II)-phenylalanine-6-methyltetrahydropterin complex with O(2) reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mössbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔE(Q)| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)═O intermediate with a rate constant of 19 mM(-1) s(-1), the Fe(IV)═O intermediate hydroxylates phenylalanine with a rate constant of 42 s(-1), and rate-limiting product release occurs with a rate constant of 6 s(-1) at 5 °C.

  16. Nucleotide sequence conservation of novel and established cis-regulatory sites within the tyrosine hydroxylase gene promoter.

    Science.gov (United States)

    Wang, Meng; Banerjee, Kasturi; Baker, Harriet; Cave, John W

    2015-02-01

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis and its gene proximal promoter ( electromobility shift assays showed that brain-region specific complexes assemble on these motifs. These studies also identified a non-canonical CRE binding (CREB) protein recognition element in the proximal promoter. Together, these studies provide a detailed analysis of evolutionary conservation within the TH promoter and identify potential cis-regulatory motifs that underlie a core set of regulatory mechanisms in mammals.

  17. On the release of catecholamines and dopamine-beta-hydroxylase evoked by ouabain in the perfused cat adrenal gland.

    OpenAIRE

    Garcia, A. G.; Hernandez, M.; Horga, J. F.; Sanchez-Garcia, P.

    1980-01-01

    1 Secretion of catecholamines (CA) and dopamine-beta-hydroxylase (DBH) activity from the retrogradely perfused cat adrenal gland was studied following ouabain infusion. Perfusion with ouabain (10(-4) M) for 10 min caused a gradual release of CA in the effluent which reached its peak 30 min after the ouabain pulse, and was maintained constant for at least 1 h. The effect of ouabain seemed to be irreversible. 2 Mecamylamine, while blocking the CA secretory effects of acetylcholine (ACh) perfusi...

  18. Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum

    OpenAIRE

    Ibáñez-Sandoval, Osvaldo; Tecuapetla, Fatuel; Unal, Bengi; Shah, Fulva; Koós, Tibor; Tepper, James M.

    2010-01-01

    Whole-cell recordings were obtained from tyrosine hydroxylase-expressing (TH+) neurons in striatal slices from bacterial artificial chromosome transgenic mice that synthesize enhanced green fluorescent protein (EGFP) selectively in neurons expressing TH transcriptional regulatory sequences. Stereological cell counting indicated that there were ~2700 EGFP–TH+ neurons/striatum. Whole-cell recordings in striatal slices demonstrated that EGFP–TH+ neurons comprise four electrophysiologically disti...

  19. Targeted delivery of the hydroxylase inhibitor DMOG provides enhanced efficacy with reduced systemic exposure in a murine model of colitis.

    Science.gov (United States)

    Tambuwala, Murtaza M; Manresa, Mario C; Cummins, Eoin P; Aversa, Vincenzo; Coulter, Ivan S; Taylor, Cormac T

    2015-11-10

    Targeting hypoxia-sensitive pathways has recently been proposed as a new therapeutic approach to the treatment of intestinal inflammation. HIF-hydroxylases are enzymes which confer hypoxic-sensitivity upon the hypoxia-inducible factor (HIF), a major regulator of the adaptive response to hypoxia. Previous studies have shown that systemic (intraperitoneal) administration of hydroxylase inhibitors such as dimethyloxalylglycine (DMOG) is profoundly protective in multiple models of colitis, however the therapeutic potential of this approach is limited due to potential side-effects associated with systemic drug exposure and the fact that orally delivered DMOG is ineffective (likely due to drug inactivation by gastric acid). In order to overcome these issues, we formulated DMOG in a liquid emulsion drug delivery system which, when coated with specific polymer coatings, permits oral delivery of a reduced dose which is released locally throughout the colon. This colon-targeted DMOG formulation demonstrated increased relative colonic bioactivity with reduced systemic exposure and provided a similar degree of protection to systemic (intraperitoneal) administration at a 40-fold lower dose in DSS-induced colitis. In summary, targeted delivery of DMOG to the colon provides local protection resulting in enhanced efficacy with reduced systemic exposure in the treatment of colitis. This novel approach to targeting hydroxylase inhibitors to specific diseased regions of the GI tract may improve it's potential as a new therapeutic in inflammatory bowel diseases such as ulcerative colitis.

  20. miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga.

    Science.gov (United States)

    De Lella Ezcurra, Ana Laura; Bertolin, Agustina Paola; Kim, Kevin; Katz, Maximiliano Javier; Gándara, Lautaro; Misra, Tvisha; Luschnig, Stefan; Perrimon, Norbert; Melani, Mariana; Wappner, Pablo

    2016-05-01

    Cellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses.

  1. A new process for obtaining hydroxytyrosol using transformed Escherichia coli whole cells with phenol hydroxylase gene from Geobacillus thermoglucosidasius.

    Science.gov (United States)

    Orenes-Piñero, Esteban; García-Carmona, Francisco; Sánchez-Ferrer, Alvaro

    2013-08-15

    Phenol hydroxylase gene cloning from the thermophilic bacteria Geobacillus thermoglucosidasius was used to develop an effective method to convert tyrosol into the high-added-value compound hydroxytyrosol by hydroxylation. Phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2 genes and strictly dependent on NADH and FAD. These two genes were subcloned together as a 2 kb fragment into Escherichia coli Rosetta cells, and the transformants were able to grow and effectively transform up to 5 mM of phenol and tyrosol using IPTG (isopropyl-β-D-thiogalactopyranoside) as inducer. In addition, when a new fragment with a 340 pb upstream pheA1 gene was subcloned, a similar biotransformation rate was attained without IPTG, confirming that this fragment encodes for a phenol hydroxylase promoter that can be recognised by E. coli. Both transformants brought about the total bioconversion of monophenols at a high concentration (5 mM), which represents an increase, both in concentration and in yield, compared with that previously described in the bibliography. The use of the transformant with its constitutive promoter was more interesting from a biotechnological point of view, since it is not necessary to use IPTG. It also gave rise to greater operational stability.

  2. Point mutation of Arg440 to his in cytochrome P450c17 causes severe 17{alpha}-hydroxylase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Fardella, C.E.; Hum, D.W.; Miller, W.L. [Univ. of California, San Francisco, CA (United States); Homoki, J. [Univ. of Ulm (Germany)

    1994-07-01

    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-1 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.

  3. Effect of Water Miscible Organic Solvents on p-Nitrophenol Hydroxylase (CYP2E1) Activity in Rat Liver Microsomes.

    Science.gov (United States)

    Patil, Pranali G; Kamble, S H; Shah, T S; Iyer, K R

    2015-01-01

    Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400) on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration). Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated) reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.

  4. Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?

    Directory of Open Access Journals (Sweden)

    Gudiña Eduardo

    2008-02-01

    Full Text Available Abstract Astaxanthin is a xanthophyll of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from β-carotene by the sequential action of two enzymes: a β-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3' positions of each of the two β-ionone rings of β-carotene, and a β-carotene ketolase that introduces keto groups at carbons 4 and 4' of the β-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of β-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementation studies of X. dendrorhous mutants and expression analysis in Mucor circinelloides reveals that the CrtS enzyme is a β-carotene hydroxylase of the P-450 monooxygenase family that converts β-carotene to the hydroxylated derivatives β-cryptoxanthin and zeaxanthin, but it does not form astaxanthin or the ketolated intermediates in this fungus. A bifunctional β-carotene hydroxylase-ketolase activity has been proposed for the CrtS protein. The evidence for and against this hypothesis is analyzed in detail in this review.

  5. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction

    Energy Technology Data Exchange (ETDEWEB)

    Day, D.J.; Barany, F.; Speiser, P.W. [Cornell Univ. Medical College, New York, NY (United States)] [and others

    1995-09-01

    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 the 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.

  6. Dimerization of human lysyl hydroxylase 3 (LH3) is mediated by the amino acids 541-547.

    Science.gov (United States)

    Heikkinen, Jari; Risteli, Maija; Lampela, Outi; Alavesa, Paula; Karppinen, Marjo; Juffer, André H; Myllylä, Raili

    2011-01-01

    Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collagen and collagen-like proteins, function as dimers. However, the amino acids responsible for dimerization and the role of dimer formation in the enzymatic activities of LH have not yet been identified. We have localized the region responsible for the dimerization of lysyl hydroxylase 3 (LH3), a multifunctional enzyme of collagen biosynthesis, to a sequence of amino acids between the glycosyltransferase activity and the lysyl hydroxylase activity domains. This area is covered by amino acids 541-547 in human LH3, but contains no cysteine residues. The region is highly conserved among LH isoforms, and is also involved in the dimerization of LH1 subunits. Dimerization is required for the LH activity of LH3, whereas it is not obligatory for the glycosyltransferase activities. In order to determine whether complex formation can occur between LH molecules originating from different species, and between different LH isoforms, double expressions were generated in a baculovirus system. Heterocomplex formation between mouse and human LH3, between human LH1 and LH3 and between human LH2 and LH3 was detected by western blot analyses. However, due to the low amount of complexes formed, the in vivo function of heterocomplexes remains unclear. Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  7. Effect of water miscible organic solvents on p-nitrophenol hydroxylase (CYP2E1 activity in rat liver microsomes

    Directory of Open Access Journals (Sweden)

    Pranali G Patil

    2015-01-01

    Full Text Available Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400 on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration. Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.

  8. Allopregnanolone reinstates tyrosine hydroxylase immunoreactive neurons and motor performance in an MPTP-lesioned mouse model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Samuel O Adeosun

    Full Text Available Restorative/protective therapies to restore dopamine neurons in the substantia nigra pars compacta (SNpc are greatly needed to effectively change the debilitating course of Parkinson's disease. In this study, we tested the therapeutic potential of a neurogenic neurosteroid, allopregnanolone, in the restoration of the components of the nigrostriatal pathway in MPTP-lesioned mice by measuring striatal dopamine levels, total and tyrosine hydroxylase immunoreactive neuron numbers and BrdU-positive cells in the SNpc. An acute treatment (once/week for two weeks with allopregnanolone restored the number of tyrosine hydroxylase-positive and total cell numbers in the SNpc of MPTP-lesioned mice, even though this did not increase striatal dopamine. It was also noted that MPTP treated mice to which allopregnanolone was administered had an increase in BrdU-positive cells in the SNpc. The effects of allopregnanolone in MPTP-lesioned mice were more apparent in mice that underwent behavioral tests. Interestingly, mice treated with allopregnanolone after MPTP lesion were able to perform at levels similar to that of non-lesioned control mice in a rotarod test. These data demonstrate that allopregnanolone promotes the restoration of tyrosine hydroxylase immunoreactive neurons and total cells in the nigrostriatal tract, improves the motor performance in MPTP-treated mice, and may serve as a therapeutic strategy for Parkinson's disease.

  9. Affinity labeling of the active site and the reactive sulfhydryl associated with activation of rat liver phenylalanine hydroxylase

    Energy Technology Data Exchange (ETDEWEB)

    Gibbs, B.S.; Benkovic, S.J. (Pennsylvania State Univ., University Park (United States))

    1991-07-09

    A pterin analogue, 5-((3-azido-6-nitrobenzylidene)amino)-2,6-diamino-4-pyrimidinone (AN-BADP), was synthesized as a probe of the pterin binding site of phenylalanine hydroxylase. The photoaffinity label has been found to be a competitive inhibitor of the enzyme with respect to 6,7-dimethyltetrahydropterin, having a K{sub i} of 8.8{plus minus}1.1 {mu}M. The irreversible labeling of phenylalanine hydroxylase by the photoaffinity label upon irradiation is both concentration and time dependent. phenylalanine hydroxylase is covalently labeled with a stoichiometry of 0.87{plus minus}0.08 mol of label/enzyme subunit. 5-Deaza-6-methyltetrahydropterin protects against inactivation and both 5-deaza-6-methyltetrahydropterin and 6-methyltetrahydropterin protect against covalent labeling, indicating that labeling occurs at the pterin binding site. Three tryptic peptides were isolated from ({sup 3}H)ANBADP-photolabeled enzyme and sequenced. All peptides indicated the sequence Thr-Leu-Lys-Ala-Leu-Tyr-Lys (residues 192-198). The residues labeled with ({sup 3}H)ANBADP were Lys198 and Lys194, with the majority of the radioactivity being associated with Lys198. Tryptic and chymotryptic peptides were isolated from fluorescein-labeled enzyme and sequenced. The modified residue was identified as Cys236.

  10. Further studies on the substrate spectrum of phytanoyl-CoA hydroxylase: implications for Refsum disease?

    Science.gov (United States)

    Foulon, Veerle; Asselberghs, Stanny; Geens, Wendy; Mannaerts, Guy P; Casteels, Minne; Van Veldhoven, Paul P

    2003-12-01

    Refsum disease is a peroxisomal disorder characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy, ataxia, and an accumulation of phytanic acid in plasma and tissues. Approximately 45% of cases are caused by mutations in phytanoyl-CoA hydroxylase (PAHX), the enzyme catalyzing the second step in the peroxisomal alpha-oxidation of 3-methyl-branched fatty acids. To study the substrate specificity of human PAHX, different 3-alkyl-branched substrates were synthesized and incubated with a recombinant polyhistidine-tagged protein. The enzyme showed activity not only toward racemic phytanoyl-CoA and the isomers of 3-methylhexadecanoyl-CoA, but also toward a variety of other mono-branched 3-methylacyl-CoA esters with a chain length down to seven carbon atoms. Furthermore, PAHX hydroxylated a 3-ethylacyl-CoA quite well, whereas a 3-propylacyl-CoA was a poor substrate. Hydroxylation of neither 2- or 4-methyl-branched acyl-CoA esters, nor long or very long straight-chain acyl-CoA esters could be detected. The results presented in this paper show that the substrate specificity of PAHX, with regard to the length of both the acyl-chain and the branch at position 3, is broader than expected. Hence, Refsum disease might be characterized by an accumulation of not only phytanic acid but also other 3-alkyl-branched fatty acids.

  11. Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene.

    Science.gov (United States)

    Jansen, G A; Ofman, R; Ferdinandusse, S; Ijlst, L; Muijsers, A O; Skjeldal, O H; Stokke, O; Jakobs, C; Besley, G T; Wraith, J E; Wanders, R J

    1997-10-01

    Refsum disease is an autosomal-recessively inherited disorder characterized clinically by a tetrad of abnormalities: retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia and elevated protein levels in the cerebrospinal fluid (CSF) without an increase in the number of cells in the CSF. All patients exhibit accumulation of an unusual branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), in blood and tissues. Biochemically, the disease is caused by the deficiency of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal protein catalyzing the first step in the alpha-oxidation of phytanic acid. We have purified PhyH from rat-liver peroxisomes and determined the N-terminal amino-acid sequence, as well as an additional internal amino-acid sequence obtained after Lys-C digestion of the purified protein. A search of the EST database with these partial amino-acid sequences led to the identification of the full-length human cDNA sequence encoding PhyH: the open reading frame encodes a 41.2-kD protein of 338 amino acids, which contains a cleavable peroxisomal targeting signal type 2 (PTS2). Sequence analysis of PHYH fibroblast cDNA from five patients with Refsum disease revealed distinct mutations, including a one-nucleotide deletion, a 111-nucleotide deletion and a point mutation. This analysis confirms our finding that Refsum disease is caused by a deficiency of PhyH.

  12. Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Ruegger, M.; Meyer, K.; Cusumano, J.C.; Chapple, C. [Purdue Univ., West Lafayette, IN (United States). Dept. of Biochemistry

    1999-01-01

    Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapolymalate, and sinapolycholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here the authors show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated that F5H expression in leaves is dependent on sequences 3{prime} of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required for F5H transcript or sinapolycholine accumulation in embryos.

  13. Cognitive outcome in adult women affected by congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    Science.gov (United States)

    Malouf, Matthew A; Migeon, Claude J; Carson, Kathryn A; Petrucci, Loredana; Wisniewski, Amy B

    2006-01-01

    Some research suggests that girls with congenital adrenal hyperplasia (CAH), who are exposed to higher than normal levels of prenatal androgens, perform better on spatial tasks, worse on verbal tasks and have a greater incidence of left-handedness than unaffected controls, all of which suggests the development of a more male-typical cognitive pattern. However, research in all three areas has produced inconsistent findings. To determine if prenatal androgen exposure has an organizing effect on female cognitive development and to what extent. 24 women, 21-71 years, with either the salt-losing (SL) or simple virilizing (SV) forms of CAH due to 21-hydroxylase deficiency, and 18 controls, 21-73 years, who were unaffected female relatives or women with polycystic ovary syndrome, were assessed with IQ, handedness, executive function, verbal learning and memory, non-verbal learning and memory, perceptual speed, visuospatial processing and visuomotor ability measures. The battery included tests known to elicit sex differences and control measures. The findings did not support the hypothesis that women with CAH develop a more male-typical cognitive pattern. This study differs from others in the older age of its participants, grouping by SL/SV diagnosis and assessment of medical treatment and compliance as determined through hormone assays. Our findings provide additional support for the conclusion that, in adult women with CAH, previous prenatal androgen exposure does not enhance spatial abilities, impair verbal abilities nor alter hand preferences in a long-lasting way.

  14. Effects of disulfiram and dopamine beta-hydroxylase knockout on cocaine-induced seizures

    Science.gov (United States)

    Gaval-Cruz, Meriem; Schroeder, Jason P.; Liles, L. Cameron; Javors, Martin A.; Weinshenker, David

    2008-01-01

    The antialcoholism drug disulfiram has shown recent promise as a pharmacotherapy for treating cocaine dependence, probably via inhibition of dopamine β-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE). We previously showed that DBH knockout (Dbh -/-) mice, which lack NE, are susceptible to seizures and are hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, suggesting that disulfiram might exacerbate cocaine-induced seizures (CIS) by inhibiting DBH. To test this, we examined CIS in wild-type and Dbh -/- mice following administration of disulfiram or the selective DBH inhibitor nepicastat. We found that Dbh genotype had no effect on CIS probability or frequency, whereas disulfiram, but not nepicastat, increased the probability of having CIS in both wild-type and Dbh -/- mice. Both disulfiram and nepicastat increased CIS frequency in wild-type but not Dbh -/- mice. There were no genotype or treatment effects on serum cocaine levels, except for an increase in disulfiram-treated Dbh -/- mice at the highest dose of cocaine. These results suggest that disulfiram enhances CIS via two distinct mechanisms: it both increases CIS frequency by inhibiting DBH and increases CIS frequency in a DBH-independent manner. PMID:18329701

  15. Pharmacogenetic Randomized Trial for Cocaine Abuse: Disulfiram and dopamine β-hydroxylase

    Science.gov (United States)

    Kosten, Thomas R.; Wu, Grace; Huang, Wen; Harding, Mark J.; Hamon, Sara C.; Lappalainen, Jaakko; Nielsen, David A.

    2012-01-01

    Background Disulfiram has been an effective cocaine addiction pharmacotherapy, and one of its possible mechanisms of efficacy is through copper chelation and inhibition of an enzyme involved in catecholamine metabolism, dopamine β-hydroxylase (DβH), which converts dopamine to norepinephrine. A variant in the gene encoding DβH leads to reduced DβH activity and as such, disulfiram may not be an effective treatment of cocaine dependence for individuals with this variant. This study explored that potential matching. Methods Seventy-four cocaine and opioid co-dependent (DSM-V) subjects were stabilized on methadone for two weeks and subsequently randomized into disulfiram (250 mg/day, N =34) and placebo groups (N =40) for 10 weeks. We genotyped the DBH gene polymorphism, −1021C/T (rs1611115), that reduces DβH enzyme levels and evaluated its role for increasing cocaine free urines with disulfiram. Results Using repeated measures analysis of variance, corrected for population structure, disulfiram pharmacotherapy reduced cocaine positive urines from 80% to 62% (p = .0001), and this disulfiram efficacy differed by DBH genotype group. Patients with the normal DβH level genotype dropped from 84% to 56% on disulfiram (p = .0001), while those with the low DBH level genotype showed no disulfiram effect. Conclusions This study indicates that a patient’s DBH genotype could be used to identify a subset of individuals for which disulfiram treatment may be an effective pharmacotherapy for cocaine dependence. PMID:22906516

  16. Targeting Oxygen-Sensing Prolyl Hydroxylase for Metformin-Associated Lactic Acidosis Treatment.

    Science.gov (United States)

    Oyaizu-Toramaru, Tomoko; Suhara, Tomohiro; Hayakawa, Noriyo; Nakamura, Takashi; Kubo, Akiko; Minamishima, Shizuka; Yamaguchi, Kyoji; Hishiki, Takako; Morisaki, Hiroshi; Suematsu, Makoto; Minamishima, Yoji Andrew

    2017-08-15

    Metformin is one of the most widely used therapeutics for type 2 diabetes mellitus and also has anticancer and antiaging properties. However, it is known to induce metformin-associated lactic acidosis (MALA), a severe medical condition with poor prognosis, especially in individuals with renal dysfunction. Inhibition of prolyl hydroxylase (PHD) is known to activate the transcription factor hypoxia-inducible factor (HIF) that increases lactate efflux as a result of enhanced glycolysis, but it also enhances gluconeogenesis from lactate in the liver that contributes to reducing circulating lactate levels. Here, we investigated the outcome of pharmaceutical inhibition of PHD in mice with MALA induced through the administration of metformin per os and an intraperitoneal injection of lactic acid. We found that the PHD inhibitors significantly increased the expression levels of genes involved in gluconeogenesis in the liver and the kidney and significantly improved the survival of mice with MALA. Furthermore, the PHD inhibitor also improved the rate of survival of MALA induced in mice with chronic kidney disease (CKD). Thus, PHD represents a new therapeutic target for MALA, which is a critical complication of metformin therapy. Copyright © 2017 American Society for Microbiology.

  17. Effect of Tryptophan Hydroxylase-2 rs7305115 SNP on suicide attempts risk in major depression

    Directory of Open Access Journals (Sweden)

    Zhang Yuqi

    2010-08-01

    Full Text Available Abstract Background Suicide and major depressive disorders (MDD are strongly associated, and genetic factors are responsible for at least part of the variability in suicide risk. We investigated whether variation at the tryptophan hydroxylase-2 (TPH2 gene rs7305115 SNP may predispose to suicide attempts in MDD. Methods We genotyped TPH2 gene rs7305115 SNP in 215 MDD patients with suicide and matched MDD patients without suicide. Differences in behavioral and personality traits according to genotypic variation were investigated by logistic regression analysis. Results There were no significant differences between MDD patients with suicide and controls in genotypic (AG and GG frequencies for rs7305115 SNP, but the distribution of AA genotype differed significantly (14.4% vs. 29.3%, p p p Conclusions The study suggested that hopelessness, negative life events and family history of suicide were risk factors of attempted suicide in MDD while the TPH2 rs7305115A remained a significant protective predictor of suicide attempts.

  18. Molecular docking of bacosides with tryptophan hydroxylase: a model to understand the bacosides mechanism.

    Science.gov (United States)

    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.

  19. Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea.

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    Bramwell G Lambrus

    Full Text Available Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP, or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.

  20. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

    Science.gov (United States)

    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.

  1. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

    Directory of Open Access Journals (Sweden)

    Zhenyi Liu

    Full Text Available The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH-Cre and dopamine active transporter (DAT or Slc6a3-Cre, by using a combination of immunohistochemistry (IHC and mRNA fluorescence in situ hybridization (FISH as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease.

  2. Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death.

    Science.gov (United States)

    Tennant, D A; Frezza, C; MacKenzie, E D; Nguyen, Q D; Zheng, L; Selak, M A; Roberts, D L; Dive, C; Watson, D G; Aboagye, E O; Gottlieb, E

    2009-11-12

    Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with alpha-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized alpha-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular alpha-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized alpha-ketoglutarate can permeate multiple layers of cells, reducing HIF1alpha levels and its target genes in vivo.

  3. The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution.

    Science.gov (United States)

    Vendelboe, Trine V; Harris, Pernille; Zhao, Yuguang; Walter, Thomas S; Harlos, Karl; El Omari, Kamel; Christensen, Hans E M

    2016-04-01

    The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system.

  4. Gibberellin (GA) biosynthesis in elongating internodes Zea mays (maize): The 3. beta. -hydroxylase

    Energy Technology Data Exchange (ETDEWEB)

    Spray, C.R.; Phinney, B.O. (Univ. of California, Los Angeles (USA)); Gaskin, P.; MacMillan, J. (Univ. of Bristol (England))

    1989-04-01

    The early-13-hydroxylation pathway for GA biosynthesis in maize leads to GA{sub 1}, the main GA responsible for shoot elongation. Growth response data, double-labeled feeding studies and endogenous GA levels suggest that the dwarf-1 mutant blocks the step GA{sub 20}to GA{sub 1}. We are screening for a system from maize from which we can purify the 3{beta}-hydroxylase and study its physical and biological properties. We find that diced internodal tissues will metabolize ({sup 13}C, {sup 3}H)-GA{sub 20} to ({sup 13}C, {sup 3}H)-GA{sub 1} and ({sup 13}C, {sup 3}H)-GA{sub 29} with metabolism as high as 80%. A cell free system from this material will give 5% metabolism. In a typical experiment 1g of internodal tissue is frozen in liquid nitrogen and macerated in 0.1M Tris plus cofactors. The homogenate is centrifuged at 15000 x g for 30 min at 4{degrees}C and the supernatant used for metabolic studies.

  5. Regulation of tryptophan hydroxylase expression by a retinal circadian oscillator in vitro.

    Science.gov (United States)

    Green, C B; Cahill, G M; Besharse, J C

    1995-04-24

    Many aspects of retinal physiology are controlled by a circadian clock including at least two steps in the melatonin synthetic pathway: the activity of the enzyme, N-acetyltransferase (NAT), and mRNA levels of the rate-limiting enzyme trytophan hydroxylase (TPH). Light and dopamine (through D2-like dopamine receptors) can phase shift the clock, and can also acutely inhibit NAT activity, resulting in supressed melatonin synthesis. In this paper, we show that eyecups cultured in constant darkness maintain a clock-controlled rhythm in TPH mRNA, with low levels in early day, rising to a peak in early night. Both eyecups and isolated retinas, cultured in light during the day, also exhibit a similar increase in TPH mRNA levels, indicating that this expression is not acutely inhibited by light. Treatment with light or quinpirole (D2 dopamine receptor agonist) in early night, at a time and dose that acutely inhibits NAT activity, does not change levels of TPH mRNA. Addition of eticlopride (D2 dopamine receptor antagonist) during the day, also has no effect on the normal daytime increase in TPH message levels. Therefore, TPH mRNA level is controlled by a circadian clock located within the eye, but acute effects of light or dopamine are not detected.

  6. Detection and characterization of p-coumaric acid hydroxylase in mung bean, Vigna mungo, seedlings.

    Science.gov (United States)

    Kojima, M; Takeuchi, W

    1989-02-01

    A new p-coumaric acid (4-hydroxycinnamic acid) hydroxylase was detected in mung bean seedlings treated with tentoxin, a fungal toxin, in which polyphenol oxidase that hydroxylates a wide variety of monophenols in vitro was completely eliminated. The enzyme required molecular oxygen and showed a pH optimum of 5.0. The enzyme acted only on p-coumaric acid (Km, 3.0 X 10(-5) M), while its specificity for the electron donor was rather broad. The Km value for NADPH (1.5 X 10(-4) M) was much lower than that for L-ascorbic acid (1.0 X 10(-2) M), although the Vmax value was almost the same with both electron donors. The enzyme was potently inhibited by beta-mercaptoethanol (Ki, 3.5 X 10(-6) M) and diethyldithiocarbamate (Ki, 2.3 X 10(-4) M), but was insensitive to p-chloromercuribenzoate. The enzyme was localized in the cell organelles which sedimented between mitochondria and endplasmic reticulum on sucrose density gradient centrifugation. The enzyme activity in the seedling was changed in response to induction by light in a manner suggesting its involvement in biosynthesis of phenolic compounds in mung bean seedlings.

  7. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

    Science.gov (United States)

    Chowdhury, Rasheduzzaman; Leung, Ivanhoe K. H.; Tian, Ya-Min; Abboud, Martine I.; Ge, Wei; Domene, Carmen; Cantrelle, François-Xavier; Landrieu, Isabelle; Hardy, Adam P.; Pugh, Christopher W.; Ratcliffe, Peter J.; Claridge, Timothy D. W.; Schofield, Christopher J.

    2016-01-01

    The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors. PMID:27561929

  8. Effect of LPS treatment on tyrosine hydroxylase expression and Parkinson-like behaviors.

    Science.gov (United States)

    Girard-Joyal, Olivier; Ismail, Nafissa

    2017-03-01

    Puberty is a critical period of development during which the brain undergoes reorganizing and remodeling. Exposure to stress during this period is thought to interfere with normal brain development and increase susceptibility to mental illnesses. In female mice, pubertal exposure to lipopolysaccharide (LPS), a bacterial endotoxin, has been shown to alter sexual, anxiety-like, and depression-like behaviors and cognition in an enduring manner. However, the mechanisms underlying these effects remain unknown. The present study examined age and sex difference in tyrosine hydroxylase (TH) expression and dopamine-dependent and Parkinson-like behaviors following LPS treatment. The results show that LPS treatment during adulthood causes an enduring increase in TH expression in many of the brain regions examined. In contrast, there is no change in TH expression following LPS treatment during puberty. However, pubertal LPS treatment induces enduring behavioral deficits in tests of Parkinson-like behaviors, more so in male than in female mice. These results suggest that the low levels of TH following exposure to pubertal immune challenge may predispose mice to Parkinson-like behavior. These findings add to our understanding of stress and immune responses during puberty and their impact on mental health later in life. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Time-resolved fluorescence analysis of the mobile flavin cofactor in -hydroxybenzoate hydroxylase

    Indian Academy of Sciences (India)

    Petra A W Van Den Berg; Koert Grever; Arie Van Hoek; Willem J H Van Berkel; Antonie J W G Visser

    2007-03-01

    Conformational heterogeneity of the FAD cofactor in -hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the `in’ conformation with the isoalloxazine ring located in the active site, and the `out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the `in’ and `out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.

  10. Prolyl Hydroxylase Domain-2 Inhibition Improves Skeletal Muscle Regeneration in a Male Murine Model of Obesity

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    Indranil Sinha

    2017-07-01

    Full Text Available Obesity leads to a loss of muscle mass and impaired muscle regeneration. In obese individuals, pathologically elevated levels of prolyl hydroxylase domain enzyme 2 (PHD2 limit skeletal muscle hypoxia-inducible factor-1 alpha and vascular endothelial growth factor (VEGF expression. Loss of local VEGF may further impair skeletal muscle regeneration. We hypothesized that PHD2 inhibition would restore vigorous muscle regeneration in a murine model of obesity. Adult (22-week-old male mice were fed either a high-fat diet (HFD, with 60% of calories derived from fat, or a regular diet (RD, with 10% of calories derived from fat, for 16 weeks. On day 5 following cryoinjury to the tibialis anterior muscle, newly regenerated muscle fiber cross-sectional areas were significantly smaller in mice fed an HFD as compared to RD, indicating an impaired regenerative response. Cryoinjured gastrocnemius muscles of HFD mice also showed elevated PHD2 levels (twofold higher and reduced VEGF levels (twofold lower as compared to RD. Dimethyloxalylglycine, a cell permeable competitive inhibitor of PHD2, restored VEGF levels and significantly improved regenerating myofiber size in cryoinjured mice fed an HFD. We conclude that pathologically increased PHD2 in the obese state drives impairments in muscle regeneration, in part by blunting VEGF production. Inhibition of PHD2 over activity in the obese state normalizes VEGF levels and restores muscle regenerative potential.

  11. Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Rolland

    2016-01-01

    Full Text Available The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson's disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications.

  12. Gene conversion-like events cause steroid 21-hydroxylase deficiency in congenital adrenal hyperplasia

    Energy Technology Data Exchange (ETDEWEB)

    Harada, F.; Kimura, A.; Iwanaga, T.; Shimozawa, K.; Yata, J.; Sasazuki,T.

    1987-11-01

    Genomic DNAs from twelve Japanese patients with steroid 21-hydroxylase deficiency were analyzed by Southern blot hybridization. A 3.7-kilobase (kb) Taq I and a 1.7-kb Pvu II restriction endonuclease fragment that correspond to a 21-OHase B gene were absent from the DNA of two unrelated patients with the salt-wasting form of the disease. However, a 10.5-kb Bgl II fragment corresponding to the region encompassing the 21-OHase B gene was still present in these two patients. The genes encoding 21-OHase were cloned from one of these two patients, who was homozygous by descent for HLA-A26;B39;C4A3;C4B1;DR4. Restriction endonuclease mapping as well as partial nucleotide sequencing analysis revealed that the 21-OHase B gene of the patient has been converted to the pseudogene, 21-OHase A, as far as the critical 0.5-kb sequence was concerned. Thus, the defect was due to both chromosomes each carrying two copies of 21-OHase A pseudogene and lacking functional 21-OHase B gene.

  13. Polymorphism of the tryptophan hydroxylase 2 (TPH2 gene is associated with chimpanzee neuroticism.

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    Kyung-Won Hong

    Full Text Available 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.

  14. Hepatic Cholesterol-25-Hydroxylase Overexpression Improves Systemic Insulin Sensitivity in Mice.

    Science.gov (United States)

    Noebauer, Britta; Jais, Alexander; Todoric, Jelena; Gossens, Klaus; Sutterlüty-Fall, Hedwig; Einwallner, Elisa

    2017-01-01

    Obesity is a major risk factor for several diseases including diabetes, heart disease, and some forms of cancer and due to its rapidly increasing prevalence it has become one of the biggest problems medicine is facing today. All the more surprising, a substantial percentage of obese patients are metabolically healthy when classified based on insulin resistance and systemic inflammation. Oxysterols are naturally occurring molecules that play important role in various metabolic and inflammatory processes and their levels are elevated in patients suffering from obesity and diabetes. 25-Hydroxycholesterol (25-OHC) is produced in cells from cholesterol by the enzyme cholesterol 25-hydroxylase (Ch25h) and is involved in lipid metabolism, inflammatory processes, and cell proliferation. Here, we investigated the role of hepatic Ch25h in the transition from metabolically healthy obesity to insulin resistance and diabetes. Using several different experimental approaches, we demonstrated the significance of Ch25h on the border of "healthy" and "diseased" states of obesity. Adenovirus-mediated Ch25h overexpression in mice improved glucose tolerance and insulin sensitivity and lowered HOMA-IR. Our data suggest that low hepatic Ch25h levels could be considered a risk marker for unhealthy obesity.

  15. Recent advances in diagnosis, treatment, and outcome of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    Science.gov (United States)

    Riepe, Felix G; Sippell, Wolfgang G

    2007-12-01

    Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal-recessive disease causing cortisol deficiency, aldosterone deficiency and hyperandrogenism. Diagnosis of 21-OHD is confirmed by steroid analysis in newborn screening or later on. Standard medical treatment consists of oral glucocorticoid and mineralocorticoid administration in order to suppress adrenal androgens and to compensate for adrenal steroid deficiencies. However, available treatment is far from ideal, and not much is known about the long-term outcome in CAH as trials in patients in adulthood or old age are rare. Here we briefly describe the pathophysiology, clinical picture, genetics and epidemiology of 21-OHD. This is followed by a comprehensive review of the recent advances in diagnosis, treatment and outcome. Novel insights have been gained in the fields of newborn screening, specific steroid measurement utilizing mass spectrometry, genetics, glucocorticoid stress dosing, additive medical therapy, prenatal treatment, side-effects of medical treatment, adrenomedullary involvement, metabolic morbidity, fertility and gender identity. However, many issues are still unresolved, and novel questions, which will have to be answered in the future, arise with every new finding.

  16. CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase.

    Science.gov (United States)

    Ly, Thuy T B; Khatri, Yogan; Zapp, Josef; Hutter, Michael C; Bernhardt, Rita

    2012-07-01

    Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and β-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and β-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-β-ionone and 13-hydroxy-nootkatone, were confirmed by (1)H and (13)C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K ( m ) and k (cat) values were calculated. The results of docking α-/β-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.

  17. Lipidomic profiling of tryptophan hydroxylase 2 knockout mice reveals novel lipid biomarkers associated with serotonin deficiency.

    Science.gov (United States)

    Weng, Rui; Shen, Sensen; Burton, Casey; Yang, Li; Nie, Honggang; Tian, Yonglu; Bai, Yu; Liu, Huwei

    2016-04-01

    Serotonin is an important neurotransmitter that regulates a wide range of physiological, neuropsychological, and behavioral processes. Consequently, serotonin deficiency is involved in a wide variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, schizophrenia, and depression. The pathophysiological mechanisms underlying serotonin deficiency, particularly from a lipidomics perspective, remain poorly understood. This study therefore aimed to identify novel lipid biomarkers associated with serotonin deficiency by lipidomic profiling of tryptophan hydroxylase 2 knockout (Tph2-/-) mice. Using a high-throughput normal-/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (NP/RP 2D LC-QToF-MS) method, 59 lipid biomarkers encompassing glycerophospholipids (glycerophosphocholines, lysoglycerophosphocholines, glycerophosphoethanolamines, lysoglycerophosphoethanolamines glycerophosphoinositols, and lysoglycerophosphoinositols), sphingolipids (sphingomyelins, ceramides, galactosylceramides, glucosylceramides, and lactosylceramides) and free fatty acids were identified. Systemic oxidative stress in the Tph2-/- mice was significantly elevated, and a corresponding mechanism that relates the lipidomic findings has been proposed. In summary, this work provides preliminary findings that lipid metabolism is implicated in serotonin deficiency.

  18. Cloning and characterization of cinnamate-4-hydroxylase gene from Rubus occidentalis L

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Mi; Lee, Seung Sik; An, Byung Chull; Barampuram, Shyamkuma; Kim, Jae Sung; Chung, Byung Yeoup [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Cho, Jae Young [Dept. of Applied Life Sciences, Chonbuk National University, Jeonju (Korea, Republic of); Lee, In Chul [Senior Industry Cluster Agency, Youngdong University, Youngdong (Korea, Republic of)

    2008-08-15

    Cinnamate-4-hydroxylase (C4H) is a key enzyme of phenylpropanoid pathway, which leads a variety of secondary metabolites to participate in differentiation and protection of plant against environmental stresses. In this study, we isolated a full-length cDNA of the C4H gene from a black raspberry (Rubus occidentalis L.), using a reverse transcriptase-PCR and rapid amplification of the cDNA ends (RACE)-PCR. The full-length cDNA of the RocC4H gene contained a 1,515 bp open reading frame (ORF) encoding a 504 amino acid protein with a calculated molecular weight of about 57.9 kDa and an isoelectric point (pI) value of 9.1. The genomic DNA analysis revealed that RocC4H gene had three exons and two introns. By multiple sequence alignment, RocC4H protein was highly homologous with other plant C4Hs, and the cytochrome P450-featured motifs, such as the heme-binding domain, the T-containing binding pocket motif (AAIETT), the ERR triad, and the tetrapeptide (PPGP) hinge motif, were highly conserved. Southern blot analysis revealed that RocC4H is a single copy gene in R. occidentalis.

  19. Increased expression of tyrosine hydroxylase and anomalous neurites in catecholaminergic neurons of ATF-2 null mice.

    Science.gov (United States)

    Kojima, Masayo; Suzuki, Takahiro; Maekawa, Toshio; Ishii, Shunsuke; Sumi-Ichinose, Chiho; Nomura, Takahide; Ichinose, Hiroshi

    2008-02-15

    ATF-2/CRE-BP1 was originally identified as a cAMP-responsive element (CRE) binding protein abundant in the brain. We previously reported that phosphorylation of ATF-2 increased the expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme for catecholamine biosynthesis, directly acting on the CRE in the promoter region of the TH gene in PC12D cells (Suzuki et al. [2002] J. Biol. Chem. 277:40768-40774). To examine the role of ATF-2 on transcriptional control of the TH gene in the brain, we investigated the TH expression in ATF-2-/- mice. We found that TH expression was greatly increased in medulla oblongata and locus ceruleus of the ATF-2-deficient embryos. Ectopic expression of TH was observed in the raphe magnus nucleus, where serotonergic neural cell bodies are located. Interestingly, A10 dorsal neurons were lost in the embryos of ATF-2-/- mice. There was no difference in the TH immunoreactivity in the olfactory bulb. The data showed that alteration in TH expression by absence of ATF-2 gradually declined from caudal to rostral part of the brain. We also found anomalous neurite extension in catecholaminergic neurons of ATF-2 null mice, i.e., increased dendritic arborization and shortened axons. These data suggest that ATF-2 plays critical roles for proper expression of the TH gene and for neurite extension of catecholaminergic neurons, possibly through a repressor-like action. (c) 2007 Wiley-Liss, Inc.

  20. In-crystal reaction cycle of a toluene-bound diiron hydroxylase

    Science.gov (United States)

    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.

  1. Approach to the adult with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    Science.gov (United States)

    Merke, Deborah P

    2008-03-01

    Congenital adrenal hyperplasia (CAH) describes a group of autosomal recessive disorders where there is impairment of cortisol biosynthesis. CAH due to 21-hydroxylase deficiency accounts for 95% of cases and shows a wide range of clinical severity. Treatment of the classic or severe form of CAH is targeted at replacing cortisol and aldosterone and effectively controlling excess androgen symptoms by using the lowest possible glucocorticoid dose. Treatment of the mild or nonclassic form is targeted at controlling excess androgen symptoms and may or may not involve glucocorticoid therapy. Hydrocortisone is the treatment of choice for children, but there is no consensus on how patients should be treated as adults. Current glucocorticoid therapy is suboptimal because it is often difficult to reduce excess androgen without giving excess glucocorticoid, and patients may experience hypercortisolism, androgen excess, or a combination of these states. Treatment of CAH, especially in the adult patient, remains controversial given the lack of prospective randomized controlled trials comparing treatment regimens. Nevertheless, patients benefit from careful individualized therapy with avoidance of Cushingoid side effects and optimization of reproductive, sexual, and bone health.

  2. Hormonal treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    Science.gov (United States)

    Bachelot, A; Chakhtoura, Z; Rouxel, A; Dulon, J; Touraine, P

    2007-09-01

    During childhood, the main aims of the medical treatment of congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency, are to prevent salt loss and virilization and to achieve normal stature and normal puberty. As such, there is a narrow therapeutic window through which the intended results can be achieved. In adulthood, the clinical management has received little attention, but recent studies have shown the relevance of long-term follow-up of these patients. Indeed, long-term evaluation of adult CAH patients enables the identification of multiple clinical, hormonal and metabolic abnormalities as bone mineral density alteration, overweight and disturbed reproductive functions. In women with classic CAH, low fertility rate is reported, and is probably the consequence of multiple factors, including neuroendocrine and hormonal factors, feminizing surgery, and psychological factors. Men with CAH may present hypogonadism either through the effect of adrenal rests or from suppression of gonadotropins resulting in infertility. These patients should therefore be carefully followed-up, from childhood through to adulthood, to avoid these complications and to ensure treatment compliance and tight control of the adrenal androgens, by multidisciplinary teams who have knowledge of CAH.

  3. Evaluation of impact of steroid replacement treatment on bone health in children with 21-hydroxylase deficiency.

    Science.gov (United States)

    Delvecchio, M; Soldano, L; Lonero, A; Ventura, A; Giordano, P; Cavallo, L; Grano, M; Brunetti, G; Faienza, M F

    2015-04-01

    There are conflicting data regarding the potential impact of chronic glucocorticoid (GC) therapy on the bone mineral density of patients with congenital adrenal hyperplasia (CAH). Previous studies performed by dual-energy X-ray absorptiometry reported conflicting results. The purpose of this study was to assess the impact of chronic GC replacement treatment in children with classical and non classical CAH due to 21-hydroxylase deficiency (21-OHD) by quantitative ultrasonometry (QUS), an easy, cheap, and radiation-free technique. The study population consisted of nineteen 21-OHD patients (nine males) on lifelong GC treatment. Anthropometric, hormonal, and treatment data were recorded for each patient, and bone quality was assessed by QUS measurements. QUS findings (amplitude-dependent speed of sound and bone transmission time) were normal in 21-OHD patients and did not correlate with duration of treatment, daily, total, and yearly hydrocortisone dose. Furthermore, no significant correlation was found between QUS findings and 17α-hydroxy progesterone, Δ4-androstenedione, and testosterone levels. In conclusion, our results provide reassurance that currently used replacement doses of GC do not have a major impact on bone in patients with CAH. QUS seems to be a reliable tool for screening of bone health in children with 21-OHD.

  4. Classical forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in adults.

    Science.gov (United States)

    Bachelot, Anne; Chakthoura, Zeina; Rouxel, Agnès; Dulon, Jérome; Touraine, Philippe

    2008-01-01

    During childhood, the main aims of the medical treatment of congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase are to prevent salt loss and virilization and to attain normal stature and normal puberty. As such, there is a narrow therapeutic window through which the intended results can be achieved. In adulthood, the clinical management has received little attention, but recent studies have shown the relevance of long-term follow-up of these patients. The aims here are to review the multiple clinical, hormonal and metabolic abnormalities that could be found in adult CAH patients as such a decrease in bone mineral density, overweight and disturbed reproductive functions. In women with classic CAH, a low fertility rate is reported, and is probably the consequence of multiple factors including neuroendocrine and hormonal factors, feminizing surgery, and psychological factors. Men with CAH may present hypogonadism either through the effect of adrenal rests or from suppression of gonadotropins resulting in infertility. Therefore a multidisciplinary team with knowledge of CAH should carefully follow up these patients, from childhood through to adulthood, to avoid these complications and to ensure treatment compliance and tight control of the adrenal androgens.

  5. Sleep deprivation affects sexual behavior and tyrosine hydroxylase (TH) levels in sexually experienced male rats.

    Science.gov (United States)

    Damasceno, Fabio; Skinner, Gabriela O; Cordeiro, Júlia F; Ferraz, Marcos R; Almeida, Olga M M S

    2008-06-09

    Paradoxical sleep deprivation (PSD) produces alterations in dopaminergic systems and also modifies sexual behavior. In this work we evaluated PSD effects on the sexual response and tyrosine hydroxylase (TH) expression in dopaminergic pathways related to sexual behavior of naive and sexual experienced rats. Male Wistar rats had their sexual behavior evaluated in 6 copulatory tests, with a 4 days interval. In these tests, the animals interacted with a receptive female and parameters that compose each component of the male sexual reply (initiation, arousal and ejaculation) were evaluated. After the 5th test, the animals were randomly divided in 2 groups, control and PSD, and 96 h later they were submitted to the last copulatory test. PSD facilitated the excitatory and the ejaculatory component, increasing the copulatory efficiency. In addition, reduced mount frequency and ejaculation latency were observed. The temporal patterning of the sexual behavior was modified, with reduction in the number of mount bouts. PSD per se was not able to modify TH levels, but in PSD sexual trained rats, an increase in the number of TH-immunoreactive cellular bodies in all dopaminergic areas evaluated was detected. Our data suggest that PSD facilitates the sexual response and this facilitation combined to sexual training could be the consequence of increased TH levels in dopaminergic pathways related to sexual reply.

  6. FoxO1 in dopaminergic neurons regulates energy homeostasis and targets tyrosine hydroxylase

    Science.gov (United States)

    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

  7. Genetic Diversity and Balancing Selection within the Human Phenylalanine Hydroxylase (PAH Gene Region in Iranian Population

    Directory of Open Access Journals (Sweden)

    J Mowla

    2012-04-01

    Full Text Available Background:Genetic diversity of three polymorphic markers in the phenylalanine hydroxylase(PAH gene region including PvuII(a, PAHSTR and MspI were investigated.Methods:Unrelated individuals (n=139 from the Iranian populations were genotyped using primers specific to PAH gene markers including PvuII(a,MspI and PAHSTR. The amplified products for PvuII(a,MspI were digested using the appropriate restriction enzymes and separated on 1.5% agarose. The PAHSTR alleles were identified using polyacrylamide gel electrophoresis followed by silver staining. The exact size of the STR alleles was determined by sequencing. The allele frequency and population status of the alleles were estimated using PHASE, FBAT and GENEPOP software.Results: The estimated degree of heterozygosity for PAHSTR, MspI and PvuII (a was 66%, 56% and 58%, respectively. The haplotype estimation analysis of the markers resulted in nine informative haplotypes with frequencies ≥5%.Moreover,the results obtained from Ewens-Watterson test for neutrality suggested that the markers were under balancing selection in the Iranian population.Conclusion:These findings suggested the presence of genetic diversity at these three markers in the PAH gene region. Therefore, the markers could be considered as functional markers for linkage analysis of the PAH gene mutations in the Iranian families with the PKU disease.

  8. Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase.

    Science.gov (United States)

    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.

  9. Immunolocalization of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase in differentiating xylem of poplar.

    Science.gov (United States)

    Sato, Takahiko; Takabe, Keiji; Fujita, Minoru

    2004-01-01

    Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and cinnamate-4-hydroxylase (C4H; EC 1.14.13.11) are pivotal enzymes involved in lignification. We synthesized peptides as the epitopes according to the amino acid sequences of these enzymes, coupled them with hemocyanin, and injected them into mice. The antiserums against peptides of PAL and C4H specifically detected PAL and C4H in the crude enzymes extracted from differentiating xylem of poplar, respectively. PAL and C4H were localized in differentiating xylem of poplar. PAL labeling was mainly localized in the cytosol, and somewhat localized on the rough-endoplasmic reticulum (r-ER) and the Golgi apparatus. In contrast, C4H was mainly observed on r-ER and the Golgi apparatus. These findings suggest that conversion of phenylalanine to cinnamic acid occurs in the cytosol and the following reaction occurs near the membrane of r-ER and the Golgi apparatus. The possibility of coordinated localization of PAL and C4H is discussed.

  10. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain.

    Science.gov (United States)

    Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J; Yue, Wyatt W

    2016-04-06

    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.

  11. Molecular characterization of phenylketonuria and tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Japan.

    Science.gov (United States)

    Okano, Yoshiyuki; Kudo, Satoshi; Nishi, Yasuaki; Sakaguchi, Tomoko; Aso, Kazuyoshi

    2011-04-01

    Phenylketonuria (PKU) is a heterogeneous metabolic disorder caused by a deficiency in hepatic phenylalanine hydroxylase (PAH). On the basis of phenotype/genotype correlations, determination of phenylketonuric genotype is important for classification of the clinical phenotype and treatment of PKU, including tetrahydrobiopterin therapy. We characterized the genotypes of 203 Japanese patients with PKU and hyperphenylalaninemia using the following systems: (1) denaturing high-performance liquid chromatography with a GC-clamped primer; (2) direct sequencing; and, (3) multiplex ligation-dependent probe amplification. Of 406 mutant alleles, 390 (96%) were genotyped; 65 mutations were identified, including 22 new mutations. R413P, R241C, IVS4-1g>a, R111X and R243Q were prevalent mutations. Mutations prevalent in the Japanese cohort are also common in Korean and Northern Chinese populations, suggesting same origin. The spectrum of prevalent mutations was not significantly different among six Japanese districts, indicating that Japan comprises a relatively homogeneous ethnic group. We classified the mutations by clinical phenotypes and in vivo PAH activity and estimated the mutations with potential tetrahydrobiopterin (BH(4)) responsiveness. The frequency of BH(4) responsiveness based on the genotype was 29.1% in Japanese PKU patients. A catalog of PKU genotypes would be useful for predicting clinical phenotype, deciding on the subsequent treatment of PKU including BH(4) therapy, and genetic counseling in East Asia.

  12. Gypsy Phenylketonuria: A point mutation of the phenylalanine hydroxylase gene in Gypsy families from Slovakia

    Energy Technology Data Exchange (ETDEWEB)

    Kalanin, J. [Institute for Clinical and Experical Medicine, Praha (Czechoslovakia); Takarada, Y. [Toyobo Research Center, Shiga (Japan); Kagawa, S.; Yamashita, K.; Ohtsuka, N.; Matsuoka, A. [Hyogo College of Medicine, Nishinomiya (Japan)

    1994-01-15

    A direct mutational analysis of the phenylalanine hydroxylase gene (PAH) in Gypsy families with phenylketonuria (PKU) has not yet been presented. However, they obviously represent a group at high risk for this inherited disease. The authors analyzed the PAH loci of 65 Gypsies originating from Eastern Slovakia by a combination of PCR amplification, direct sequencing and ASO hybridization. These studies uncovered 10 {open_quotes}classical PKU{close_quotes} patients to be homozygous for a R252W (CGG-TGG) transition, and 29 heterozygous carriers of this mutation. Fifteen control Caucasoid PKU patients from the Czech and Slovak Republics were selected. In this group they detected R252W mutation in two subjects (6.67% of all mutant alleles). Both were compound heterozygous for two different mutations. Previous haplotype studies of Welsh Gypsies with PKU were uninformative in the determination of heterozygosity. ASO hybridization served effectively for the consequent analyses in Gypsy PKU-related families and to identify the carriers among the unrelated subjects. 19 refs., 2 figs.

  13. The interplay between genotype, metabolic state and cofactor treatment governs phenylalanine hydroxylase function and drug response.

    Science.gov (United States)

    Staudigl, Michael; Gersting, Søren W; Danecka, Marta K; Messing, Dunja D; Woidy, Mathias; Pinkas, Daniel; Kemter, Kristina F; Blau, Nenad; Muntau, Ania C

    2011-07-01

    The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH(4) cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH(4) concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH(4) loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH(4) ratio. Finally, evaluation of data from BH(4) loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH(4)-responsive PAH deficiency.

  14. Comparative Analysis of Phenylalanine Hydroxylase Mutations Spectrum in Novosibirsk and Kemerovo regions of Western Siberia, Russia

    Directory of Open Access Journals (Sweden)

    Olga A. Baturina

    2016-03-01

    Full Text Available Results of phenylalanine hydroxylase (PAH locus molecular genotyping for 115 phenylketonuria (PKU patients and their family members from Novosibirsk and Kemerovo regions of Western Siberia are presented. The direct exons and adjacent introns regions sequencing was used to identify PKU-associated mutations. Mutations typical for Europe (p.R158Q, p.R252W, p.P281L, IVS10-11G>A, p.R408W, IVS12+1G>A and typical for South-Eastern Asia and Turkey (p.R261Q и p.R243Q were identified as well as a bunch of rare mutations (IVS2+5G>A, p.R155H, p.Y168H, p.W187R, E221_D222>Efs, p.A342T, p.Y386C, IVS11+1G>C. The p.R408W mutation was prevailing. Mutations spectrum for Novosibirsk region appeared to be more diverse than one for Kemerovo region.

  15. Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice.

    Science.gov (United States)

    Yew, Nelson S; Dufour, Emmanuelle; Przybylska, Malgorzata; Putelat, Julie; Crawley, Cristin; Foster, Meta; Gentry, Sarah; Reczek, David; Kloss, Alla; Meyzaud, Aurélien; Horand, Françoise; Cheng, Seng H; Godfrin, Yann

    2013-08-01

    Enzyme replacement therapy is often hampered by the rapid clearance and degradation of the administered enzyme, limiting its efficacy and requiring frequent dosing. Encapsulation of therapeutic molecules into red blood cells (RBCs) is a clinically proven approach to improve the pharmacokinetics and efficacy of biologics and small molecule drugs. Here we evaluated the ability of RBCs encapsulated with phenylalanine hydroxylase (PAH) to metabolize phenylalanine (Phe) from the blood and confer sustained enzymatic activity in the circulation. Significant quantities of PAH were successfully encapsulated within murine RBCs (PAH-RBCs) with minimal loss of endogenous hemoglobin. While intravenously administered free PAH enzyme was rapidly eliminated from the blood within a few hours, PAH-RBCs persisted in the circulation for at least 10days. A single injection of PAH-RBCs was able to decrease Phe levels by nearly 80% in normal mice. These results demonstrate the ability of enzyme-loaded RBCs to metabolize circulating amino acids and highlight the potential to treat disorders of amino acid metabolism.

  16. Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments.

    Science.gov (United States)

    Roberts, Kenneth M; Pavon, Jorge Alex; Fitzpatrick, Paul F

    2013-02-12

    Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH(4)) and O(2). 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-BH(4)-phenylalanine complex begins with the rapid binding of BH(4) (K(d) = 65 μM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (K(d) = 130 μM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O(2) rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected 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 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines k(cat). Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.

  17. Genotype-phenotype correlations analysis of mutations in the phenylalanine hydroxylase (PAH) gene.

    Science.gov (United States)

    Bercovich, Dani; Elimelech, Arava; Zlotogora, Joel; Korem, Sigal; Yardeni, Tal; Gal, Nurit; Goldstein, Nurit; Vilensky, Bela; Segev, Roni; Avraham, Smadar; Loewenthal, Ron; Schwartz, Gerard; Anikster, Yair

    2008-01-01

    The aims of our research were to define the genotype-phenotype correlations of mutations in the phenylalanine hydroxylase (PAH) gene that cause phenylketonuria (PKU) among the Israeli population. The mutation spectrum of the PAH gene in PKU patients in Israel is described, along with a discussion on genotype-phenotype correlations. By using polymerase chain reaction/denaturing high-performance liquid chromatography (PCR/dHPLC) and DNA sequencing, we screened all exons of the PAH gene in 180 unrelated patients with four different PKU phenotypes [classic PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP)]. In 63.2% of patient genotypes, the metabolic phenotype could be predicted, though evidence is also found for both phenotypic inconsistencies among subjects with more than one type of mutation in the PAH gene. Data analysis revealed that about 25% of patients could participate in the future in (6R)-L: -erythro-5, 6, 7, 8-tetrahydrobiopterin (BH4) treatment trials according to their mutation genotypes. This study enables us to construct a national database in Israel that will serve as a valuable tool for genetic counseling and a prognostic evaluation of future cases of PKU.

  18. Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan.

    Science.gov (United States)

    Lin, Yuheng; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

    2014-07-18

    5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in Escherichia coli. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan to 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1-1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.

  19. Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase.

    Science.gov (United States)

    Zhang, Shengnan; Fitzpatrick, Paul F

    2016-04-01

    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.

  20. Cinnamate 4-Hydroxylase (C4H) genes from Leucaena leucocephala: a pulp yielding leguminous tree.

    Science.gov (United States)

    Kumar, Santosh; Omer, Sumita; Patel, Krunal; Khan, Bashir M

    2013-02-01

    Leucaena leucocephala is a leguminous tree species accounting for one-fourth of raw material supplied to paper and pulp industry in India. Cinnamate 4-Hydroxylase (C4H, EC 1.14.13.11) is the second gene of phenylpropanoid pathway and a member of cytochrome P450 family. There is currently intense interest to alter or modify lignin content of L. leucocephala. Three highly similar C4H alleles of LlC4H1 gene were isolated and characterized. The alleles shared more than 98 % sequence identity at amino acid level to each other. Binding of partial promoter of another C4H gene LlC4H2, to varying amounts of crude nuclear proteins isolated from leaf and stem tissues of L. leucocephala formed two loose and one strong complex, respectively, suggesting that the abundance of proteins that bind with the partial C4H promoter is higher in stem tissue than in leaf tissue. Quantitative Real Time PCR study suggested that among tissues of same age, root tissues had highest level of C4H transcripts. Maximum transcript level was observed in 30 day old root tissue. Among the tissues investigated, C4H activity was highest in 60 day old root tissues. Tissue specific quantitative comparison of lignin from developing seedling stage to 1 year old tree stage indicated that Klason lignin increased in tissues with age.

  1. Hepatic Cholesterol-25-Hydroxylase Overexpression Improves Systemic Insulin Sensitivity in Mice

    Directory of Open Access Journals (Sweden)

    Britta Noebauer

    2017-01-01

    Full Text Available Obesity is a major risk factor for several diseases including diabetes, heart disease, and some forms of cancer and due to its rapidly increasing prevalence it has become one of the biggest problems medicine is facing today. All the more surprising, a substantial percentage of obese patients are metabolically healthy when classified based on insulin resistance and systemic inflammation. Oxysterols are naturally occurring molecules that play important role in various metabolic and inflammatory processes and their levels are elevated in patients suffering from obesity and diabetes. 25-Hydroxycholesterol (25-OHC is produced in cells from cholesterol by the enzyme cholesterol 25-hydroxylase (Ch25h and is involved in lipid metabolism, inflammatory processes, and cell proliferation. Here, we investigated the role of hepatic Ch25h in the transition from metabolically healthy obesity to insulin resistance and diabetes. Using several different experimental approaches, we demonstrated the significance of Ch25h on the border of “healthy” and “diseased” states of obesity. Adenovirus-mediated Ch25h overexpression in mice improved glucose tolerance and insulin sensitivity and lowered HOMA-IR. Our data suggest that low hepatic Ch25h levels could be considered a risk marker for unhealthy obesity.

  2. Copper reduces striatal protein nitration and tyrosine hydroxylase inactivation induced by MPP+ in rats.

    Science.gov (United States)

    Rubio-Osornio, M; Montes, S; Pérez-Severiano, F; Aguilera, P; Floriano-Sánchez, E; Monroy-Noyola, A; Rubio, C; Ríos, C

    2009-06-01

    Striatal administration of 1-methyl-4-phenylpyridinium (MPP(+)), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson's disease. Copper acts as a prosthetic group of several antioxidant enzymes and recent data show that copper attenuated MPP(+)-evoked neurotoxicity. We evaluated the effect of copper (as a supplement) upon proteins nitration (60 kDa) and tyrosine hydroxylase (TH) inactivation induced by MPP(+) (10 microg/8 microL) injection into the rat striatum. Copper pretreatment (10 micromol/kg i.p.) prevented both MPP(+)-induced proteins nitration and TH inactivation. Copper treatment also prevented the dopamine-depleting effect of MPP(+) injection. Those results were accompanied by a significant reduction of enzymatic activity of the constitutive nitric oxide synthase (cNOS), whereas, the protein levels of the three isoforms of NOS remained unchanged. Results indicate that the effect of copper against MPP(+)-induced proteins nitration and TH inactivation in the striatum of rat may be mediated by a reduction of cNOS activity.

  3. Localization of tyrosine hydroxylase immunoreactive neurons in the forebrain of the guppy Poecilia reticulata.

    Science.gov (United States)

    Parafati, M; Senatori, O; Nicotra, A

    2009-10-01

    The current study reports for the first time the distribution of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the forebrain of the guppy Poecilia reticulata. Numerous small TH-ir neurons were observed in the olfactory bulbs, located mainly in the periphery of the bulbs. The TH-ir telencephalic neurons are localized in the ventral telencephalic area where they are grouped in three distinct nuclei (Vv,Vd and Vp) composed of a small number of cells forming a continuous strip. The largest number of forebrain TH-ir neurons was observed in the diencephalon where both small and larger neurons are present. Diencephalic TH-ir neurons are subdivided in large nuclei located in the preoptic region (nSC, nPOp and nPOm), the thalamus (nDM), the pretectal region (nPPv and nAP), the hypothalamus (nPP and nRP) and the posterior tuberculum (nPT). Many diencephalic nuclei are distributed in periventricular regions and no TH-ir cells were observed in the paraventricular organ. A comparative analysis indicates that the present observations are consistent with the general pattern of TH-ir neurons distribution reported for the forebrain of other teleosts, but with some interspecies variability present, mainly in the diencephalon. This paper also provides valuable neuroanatomical information for P. reticulata, a teleost frequently used in toxicological tests, for future studies investigating the effects of environmental pollutants on the catecholaminergic system.

  4. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

    Science.gov (United States)

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease.

  5. Role of prolyl hydroxylase domain proteins in the regulation of insulin secretion.

    Science.gov (United States)

    Huang, Mei; Paglialunga, Sabina; Wong, Julia M-K; Hoang, Monica; Pillai, Renjitha; Joseph, Jamie W

    2016-03-01

    Type 2 diabetes is associated with impaired nutrient-regulated anaplerosis and insulin secretion in pancreatic β-cells. One key anaplerotic substrate that may be involved in regulating insulin release is α-ketoglutarate (αKG). Since prolyl hydroxylase domain proteins (PHDs) can metabolize cytosolic αKG, we sought to explore the role of this enzyme in the regulation of β-cell function. The oxygen-sensing PHDs regulate the stability of hypoxia-inducible factor 1α (HIF1α) as well as other proline-containing proteins by catalyzing the hydroxylation of proline residues. This reaction is dependent on sufficient levels of oxygen, iron, and αKG. In the present study, we utilized both pharmacological and genetic approaches to assess the impact of inhibiting PHD activity on β-cell function. We demonstrate that ethyl-3,4-dihydroxybenzoate (EDHB), a PHD inhibitor, significantly blunted glucose-stimulated insulin secretion (GSIS) from 832/13 clonal cells, rat, and human islets. EDHB reduced glucose utilization, ATP/ADP ratio, and key TCA cycle intermediates such as pyruvate, citrate, fumarate, and malate. siRNA-mediated knockdown of PHD1 and PHD3 inhibited GSIS, whereas siRNA-mediated knockdown of PHD2 had no effect on GSIS. Taken together, the current results demonstrate an important role for PHDs as mediators of islet insulin secretion.

  6. Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism.

    Science.gov (United States)

    Zhang, Kewei; Halitschke, Rayko; Yin, Changxi; Liu, Chang-Jun; Gan, Su-Sheng

    2013-09-01

    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.

  7. Determination of the aromatic hydrocarbon to total hydrocarbon ratio of mineral oil in commercial lubricants.

    Science.gov (United States)

    Uematsu, Yoko; Suzuki, Kumi; Ogimoto, Mami

    2016-01-01

    A method was developed to determine the aromatic hydrocarbon to total hydrocarbon ratio of mineral oil in commercial lubricants; a survey was also conducted of commercial lubricants. Hydrocarbons in lubricants were separated from the matrix components of lubricants using a silica gel solid phase extraction (SPE) column. Normal-phase liquid chromatography (NPLC) coupled with an evaporative light-scattering detector (ELSD) was used to determine the aromatic hydrocarbon to total hydrocarbon ratio. Size exclusion chromatography (SEC) coupled with a diode array detector (DAD) and a refractive index detector (RID) was used to estimate carbon numbers and the presence of aromatic hydrocarbons, which supplemented the results obtained by NPLC/ELSD. Aromatic hydrocarbons were not detected in 12 lubricants specified for use for incidental food contact, but were detected in 13 out of 22 lubricants non-specified for incidental food contact at a ratio up to 18%. They were also detected in 10 out of 12 lubricants collected at food factories at a ratio up to 13%. The centre carbon numbers of hydrocarbons in commercial lubricants were estimated to be between C16 and C50.

  8. Volatile hydrocarbons inhibit methanogenic crude oil degradation

    Directory of Open Access Journals (Sweden)

    Angela eSherry

    2014-04-01

    Full Text Available Methanogenic degradation of crude oil in subsurface sediments occurs slowly, but without the need for exogenous electron acceptors, is sustained for long periods and has enormous economic and environmental consequences. Here we show that volatile hydrocarbons are inhibitory to methanogenic oil biodegradation by comparing degradation of an artificially weathered crude oil with volatile hydrocarbons removed, with the same oil that was not weathered. Volatile hydrocarbons (nC5-nC10, methylcyclohexane, benzene, toluene and xylenes were quantified in the headspace of microcosms. Aliphatic (n-alkanes nC12-nC34 and aromatic hydrocarbons (4-methylbiphenyl, 3-methylbiphenyl, 2-methylnaphthalene, 1-methylnaphthalene were quantified in the total hydrocarbon fraction extracted from the microcosms. 16S rRNA genes from key microorganisms known to play an important role in methanogenic alkane degradation (Smithella and Methanomicrobiales were quantified by quantitative PCR. Methane production from degradation of weathered oil in microcosms was rapid (1.1 ± 0.1 µmol CH4/g sediment/day with stoichiometric yields consistent with degradation of heavier n-alkanes (nC12-nC34. For non-weathered oil, degradation rates in microcosms were significantly lower (0.4 ± 0.3 µmol CH4/g sediment/day. This indicated that volatile hydrocarbons present in the non-weathered oil inhibit, but do not completely halt, methanogenic alkane biodegradation. These findings are significant with respect to rates of biodegradation of crude oils with abundant volatile hydrocarbons in anoxic, sulphate-depleted subsurface environments, such as contaminated marine sediments which have been entrained below the sulfate-reduction zone, as well as crude oil biodegradation in petroleum reservoirs and contaminated aquifers.

  9. Foaming of mixtures of pure hydrocarbons

    Science.gov (United States)

    Robinson, J. V.; Woods, W. W.

    1950-01-01

    Mixtures of pure liquid hydrocarbons are capable of foaming. Nine hydrocarbons were mixed in pairs, in all possible combinations, and four proportions of each combination. These mixtures were sealed in glass tubes, and the foaming was tested by shaking. Mixtures of aliphatic with other aliphatic hydrocarbons, or of alkyl benzenes with other alkyl benzenes, did not foam. Mixtures of aliphatic hydrocarbons with alkyl benzenes did foam. The proportions of the mixtures greatly affected the foaming, the maximum foaming of 12 of 20 pairs being at the composition 20 percent aliphatic hydrocarbon, 80 percent alkyl benzene. Six seconds was the maximum foam lifetime of any of these mixtures. Aeroshell 120 lubricating oil was fractionated into 52 fractions and a residue by extraction with acetone in a fractionating extractor. The index of refraction, foam lifetime, color, and viscosity of these fractions were measured. Low viscosity and high index fractions were extracted first. The viscosity of the fractions extracted rose and the index decreased as fractionation proceeded. Foam lifetimes and color were lowest in the middle fractions. Significance is attached to the observation that none of the foam lifetimes of the fractions or residue is as high as the foam lifetime of the original Aeroshell, indicating that the foaming is not due to a particular foaming constituent, but rather to the entire mixture.

  10. Syngas Upgrading to Hydrocarbon Fuels Technology Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Talmadge, M.; Biddy, Mary J.; Dutta, Abhijit; Jones, Susanne B.; Meyer, Pimphan A.

    2013-03-31

    In support of the Bioenergy Technologies Office, the National Renewable Energy Laboratory (NREL) and the Pacific Northwest National Laboratory (PNNL) are undertaking studies of biomass conversion technologies to hydrocarbon fuels to identify barriers and target research toward reducing conversion costs. Process designs and preliminary economic estimates for each of these pathway cases were developed using rigorous modeling tools (Aspen Plus and Chemcad). These analyses incorporated the best information available at the time of development, including data from recent pilot and bench-scale demonstrations, collaborative industrial and academic partners, and published literature and patents. This pathway case investigates the upgrading of biomass derived synthesis gas (‘syngas’) to hydrocarbon biofuels. While this specific discussion focuses on the conversion of syngas via a methanol intermediate to hydrocarbon blendstocks, there are a number of alternative conversion routes for production of hydrocarbons through a wide array of intermediates from syngas. Future work will also consider the variations to this pathway to determine the most economically viable and risk adverse conversion route. Technical barriers and key research needs have been identified that should be pursued for the syngas to hydrocarbon pathway to be competitive with petroleum-derived gasoline, diesel and jet range blendstocks.

  11. Velocity Dependence of Friction of Confined Hydrocarbons

    DEFF Research Database (Denmark)

    Sivebæk, Ion Marius; Samoilov, Vladimir N.; Persson, Bo N. J.

    2010-01-01

    We present molecular dynamics friction calculations for confined hydrocarbon “polymer” solids with molecular lengths from 20 to 1400 carbon atoms. Two cases are considered: (a) polymer sliding against a hard substrate and (b) polymer sliding on polymer. We discuss the velocity dependence of the f......We present molecular dynamics friction calculations for confined hydrocarbon “polymer” solids with molecular lengths from 20 to 1400 carbon atoms. Two cases are considered: (a) polymer sliding against a hard substrate and (b) polymer sliding on polymer. We discuss the velocity dependence...... in the polymer film is always close to the thermostat temperature. In the first setup (a), for hydrocarbons with molecular lengths from 60 to 1400 carbon atoms, the shear stresses are nearly independent of molecular length, but for the shortest hydrocarbon C20H42 the frictional shear stress is lower. In all...... cases the frictional shear stress increases monotonically with the sliding velocity. For polymer sliding on polymer (case b) the friction is much larger, and the velocity dependence is more complex. For hydrocarbons with molecular lengths from 60 to 140 C atoms, the number of monolayers of lubricant...

  12. Paleozoic Hydrocarbon-Seep Limestones

    Science.gov (United States)

    Peckmann, J.

    2007-12-01

    To date, five Paleozoic hydrocarbon-seep limestones have been recognized based on carbonate fabrics, associated fauna, and stable carbon isotopes. These are the Middle Devonian Hollard Mound from the Antiatlas of Morocco [1], Late Devonian limestone lenses with the dimerelloid brachiopod Dzieduszyckia from the Western Meseta of Morocco [2], Middle Mississippian limestones with the dimerelloid brachiopod Ibergirhynchia from the Harz Mountains of Germany [3], Early Pennsylvanian limestones from the Tantes Mound in the High Pyrenees of France [4], and Late Pennsylvanian limestone lenses from the Ganigobis Shale Member of southern Namibia [5]. Among these examples, the composition of seepage fluids varied substantially as inferred from delta C-13 values of early diagenetic carbonate phases. Delta C-13 values as low as -50 per mil from the Tantes Mound and -51 per mil from the Ganigobis limestones reveal seepage of biogenic methane, whereas values of -12 per mil from limestones with Dzieduszyckia associated with abundant pyrobitumen agree with oil seepage. Intermediate delta C-13 values of carbonate cements from the Hollard Mound and Ibergirhynchia deposits probably reflect seepage of thermogenic methane. It is presently very difficult to assess the faunal evolution at seeps in the Paleozoic based on the limited number of examples. Two of the known seeps were typified by extremely abundant rhynchonellide brachiopods of the superfamily Dimerelloidea. Bivalve mollusks and tubeworms were abundant at two of the known Paleozoic seep sites; one was dominated by bivalve mollusks (Hollard Mound, Middle Devonian), another was dominated by tubeworms (Ganigobis Shale Member, Late Pennsylvanian). The tubeworms from these two deposits are interpreted to represent vestimentiferan worms, based on studies of the taphonomy of modern vestimentiferans. However, this interpretation is in conflict with the estimated evolutionary age of vestimentiferans based on molecular clock methods

  13. Electrochemical Routes towards Sustainable Hydrocarbon Fuels

    DEFF Research Database (Denmark)

    Mogensen, Mogens Bjerg

    2012-01-01

    The potential of renewable energy and possible solution to the intermittency problem of renewable energy sources like sun and wind are explained. The densest storage of energy is in the form of hydrocarbons. The most suitable method of conversion and storage within a foreseeable future is electro......The potential of renewable energy and possible solution to the intermittency problem of renewable energy sources like sun and wind are explained. The densest storage of energy is in the form of hydrocarbons. The most suitable method of conversion and storage within a foreseeable future...... is electrolysis followed by conversion into synthetic hydrocarbons, alcohols or ethers. Several types of electrolysers exist. The various types are listed together with a short description of principle and status. It is argued that electrolysis will at least become part of large sustainable energy systems...

  14. Reappraisal of hydrocarbon biomarkers in Archean rocks

    Science.gov (United States)

    French, Katherine L.; Hallmann, Christian; Hope, Janet M.; Schoon, Petra L.; Zumberge, J. Alex; Hoshino, Yosuke; Peters, Carl A.; George, Simon C.; Love, Gordon D.; Brocks, Jochen J.; Buick, Roger; Summons, Roger E.

    2015-05-01

    Hopanes and steranes found in Archean rocks have been presented as key evidence supporting the early rise of oxygenic photosynthesis and eukaryotes, but the syngeneity of these hydrocarbon biomarkers is controversial. To resolve this debate, we performed a multilaboratory study of new cores from the Pilbara Craton, Australia, that were drilled and sampled using unprecedented hydrocarbon-clean protocols. Hopanes and steranes in rock extracts and hydropyrolysates from these new cores were typically at or below our femtogram detection limit, but when they were detectable, they had total hopane (oxygenic photosynthesis and eukaryotes by ∼2.7 billion years ago. Although suitable Proterozoic rocks exist, no currently known Archean strata lie within the appropriate thermal maturity window for syngenetic hydrocarbon biomarker preservation, so future exploration for Archean biomarkers should screen for rocks with milder thermal histories.

  15. EVALUATION OF PETROLEUM HYDROCARBONS ELUTION FROM SOIL

    Directory of Open Access Journals (Sweden)

    Janina Piekutin

    2015-06-01

    Full Text Available The paper presents studies on oil removal from soil by means of water elution with a help of shaking out the contaminants from the soil. The tests were performed on simulated soil samples contaminated with a mixture of petroleum hydrocarbons. The study consisted in recording the time influence and the number of elution cycles to remove contaminants from the soil. The samples were then subject to the determination of petroleum hydrocarbons, aliphatic hydrocarbons, and BTEX compounds (benzene, toluene, ethylbenzene, xylene. Due to adding various concentrations of petroleum into particular soil samples and applying different shaking times, it was possible to find out the impact of petroleum content and sample shaking duration on the course and possibility of petroleum substances removal by means of elution process.

  16. Hydrocarbon Degrading Bacteria: Isolation and Identification

    Directory of Open Access Journals (Sweden)

    Lies Indah Sutiknowati

    2007-11-01

    Full Text Available There is little information how to identify hydrocarbon degrading bacteria for bioremediation of marine oil spills. We have used gravel which contaminated oil mousse from Beach Simulator Tank, in Marine Biotechnology Institute, Kamaishi, Japan, and grown on enrichment culture. Biostimulation with nutrients (N and P was done to analyze biodegradation of hydrocarbon compounds: Naphthalene, Phenanthrene, Trichlorodibenzofuran and Benzo[a]pyrene. Community of bacteria from enrichment culture was determined by DGGE. Isolating and screening the bacteria on inorganic medium contain hydrocarbon compounds and determination of bacteria by DAPI (number of cells and CFU. DNA was extracted from colonies of bacteria and sequence determination of the 16S rDNA was amplified by primers U515f and U1492r. Twenty nine strains had been sequence and have similarity about 90-99% to their closest taxa by homology Blast search and few of them have suspected as new species.

  17. Using supercritical fluids to refine hydrocarbons

    Science.gov (United States)

    Yarbro, Stephen Lee

    2014-11-25

    This is a method to reactively refine hydrocarbons, such as heavy oils with API gravities of less than 20.degree. and bitumen-like hydrocarbons with viscosities greater than 1000 cp at standard temperature and pressure using a selected fluid at supercritical conditions. The reaction portion of the method delivers lighter weight, more volatile hydrocarbons to an attached contacting device that operates in mixed subcritical or supercritical modes. This separates the reaction products into portions that are viable for use or sale without further conventional refining and hydro-processing techniques. This method produces valuable products with fewer processing steps, lower costs, increased worker safety due to less processing and handling, allow greater opportunity for new oil field development and subsequent positive economic impact, reduce related carbon dioxide, and wastes typical with conventional refineries.

  18. Artificial Hydrocarbon Networks Fuzzy Inference System

    Directory of Open Access Journals (Sweden)

    Hiram Ponce

    2013-01-01

    Full Text Available This paper presents a novel fuzzy inference model based on artificial hydrocarbon networks, a computational algorithm for modeling problems based on chemical hydrocarbon compounds. In particular, the proposed fuzzy-molecular inference model (FIM-model uses molecular units of information to partition the output space in the defuzzification step. Moreover, these molecules are linguistic units that can be partially understandable due to the organized structure of the topology and metadata parameters involved in artificial hydrocarbon networks. In addition, a position controller for a direct current (DC motor was implemented using the proposed FIM-model in type-1 and type-2 fuzzy inference systems. Experimental results demonstrate that the fuzzy-molecular inference model can be applied as an alternative of type-2 Mamdani’s fuzzy control systems because the set of molecular units can deal with dynamic uncertainties mostly present in real-world control applications.

  19. Carriers of 21-hydroxylase deficiency are not at increased risk for hyperandrogenism.

    Science.gov (United States)

    Knochenhauer, E S; Cortet-Rudelli, C; Cunnigham, R D; Conway-Myers, B A; Dewailly, D; Azziz, R

    1997-02-01

    A deficiency of 21-hydroxylase activity is one of the most commonly inherited genetic disorders in man, with heterozygosity for CYP21 mutations affecting approximately 1 in 60 of the non-Jewish Caucasian population. We have hypothesized that heterozygosity for CYP21 mutations in women increases their risk of developing clinically evident hyperandrogenism, and that this risk is related to the severity of the mutation of CYP21 and/or the 17-hydroxyprogesterone (17-OHP) response to ACTH stimulation. To test these hypotheses, we studied 38 obligate carriers for 21-hydroxylase deficiency (i.e. mothers of children with congenital adrenal hyperplasia or nonclassic congenital adreanl hyperplasia), comparing them to 27 weight-, parity-, and age-matched controls. Premenopausal carriers, not receiving hormonal treatment (n = 27), had higher mean total and free testosterone [T; 2.02 +/- 0.55 vs. 1.56 +/- 0.65 nmol/L (P < 0.007) and 0.018 +/- 0.007 vs. 0.012 +/- 0.006 nmol/L (P < 0.007), respectively] and lower mean sex hormone-binding globulin (214 +/- 62 vs. 277 +/- 129 nmol/L; P < 0.03) levels compared to controls. There was no difference in the mean basal levels of dehydroepiandrosterone sulfate, androstenedione (A4), or 17-OHP between carriers and controls. As expected, carriers exhibited higher stimulated and net increment 17-OHP levels than controls [21.1 +/- 27.1 vs. 6.2 +/- 3.1 nmol/L (P < 0.01) and 19.0 +/- 26.5 vs. 4.4 +/- 2.8 nmol/L (P < 0.009), respectively]. However, no difference was observed in the response of A4 to ACTH-(1-24) stimulation. Of the 27 carriers studied biochemically, 2 (7.4%) had a stimulated 17-OHP value between 30.3-60.6 nmol/L, and 1 (3.7%) had a 17-OHP level above 60.6 nmol/L, suggestive of nonclassic adrenal hyperplasia. Of all carriers studied genetically (n = 36), 50.0% (18 of 36) had 1, 33% (12 of 36) had 2, and 16.7% (6 of 36) had 3 or more mutations. In 27.8% (10 of 36) of carriers, the mutations were contiguous, consistent with a large

  20. Bioremediation of Petroleum Hydrocarbon Contaminated Sites

    Energy Technology Data Exchange (ETDEWEB)

    Fallgren, Paul

    2009-03-30

    Bioremediation has been widely applied in the restoration of petroleum hydrocarbon-contaminated. Parameters that may affect the rate and efficiency of biodegradation include temperature, moisture, salinity, nutrient availability, microbial species, and type and concentration of contaminants. Other factors can also affect the success of the bioremediation treatment of contaminants, such as climatic conditions, soil type, soil permeability, contaminant distribution and concentration, and drainage. Western Research Institute in conjunction with TechLink Environmental, Inc. and the U.S. Department of Energy conducted laboratory studies to evaluate major parameters that contribute to the bioremediation of petroleum-contaminated drill cuttings using land farming and to develop a biotreatment cell to expedite biodegradation of hydrocarbons. Physical characteristics such as soil texture, hydraulic conductivity, and water retention were determined for the petroleum hydrocarbon contaminated soil. Soil texture was determined to be loamy sand to sand, and high hydraulic conductivity and low water retention was observed. Temperature appeared to have the greatest influence on biodegradation rates where high temperatures (>50 C) favored biodegradation. High nitrogen content in the form of ammonium enhanced biodegradation as well did the presence of water near field water holding capacity. Urea was not a good source of nitrogen and has detrimental effects for bioremediation for this site soil. Artificial sea water had little effect on biodegradation rates, but biodegradation rates decreased after increasing the concentrations of salts. Biotreatment cell (biocell) tests demonstrated hydrocarbon biodegradation can be enhanced substantially when utilizing a leachate recirculation design where a 72% reduction of hydrocarbon concentration was observed with a 72-h period at a treatment temperature of 50 C. Overall, this study demonstrates the investigation of the effects of

  1. Structure of human phytanoyl-CoA 2-hydroxylase identifies molecular mechanisms of Refsum disease.

    Science.gov (United States)

    McDonough, Michael A; Kavanagh, Kathryn L; Butler, Danica; Searls, Timothy; Oppermann, Udo; Schofield, Christopher J

    2005-12-01

    Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the initial alpha-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG oxygenases, PAHX possesses a double-stranded beta-helix core, which supports three iron binding ligands (His(175), Asp(177), and His(264)); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys(191)) 6.7 A from the Fe(II) and two further histidine residues (His(155) and His(281)) at its active site distinguishes it from that of the other human 2OG oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro(173), His(175), Gln(176), Asp(177), and His(220)) and 2OG binding sites (Trp(193), Glu(197), Ile(199), Gly(204), Asn(269), and Arg(275)). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.

  2. Molecular Cloning and Yeast Expression of Cinnamate 4-Hydroxylase from Ornithogalum saundersiae Baker

    Directory of Open Access Journals (Sweden)

    Jian-Qiang Kong

    2014-01-01

    Full Text Available OSW-1, isolated from the bulbs of Ornithogalum saundersiae Baker, is a steroidal saponin endowed with considerable antitumor properties. Biosynthesis of the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically via the phenylpropanoid biosynthetic pathway, but molecular biological characterization of the related genes has been insufficient. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11, catalyzing the hydroxylation of trans-cinnamic acid to p-coumaric acid, plays a key role in the ability of phenylpropanoid metabolism to channel carbon to produce the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1. Molecular isolation and functional characterization of the C4H genes, therefore, is an important step for pathway characterization of 4-methoxybenzoyl group biosynthesis. In this study, a gene coding for C4H, designated as OsaC4H, was isolated according to the transcriptome sequencing results of Ornithogalum saundersiae. The full-length OsaC4H cDNA is 1,608-bp long, with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 55-bp 5′ non-coding region and a 35-bp 3'-untranslated region. OsaC4H was functionally characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of trans-cinnamic acid to p-coumaric acid, which was identified by high performance liquid chromatography with diode array detection (HPLC-DAD, HPLC-MS and nuclear magnetic resonance (NMR analysis. The identification of the OsaC4H gene was expected to open the way to clarification of the biosynthetic pathway of OSW-1.

  3. Human placenta expresses both peripheral and neuronal isoform of tryptophan hydroxylase.

    Science.gov (United States)

    Laurent, Laetitia; Deroy, Kathy; St-Pierre, Joey; Côté, Francine; Sanderson, J Thomas; Vaillancourt, Cathy

    2017-09-01

    The role of placental serotonin has been an active topic of research notably because of its crucial role in brain development. However, which cell types synthesize serotonin in human placenta remains unknown. Moreover, it is not known if the two tryptophan hydroxylase isoforms (TPH1 and TPH2), the rate-limiting enzymes in serotonin biosynthesis, are expressed in placenta. Human placentas were obtained in first trimester or at term, and trophoblast cells were isolated and purified using a magnetic cell sorter and placed in primary culture. The tissue sublocalization of each TPH was determined by immunohistochemistry. TPH expression in primary villous trophoblasts was determined by PCR and immunoblotting, and serotonin secretion by LC-MS/MS. Villous cytotrophoblasts, syncytiotrophoblast, fetal capillaries, extravillous cytotrophoblasts, and decidual cells co-expressed TPH1 and TPH2. Moreover, mRNA and protein levels of both TPHs were detected in human primary trophoblast as well as in mouse placental tissues. Finally, human trophoblast cells were shown to produce serotonin de novo. This study demonstrates that both TPH1 and TPH2 are expressed in human and mouse placenta throughout pregnancy and helps to better understand the placental serotonin system, which is crucial for healthy pregnancy and fetal development. It is therefore important to further understand regulation of the placental serotonin system and how its disruption during pregnancy may impact the developing fetus and subsequent child programming. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  4. Disulfiram Attenuates Drug-Primed Reinstatement of Cocaine Seeking via Inhibition of Dopamine β-Hydroxylase

    Science.gov (United States)

    Schroeder, Jason P; Cooper, Debra A; Schank, Jesse R; Lyle, Megan A; Gaval-Cruz, Meriem; Ogbonmwan, Yvonne E; Pozdeyev, Nikita; Freeman, Kimberly G; Iuvone, P Michael; Edwards, Gaylen L; Holmes, Philip V; Weinshenker, David

    2010-01-01

    The antialcoholism medication disulfiram (Antabuse) inhibits aldehyde dehydrogenase (ALDH), which results in the accumulation of acetaldehyde upon ethanol ingestion and produces the aversive ‘Antabuse reaction' that deters alcohol consumption. Disulfiram has also been shown to deter cocaine use, even in the absence of an interaction with alcohol, indicating the existence of an ALDH-independent therapeutic mechanism. We hypothesized that disulfiram's inhibition of dopamine β-hydroxylase (DBH), the catecholamine biosynthetic enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons, underlies the drug's ability to treat cocaine dependence. We tested the effects of disulfiram on cocaine and food self-administration behavior and drug-primed reinstatement of cocaine seeking in rats. We then compared the effects of disulfiram with those of the selective DBH inhibitor, nepicastat. Disulfiram, at a dose (100 mg/kg, i.p.) that reduced brain NE by ∼40%, did not alter the response for food or cocaine on a fixed ratio 1 schedule, whereas it completely blocked cocaine-primed (10 mg/kg, i.p.) reinstatement of drug seeking following extinction. A lower dose of disulfiram (10 mg/kg) that did not reduce NE had no effect on cocaine-primed reinstatement. Nepicastat recapitulated the behavioral effects of disulfiram (100 mg/kg) at a dose (50 mg/kg, i.p.) that produced a similar reduction in brain NE. Food-primed reinstatement of food seeking was not impaired by DBH inhibition. Our results suggest that disulfiram's efficacy in the treatment of cocaine addiction is associated with the inhibition of DBH and interference with the ability of environmental stimuli to trigger relapse. PMID:20736996

  5. Predicting the onset of Addison's disease: ACTH, renin, cortisol and 21-hydroxylase autoantibodies

    Science.gov (United States)

    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

  6. Dopamine pathology in schizophrenia: Analysis of total and phosphorylated tyrosine hydroxylase in the substantia nigra

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    EMMA ePEREZ-COSTAS

    2012-04-01

    Full Text Available Despite the importance of dopamine neurotransmission in schizophrenia, very few studies have addressed anomalies in the mesencephalic dopaminergic neurons of the substantia nigra/ventral tegmental area (SN/VTA. Tyrosine hydroxylase (TH is the rate-limiting enzyme for the production of dopamine, and a possible contributor to the anomalies in the dopaminergic neurotransmission observed in schizophrenia.In this study, we had three objectives: 1 Compare TH expression (mRNA and protein in the SN/VTA of schizophrenia and control postmortem samples. 2 Assess the effect of antipsychotic medications on the expression of TH in the SN/VTA. 3 Examine possible regional differences in TH expression anomalies within the SN/VTA.To achieve these objectives three independent studies were conducted: 1 A pilot study to compare TH mRNA and TH protein levels in the SN/VTA of postmortem samples from schizophrenia and controls. 2 A chronic treatment study was performed in rodents to assess the effect of antipsychotic medications in TH protein levels in the SN/VTA. 3 A second postmortem study was performed to assess TH and phosphorylated TH protein levels in two types of samples: schizophrenia and control samples containing the entire rostro-caudal extent of the SN/VTA, and schizophrenia and control samples containing only mid-caudal regions of the SN/VTA.Our studies showed impairment in the dopaminergic system in schizophrenia that could be mainly (or exclusively located in the rostral region of the SN/VTA. Our studies also showed that TH protein levels were significantly abnormal in schizophrenia, while mRNA expression levels were not affected, indicating that TH pathology in this region may occur posttranscriptionally. Lastly, our antipsychotic animal treatment study showed that TH protein levels were not significantly affected by antipsychotic treatment, indicating that these anomalies are an intrinsic pathology rather than a treatment effect.

  7. Aspartate-β-hydroxylase Induces Epitope-specific T Cell Responses in Hepatocellular Carcinoma

    Science.gov (United States)

    Tomimaru, Yoshito; Mishra, Sasmita; Safran, Howard; Charpentier, Kevin P.; Martin, William; De Groot, Anne S.; Gregory, Stephen H.; Wands, Jack R.

    2015-01-01

    Hepatocellular carcinoma (HCC) has a poor prognosis due to high recurrence rate. Aspartate-β-hydroxylase (ASPH) is a highly conserved transmembrane protein, which is over expressed in HCC and promotes a malignant phenotype. The capability of ASPH protein-derived HLA Class I and II peptides to generate antigen specific CD4+ and CD8+ immune responses is unknown. Therefore, these studies aim to define the epitope specific components required for a peptide based candidate vaccine. Monocyte-derived dendritic cells (DCs) generated from the peripheral blood mononuclear cells (PBMCs) of HCC patients were loaded with ASPH protein. Helper CD4+ T cells and CD8+ cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs; T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to predict HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ ELISpot assay. ASPH protein-loaded DCs activated both CD4+ and CD8+ T cells contained within the PBMC population derived from HCC patients. Furthermore, the predicted HLA class I- and class II-restricted ASPH peptides were significantly immunogenic. Both HLA class I- and class II-restricted peptides derived from ASPH induce T cell activation in HCC. We observed that ASPH protein and related peptides were highly immunogenic in patients with HCC and produce the type of cellular immune responses required for generation of anti-tumor activity. PMID:25629522

  8. Genotype-independent decrease in plasma dopamine beta-hydroxylase activity in Alzheimer’s disease

    Science.gov (United States)

    Mustapic, Maja; Presecki, Paola; Pivac, Nela; Mimica, Ninoslav; Hof, Patrick R.; Simic, Goran; Folnegovic-Smalc, Vera; Muck-Seler, Dorotea

    2014-01-01

    The noradrenergic system is involved in the etiology and progression of Alzheimer’s disease (AD) but its role is still unclear. Dopamine beta-hydroxylase (DBH) as a catecholamine-synthesizing enzyme plays a central role in noradrenaline (NA) synthesis and turnover. Plasma DBH (pDBH) activity shows wide inheritable interindividual variability that is under genetic control. The aim of this study was to determine pDBH activity, DBH (C-970T; rs1611115) and DBH (C1603T; rs6271) gene polymorphisms in 207 patients with AD and in 90 healthy age-matched controls. Plasma DBH activity was lower, particularly in the early stage of AD, compared to values in middle and late stages of the disease, as well as to control values. Two-way ANOVA revealed significant effect of both diagnosis and DBH (C-970T) or DBH (C1603T) genotypes on pDBH activity, but without significant diagnosis×genotype interaction. No association was found between AD and DBH C-970T (OR=1.08, 95% CI 1.13–4.37; p=0.779) and C1603T (OR=0.89; 95% CI 0.36–2.20; p=0.814) genotypes controlled for age, gender, and ApoE4 allele. The decrease in pDBH activity, found in early phase of AD suggests that alterations in DBH activity represent a compensatory mechanism for the loss of noradrenergic neurons, and that treatment with selective NA reuptake inhibitors may be indicated in early stages of AD to compensate for loss of noradrenergic activity in the locus coeruleus. PMID:23416088

  9. Physical weight loading induces expression of tryptophan hydroxylase 2 in the brain stem.

    Directory of Open Access Journals (Sweden)

    Joon W Shim

    Full Text Available Sustaining brain serotonin is essential in mental health. Physical activities can attenuate mental problems by enhancing serotonin signaling. However, such activity is not always possible in disabled individuals or patients with dementia. Knee loading, a form of physical activity, has been found to mimic effects of voluntary exercise. Focusing on serotonergic signaling, we addressed a question: Does local mechanical loading to the skeleton elevate expression of tryptophan hydroxylase 2 (tph2 that is a rate-limiting enzyme for brain serotonin? A 5 min knee loading was applied to mice using 1 N force at 5 Hz for 1,500 cycles. A 5-min treadmill running was used as an exercise (positive control, and a 90-min tail suspension was used as a stress (negative control. Expression of tph2 was determined 30 min - 2 h in three brain regions --frontal cortex (FC, ventromedial hypothalamus (VMH, and brain stem (BS. We demonstrated for the first time that knee loading and treadmill exercise upregulated the mRNA level of tph2 in the BS, while tail suspension downregulated it. The protein level of tph2 in the BS was also upregulated by knee loading and downregulated by tail suspension. Furthermore, the downregulation of tph2 mRNA by tail suspension can be partially suppressed by pre-application of knee loading. The expression of tph2 in the FC and VMH was not significantly altered with knee loading. In this study we provided evidence that peripheral mechanical loading can activate central tph2 expression, suggesting that physical cues may mediate tph2-cathalyzed serotonergic signaling in the brain.

  10. Increased activity of tyrosine hydroxylase in the cerebellum of the x-irradiated dystonic rat

    Energy Technology Data Exchange (ETDEWEB)

    Dopico, A.M.; Rios, H.; Mayo, J.; Zieher, L.M. (Departamentos de Biologia Celular e Histologia y de Farmacologia y Toxicologia, Facultad de Medicina, Universidad de Buenos Aires, (Argentina))

    1990-08-01

    The exposure of the cephalic end of rats to repeated doses of x-irradiation (150 rad) immediately after birth induces a long-term increase in the noradrenaline (NA) content of cerebellum (CE) (+ 37.8%), and a decrease in cerebellar weight (65.2% of controls), which results in an increased NA concentration (+ 109%). This increase in the neurotransmitter level is accompanied by a dystonic syndrome and histological abnormalities: Purkinje cells (the target cells for NA afferents to CE) fail to arrange in a characteristic monolayer, and their primary dendritic tree appears randomly oriented. The injection of reserpine 0.9 and 1.2 mg/kg ip to adult rats for 18 h depletes cerebellar NA content in both controls (15.7 {plus minus} 4 ng/CE and 2.8 {plus minus} 1.5 ng/CE, respectively) and x-irradiated rats (17.1 {plus minus} 1 ng/CE and 8.3 {plus minus} 2 ng/CE, respectively). The activity of tyrosine hydroxylase (TH) in CE of adult rats, measured by an in vitro assay, is significantly increased in neonatally x-irradiated animals when compared to age-matched controls (16.4 {plus minus} 1.4 vs 6.32 {plus minus} 0.6 nmol CO2/h/mg prot., p less than 0.01). As observed for NA levels, a net increase in TH activity induced by the ionizing radiation is also measured: 308.9 {plus minus} 23.8 vs 408.2 {plus minus} 21.5 nmol CO2/h/CE, p less than 0.01 (controls and x-treated, respectively). These results suggest that x-irradiation at birth may induce an abnormal sprouting of noradrenergic afferents to CE. The possibility that these changes represent a response of the NA system to the dystonic syndrome is discussed.

  11. A Rare Combination: Congenital Adrenal Hyperplasia Due To 21 Hydroxylase Deficiency and Turner Syndrome

    Science.gov (United States)

    Peltek Kendirci, Havva Nur; Aycan, Zehra; Çetinkaya, Semra; Baş, Veysel Nijat; Ağladıoğlu, Sebahat Yılmaz; Önder, Aşan

    2012-01-01

    A combination of Turner syndrome (TS) and classical congenital adrenal hyperplasia (CAH) is rare. A one-day-old newborn was referred to our hospital with ambiguous genitalia. The parents were third-degree relatives. The infant’s weight was 3350g (50-75p), and the head circumference was 34.5cm (50p). The gonads were nonpalpable. Presence of a 3 cm phallus, one urogenital opening into the perineum, and incomplete labial fusion were identified. Laboratory tests revealed a classical type of CAH due to 21-hydroxylase deficiency. Karyotyping revealed a 45X0(35)/46XX(22) pattern with negative sex-determining region Y (SRY) on gene analysis. At the most recent follow-up visit, the patient appeared to be in good health - her height was 70.4 cm [-1.5 standard deviation (SD)] and her weight was 9.8 kg (0.3 SD). She was receiving hydrocortisone in a dose of 10 mg/m2/day, fludrocortisone acetate in a dose of 0.075 mg/day, and oral salt of 1 g/day. System examinations were normal. The patient’s electrolyte levels were found to be normal and she was in good metabolic control. The findings of this patient demonstrate that routine karyotyping during investigation of patients with sexual differentiation disorders can reveal TS. Additionally, signs of virilism should always be investigated at diagnosis or during physical examinations for follow-up of TS cases. [i][/i]SRY analysis should be performed primarily when signs of virilism are observed. CAH should also be considered in patients with negative [i]SRY[/i]. Conflict of interest:None declared. PMID:23261864

  12. Isolation and Expression Analysis of Two Genes Encoding Cinnamate 4-Hydroxylase from Cotton (Gossypium hirsutum)

    Institute of Scientific and Technical Information of China (English)

    NI Zhi-yong; LI Bo; Neumann MPeter; L Meng; FAN Ling

    2014-01-01

    Two genes (GhC4H1 and GhC4H2) that encode putative cotton cinnamate 4-hydroxylases that catalyze the second step in the phenylpropanoid pathway were isolated from developing cotton ifbers. GhC4H1 and GhC4H2 each contain open reading frames of 1 518 base pairs (bp) in length and both encode proteins consisting of 505 amino acid residues. They are 90.89%identical to each other at the amino acid sequence level and belong to class I of plant C4Hs. GhC4H1 and GhC4H2 genomic DNA are 2 247 and 2 161 bp long, respectively, and contain two introns located at conserved positions relative to the coding sequence. GhC4H1 and GhC4H2 promoters were isolated and found to contain many cis-elements (boxes P, L and AC-I element) previously identiifed in the promoters of other phenylpropanoid pathway genes. Histochemical staining showed GUS expression driven by the GhC4H1 and GhC4H2 promoters in ovules and ifbers tissues. GhC4H1 and GhC4H2 were also widely expressed in other cotton tissues. GhC4H2 expression reached its highest level during the elongation stage of ifber development, whereas GhC4H1 expression increased during the secondary wall development period in cotton ifbers. Our results contribute to a better understanding of the biochemical role of GhC4H1 and GhC4H2 in cotton ifber development.

  13. Alzheimer disease and type 2 diabetes mellitus: the link to tyrosine hydroxylase and probable nutritional strategies.

    Science.gov (United States)

    Aliev, Gjumrakch; Shahida, Khan; Gan, Siew Hua; Firoz, Ck; Khan, Aziz; Abuzenadah, Adel M; Kamal, Warda; Kamal, Mohammad A; Tan, Yi; Qu, Xianqin; Reale, Marcella

    2014-04-01

    Alzheimer disease (AD) and type 2 diabetes mellitus (T2DM) are chronic health disorders that affect millions of people around the world. According to recent studies, there are molecular similarities in the inflammatory pathways involved in both AD and T2DM, which opens a new avenue for researchers with different perspectives to target the cause of these diseases rather than their obvious symptoms. Several links between inflammation, cardiovascular disease, T2DM and central nervous system disorders such as AD and Parkinson's disease have been elucidated. Mutations in the hippocampal-β-amyloid precursor protein gene in genetically high-risk individuals have been shown to cause the early onset of AD symptoms. The overexpression of β-amyloid protein in the hippocampal region and the synaptotoxicity that occurs as a result have been considered a typical feature of AD and leads to neuronal loss and cognitive decline. However, the identity of the cellular components that cause the late onset of the disease seen in the majority of the cases is still unknown. Synaptic insults associated with neuronal dysfunction may involve several cascades and molecules, one of which has been hypothesized to be tyrosine hydroxylase (TH). The axons of the noradrenergic cells that project to the hippocampus appear to be affected by the β-amyloid protein, which subsequently contributes to TH loss in Alzheimer brain cells. In this review, we attempt to shed light on the important mechanisms involved in AD as well as T2DM such as inflammatory factors, abnormalities in the insulin signaling system and the possible role of the endocrine enzyme TH.

  14. Etamicastat, a new dopamine-ß-hydroxylase inhibitor, pharmacodynamics and metabolism in rat.

    Science.gov (United States)

    Loureiro, Ana I; Bonifácio, Maria João; Fernandes-Lopes, Carlos; Igreja, Bruno; Wright, Lyndon C; Soares-da-Silva, Patrício

    2014-10-05

    Despite the importance of sympathetic nervous system in pathophysiological mechanisms of cardiac heart failure and essential hypertension, therapy specifically targeting the sympathetic nervous system is currently underutilized. Etamicastat is a novel dopamine-ß-hydroxylase (DBH) inhibitor that is oxidized into BIA 5-965 and deaminated followed by oxidation to BIA 5-998, which represents 13% of total etamicastat and quantified metabolites. However, the primary metabolic pathway of etamicastat in rats was found to be the N-acetylation (BIA 5-961), which represents 44% of total etamicastat and quantified metabolites. Trace amounts of BIA 5-961 de-sulfated and S-glucuronide were also detected. All the main metabolites of etamicastat inhibited DBH with IC50 values of 306 (228, 409), 629 (534, 741), 427 (350, 522) nM for BIA 5-965, BIA 5-998 and BIA 5-961, respectively. However, only etamicastat (IC50 of 107 (94; 121) nM) was able to reduce catecholamine levels in sympathetic nervous system innervated peripheral tissues, without effect upon brain catecholamines. Quantitative whole body autoradiography revealed a limited transfer of etamicastat related radioactivity to brain tissues and the mean recovery of radioactivity was ~90% of the administered radioactive dose, eliminated primarily via renal excretion over 5 days. The absolute oral bioavailability of etamicastat was 64% of the administered dose. In conclusion, etamicastat is a peripheral selective DBH inhibitor mainly N-acetylated in the aminoethyl moiety and excreted in urine. Etamicastat main metabolites inhibit DBH, but only etamicastat demonstrated unequivocal pharmacological effects as a DBH inhibitor with impact upon the activity of the sympathetic nervous system under in vivo conditions.

  15. Prolyl hydroxylase inhibitors protect from the bone loss in ovariectomy rats by increasing bone vascularity.

    Science.gov (United States)

    Liu, Xiaodong; Tu, Yihui; Zhang, Lianfang; Qi, Jin; Ma, Tong; Deng, Lianfu

    2014-05-01

    The hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway is involved in skeletal development, bone repair, and postmenopausal osteoporosis. Inhibitors of prolyl hydroxylases (PHD) enhance vascularity, increase callus formation in a stabilized fracture model, and activate the HIF-1α/VEGF pathway. This study examined the effects of estrogen on the HIF-1α/VEGF pathway in osteoblasts and whether PHD inhibitors can protect from bone loss in postmenopausal osteoporosis. Osteoblasts were treated with estrogen, and expressions of HIF-1α and VEGF were measured at mRNA (qPCR) and protein (Western blot) levels. Further, osteoblasts were treated with inhibitors of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, and levels of VEGF mRNA and protein expression were detected. In addition, ovariectomized rats were treated with PHD inhibitors, and bone microarchitecture and bone mechanical strength were assessed using micro-CT and biomechanical analyses (lower ultimate stress, modulus, and stiffness). Blood vessel formation was measured with India Ink Perfusion and immunohistochemistry. Estrogen, in a dose- and time-dependent manner, induced VEGF expression at both mRNA and protein levels and enhanced HIF-1α protein stability. Further, the estrogen-induced VEGF expression in osteoblasts involved the PI3K/Akt pathway. PHD inhibitors increased bone mineral density, bone microarchitecture and bone mechanical strength, and promoted blood vessel formation in ovariectomized rats. In conclusion, estrogen and PHD inhibitors activate the HIF-1α/VEGF pathway in osteoblasts. PHD inhibitors can be utilized to protect bone loss in postmenopausal osteoporosis by improving bone vascularity and angiogenesis in bone marrow.

  16. Ethnic disparity in 21-hydroxylase gene mutations identified in Pakistani congenital adrenal hyperplasia patients

    Directory of Open Access Journals (Sweden)

    Jabbar Abdul

    2011-02-01

    Full Text Available Abstract Background Congenital adrenal hyperplasia (CAH is a group of autosomal recessive disorders caused by defects in the steroid 21 hydroxylase gene (CYP21A2. We studied the spectrum of mutations in CYP21A2 gene in a multi-ethnic population in Pakistan to explore the genetics of CAH. Methods A cross sectional study was conducted for the identification of mutations CYP21A2 and their phenotypic associations in CAH using ARMS-PCR assay. Results Overall, 29 patients were analyzed for nine different mutations. The group consisted of two major forms of CAH including 17 salt wasters and 12 simple virilizers. There were 14 phenotypic males and 15 females representing all the major ethnic groups of Pakistan. Parental consanguinity was reported in 65% cases and was equally distributed in the major ethnic groups. Among 58 chromosomes analyzed, mutations were identified in 45 (78.6% chromosomes. The most frequent mutation was I2 splice (27% followed by Ile173Asn (26%, Arg 357 Trp (19%, Gln319stop, 16% and Leu308InsT (12%, whereas Val282Leu was not observed in this study. Homozygosity was seen in 44% and heterozygosity in 34% cases. I2 splice mutation was found to be associated with SW in the homozygous. The Ile173Asn mutation was identified in both SW and SV forms. Moreover, Arg357Trp manifested SW in compound heterozygous state. Conclusion Our study showed that CAH exists in our population with ethnic difference in the prevalence of mutations examined.

  17. Cloning of cDNA encoding steroid 11. beta. -hydroxylase (P450c11)

    Energy Technology Data Exchange (ETDEWEB)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-10-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11..beta..-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11..beta..-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.

  18. Role for threonine 201 in the catalytic cycle of the soluble diiron hydroxylase toluene 4-monooxygenase.

    Science.gov (United States)

    Elsen, Nathaniel L; Bailey, Lucas J; Hauser, Andrew D; Fox, Brian G

    2009-05-12

    The active site residue Thr-201 in toluene 4-monooxygenase hydroxylase (T4moH) has a structural counterpart in the active sites of all diiron monooxygenases. Thus, our previous finding that mutation of this residue to Ala, Gly, or Ser had no impact on steady-state catalysis or coupling was surprising. In this work, we provide kinetic, biochemical, and structural evidence that one role of Thr-201 may be to stabilize a peroxo-level intermediate during enzyme catalysis. During reactions in the absence of substrate, T201 T4moH slowly consumed O(2) but only a negligible amount of H(2)O(2) was released. In contrast, T201A T4moH gave stoichometric release of H(2)O(2) during reaction in the absence of substrate. Both enzyme isoforms were tightly coupled during steady-state catalysis with saturating toluene and other optimal substrates and exhibited near-identical kinetic parameters. However, rapid mix single-turnover studies showed that T201A T4moH had a faster first-order rate constant for product formation than T201 T4moH did. Comparison of X-ray crystal structures of resting and reduced T201A T4moH in complex with T4moD with comparable structures of T201 T4moHD revealed changes in the positions of several key active site residues relative to the comparable structures of T201 T4moH with T4moD. This combination of catalytic and structural studies offers important new insight into the role of the role of conserved Thr-201, and its contributions to the catalytic reaction cycle.

  19. Gender role across development in adult women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

    Science.gov (United States)

    Long, Dominique N; Wisniewski, Amy B; Migeon, Claude J

    2004-10-01

    This study evaluated the degree of femininity and masculinity at different developmental stages in a group of adult women, some of whom were exposed to elevated prenatal adrenal androgens as a result of congenital adrenal hyperplasia (CAH) due to 21 hydroxylase (21-OH) deficiency. Women who had presented to the Johns Hopkins Hospital Pediatric Endocrine Clinic for treatment of CAH due to 21-OH deficiency were included. The control group consisted of sisters of CAH participants and women referred for evaluation of polycystic ovary syndrome. Study participants were given a questionnaire asking them to indicate their degree of masculinity and femininity during childhood, adolescence, and adulthood. In addition, participants were asked questions related to their play behavior during childhood, including playmate preferences, toy preferences, and admiration of male or female characters during fantasy play. Across participant groups, self-reported femininity decreased in a dose response manner, according to prenatal androgen exposure. For all groups, femininity increased through developmental stages. Women with salt-losing CAH remained less feminine than controls into adulthood. Conversely, self-reported masculinity increased in a dose-response manner, according to prenatal androgen exposure, across participant groups. Women with CAH showed a decrease in masculinity across developmental stages, such that by adulthood, there were no significant differences in masculinity between controls and the women with CAH. Women with salt-losing CAH were more likely to recall preferences for boy playmates, male-typical toys, and admiration for male characters during childhood than other study participants. Our data support the effect of both prenatal androgen exposure and socialization on gender role behavior in adult women with CAH due to 21-OH deficiency.

  20. Chronic inhibition of dopamine β-hydroxylase facilitates behavioral responses to cocaine in mice.

    Directory of Open Access Journals (Sweden)

    Meriem Gaval-Cruz

    Full Text Available The anti-alcoholism medication, disulfiram (Antabuse, decreases cocaine use in humans regardless of concurrent alcohol consumption and facilitates cocaine sensitization in rats, but the functional targets are unknown. Disulfiram inhibits dopamine β-hydroxylase (DBH, the enzyme that converts dopamine (DA to norepinephrine (NE in noradrenergic neurons. The goal of this study was to test the effects of chronic genetic or pharmacological DBH inhibition on behavioral responses to cocaine using DBH knockout (Dbh -/- mice, disulfiram, and the selective DBH inhibitor, nepicastat. Locomotor activity was measured in control (Dbh +/- and Dbh -/- mice during a 5 day regimen of saline+saline, disulfiram+saline, nepicastat+saline, saline+cocaine, disulfiram+cocaine, or nepicastat+cocaine. After a 10 day withdrawal period, all groups were administered cocaine, and locomotor activity and stereotypy were measured. Drug-naïve Dbh -/- mice were hypersensitive to cocaine-induced locomotion and resembled cocaine-sensitized Dbh +/- mice. Chronic disulfiram administration facilitated cocaine-induced locomotion in some mice and induced stereotypy in others during the development of sensitization, while cocaine-induced stereotypy was evident in all nepicastat-treated mice. Cocaine-induced stereotypy was profoundly increased in the disulfiram+cocaine, nepicastat+cocaine, and nepicastat+saline groups upon cocaine challenge after withdrawal in Dbh +/- mice. Disulfiram or nepicastat treatment had no effect on behavioral responses to cocaine in Dbh -/- mice. These results demonstrate that chronic DBH inhibition facilitates behavioral responses to cocaine, although different methods of inhibition (genetic vs. non-selective inhibitor vs. selective inhibitor enhance qualitatively different cocaine-induced behaviors.

  1. Molecular cloning and characterization of a flavanone-3-hydroxylase gene from rubus occidentalis L

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Sik; Lee, Eun Mi; An, Byung Chull; Barampuram, Shyamkumar; Kim, Jae Sung; Chung, Jae Sung [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Cho, Jae Young [Dept. of Applied Life Sciences, Chonbuk National University, Jeonju (Korea, Republic of); Lee, In Chul [Senior Industry Cluster Agency, Youngdong University, Youngdong (Korea, Republic of)

    2008-08-15

    Flavanone-3-hydroxylase (F3H) is one of the key enzymes for the biosynthesis of flavonals, anthocyanins, catechins and proanthocyanins. F3H catalyzes the 3β-hydroxylation of (2S)-flavonones to form (2R, 3R)-dihydroflavonols. In this report, we isolated a full-length cDNA of RocF3H from black raspberry (Rubus occidentalis L.) using a reverse transcriptase-PCR and rapid amplification of the cDNA ends (RACE)-PCR. The full-length cDNA of RocF3H contains a 1,098 bp open reading frame (ORF) encoding a 365 amino acid protein with a calculated molecular weight of about 41.1 kDa and isoelectric point (pI) of 5.45. The genomic DNA analysis revealed that the RocF3H gene had three exons and two introns. Comparison of the deduced amino acid sequence of the RocF3H with other F3Hs revealed that the protein is highly homologous with various plant species. The conserved amino acids ligating the ferrous iron and the residues participating in the 2-oxoglutarate binding (R-X-S) were found in RocF3H at the similar positions to other F3Hs. Southern blot analysis indicated that RocF3H exist a multi-gene family. The isolation of RocF3H gene will be helpful to further study the role of F3H gene in the biosynthesis of flavonoids in R. occidnetalis.

  2. Mutations distribution and correlation with phenotypes in steroid 21-hydroxylase deficiency Italian patients

    Energy Technology Data Exchange (ETDEWEB)

    Carrera, P.; Volorio, S.; Ferran, M. [and others

    1994-09-01

    Steroid 21-hydroxylase deficiency is recessively inherited and accounts for over 90% of the genetic disorders of steroidogenesis (CAH). We previously described the distribution of the whole deletion (14.4%) and large scale gene conversion (7.8%) at the P450c21-B locus in our population. In this study we determined the distribution of seven point mutations and searched for new mutations in patients where large rearrangements were not found. For this purpose we have studied 45 Italian families using a P450c21-B-specific PCR in combination with either dot blot analysis and allele-specific oligonucleotide hybridization or by cloning and sequencing. Molecular results have indicated a high frequency of point mutations (61%) corresponding to deleterious sequences normally present in the pseudogene. In particular, only 3 of them were prominent: the splicing mutation at codon 281 (9/16) was the most common within the non-classic form. By cloning and sequencing we detected a deletion of the C2029 residue causing a frameshift and the downstream insertion of a stop codon (2124-2126). This mutation was found in a non-classical patient who is a compound heterozygote for the mutation 281. Family genotyping revealed 5 de novo mutations, and in 8 asymptomatic parents, we detected causative mutations in both alleles. These data suggest that phenotype is not always correlated to allelic variations in P450c21-B genes. For this reason, in these families prenatal diagnosis should be performed by direct detection of mutations instead of linkage analysis.

  3. Height outcome of patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

    Institute of Scientific and Technical Information of China (English)

    Feng Yun-lin; Zhu Hui-juan; Gong Feng-ying; Li Nai-shi; Pan Hui; Lu Zhao-lin; Shi Yi-fan

    2007-01-01

    Objective: To retrospectively investigate the height outcome of patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD).Methods: The 135 CAH patients with 21-OHD diagnosed in our hospital from Jan 1980 to Oct 2006 were retrospectively analyzed.The investigated parameters included final height(FH), FH standard deviation score(FH SDS), target height SDS (TH SDS), difference between TH and FH (TH-FH), FH SDS-TH SDS, the age of onset of sexual development, and the difference between bone age and chronological age(BA-CA) when patients got the FH.Results: Among the 135 patients, female/male=108/27.Mean FH was (156.84±5.4) cm (n=14) and (150.8±6.8) cm (n=76) for males and females, respectively.Mean FH SDS was (-0.6±0.8) (n=13) and (0.2±1.2) (n=54) for males and females, respectively.Sexual development began at (5.2±1.7) years old (y/o) (n=13) and (7.9±3.2) y/o (n=43) in males and females, respectively.Conclusions: The FH of CAH patients with 21-OHD was lower than that of the normal range.Effect of the disease on the height growth in male patients was more severe than that in females.All patients began sexual development much earlier than the normal age-matched group.Male patients began their sexual development even earlier.

  4. Spatial distribution of synapses on tyrosine hydroxylase-expressing juxtaglomerular cells in the mouse olfactory glomerulus.

    Science.gov (United States)

    Kiyokage, Emi; Kobayashi, Kazuto; Toida, Kazunori

    2017-04-01

    Olfactory sensory axons converge in specific glomeruli where they form excitatory synapses onto dendrites of mitral/tufted (M/T) and juxtaglomerular (JG) cells, including periglomerular (PG), external tufted (ET), and superficial-short axon cells. JG cells consist of heterogeneous subpopulations with different neurochemical, physiological, and morphological properties. Among JG cells, previous electron microscopic (EM) studies have shown that the majority of synaptic inputs to tyrosine hydroxylase (TH)-immunoreactive neurons were asymmetrical synapses from olfactory nerve (ON) terminals. However, recent physiological results revealed that 70% of dopaminergic/γ-aminobutyric acid (GABA)ergic neurons received polysynaptic inputs via ET cells, whereas the remaining 30% received monosynaptic ON inputs. To understand the discrepancies between EM and physiological data, we used serial EM analysis combined with confocal laser scanning microscope images to examine the spatial distribution of synapses on dendrites using mice expressing enhanced green fluorescent protein under the control of the TH promoter. The majority of synaptic inputs to TH-expressing JG cells were from ON terminals, and they preferentially targeted distal dendrites from the soma. On the other hand, the numbers of non-ON inputs were fewer and targeted proximal dendrites. Furthermore, individual TH-expressing JG cells formed serial synapses, such as M/T→TH→another presumed M/T or ON→TH→presumed M/T, but not reciprocal synapses. Serotonergic fibers also associated with somatic regions of TH neurons, displaying non-ON profiles. Thus, fewer proximal non-ON synapses provide more effective inputs than large numbers of distal ON synapses and may occur on the physiologically characterized population of dopaminergic-GABAergic neurons (70%) that receive their most effective inputs indirectly via an ON→ET→TH circuit. J. Comp. Neurol. 525:1059-1074, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley

  5. Prolyl-4-hydroxylase 3 (PHD3 expression is downregulated during epithelial-to-mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    Trenton L Place

    Full Text Available Prolyl-4-hydroxylation by the intracellular prolyl-4-hydroxylase enzymes (PHD1-3 serves as a master regulator of environmental oxygen sensing. The activity of these enzymes is tightly tied to tumorigenesis, as they regulate cell metabolism and angiogenesis through their control of hypoxia-inducible factor (HIF stability. PHD3 specifically, is gaining attention for its broad function and rapidly accumulating array of non-HIF target proteins. Data from several recent studies suggest a role for PHD3 in the regulation of cell morphology and cell migration. In this study, we aimed to investigate this role by closely examining the relationship between PHD3 expression and epithelial-to-mesenchymal transition (EMT; a transcriptional program that plays a major role in controlling cell morphology and migratory capacity. Using human pancreatic ductal adenocarcinoma (PDA cell lines and Madin-Darby Canine Kidney (MDCK cells, we examined the correlation between several markers of EMT and PHD3 expression. We demonstrated that loss of PHD3 expression in PDA cell lines is highly correlated with a mesenchymal-like morphology and an increase in cell migratory capacity. We also found that induction of EMT in MDCK cells resulted in the specific downregulation of PHD3, whereas the expression of the other HIF-PHD enzymes was not affected. The results of this study clearly support a model by which the basal expression and hypoxic induction of PHD3 is suppressed by the EMT transcriptional program. This may be a novel mechanism by which migratory or metastasizing cells alter signaling through specific pathways that are sensitive to regulation by O2. The identification of downstream pathways that are affected by the suppression of PHD3 expression during EMT may provide important insight into the crosstalk between O2 and the migratory and metastatic potential of tumor cells.

  6. A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity.

    Science.gov (United States)

    Huebener, Nicole; Fest, Stefan; Strandsby, Anne; Michalsky, Elke; Preissner, Robert; Zeng, Yan; Gaedicke, Gerhard; Lode, Holger N

    2008-07-01

    Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I antigen H2-K(k) according to the prediction program SYFPEITHI and computer modeling of epitopes into the MHC class I antigen binding groove. Subsequently, a DNA minigene vaccine was generated based on the expression vector pCMV-F3Ub encoding mutated ubiquitin (Gly(76) to Ala(76)) and mTH3. Prophylactic and therapeutic efficacies of this vaccine were established following oral delivery with attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependent on ubiquitin and high affinity of the mTH epitopes to MHC class I antigens. Specifically, we showed a crucial role for minigene expression as a stable ubiquitin-Ala(76) fusion peptide for vaccine efficacy. The immune response following the mTH3 DNA minigene vaccination was mediated by CD8(+) T cells as indicated by infiltration of primary tumors and TH-specific cytolytic activity in vitro. Importantly, no cell infiltration was detectable in TH-expressing adrenal medulla, indicating the absence of autoimmunity. In summary, we show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy.

  7. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Zhichao [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Yu, Xuemei [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China); Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Yintao [Institute of Endocrinology and Diabetology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai (China); Yang, Lili [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China); Ruan, Yuanyuan; Gu, Jianxin [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Ren, Shifang, E-mail: renshifang@fudan.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Songwen, E-mail: songwenzhang@fudan.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)

    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 that 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.

  8. Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene

    Energy Technology Data Exchange (ETDEWEB)

    Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (United States)); Konecki, D.S.; Lichter-Konecki, U.

    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, 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.

  9. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    Science.gov (United States)

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    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 k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) 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.

  10. Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications.

    Science.gov (United States)

    Flydal, Marte I; Mohn, Tonje C; Pey, Angel L; Siltberg-Liberles, Jessica; Teigen, Knut; Martinez, Aurora

    2010-11-01

    Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h)=2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thórólfsson et al. in Biochemistry 41:7573-7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h=0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus ~1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds ~1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h=1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L: -Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.

  11. Mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Shanxi, China

    Directory of Open Access Journals (Sweden)

    Yong-An Zhou

    2012-01-01

    Full Text Available The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH gene was investigated in 59 children with phenylketonuria (PKU and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%, followed by Ivs7 +2T>A (5.1% and T278I (2.5%. G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.

  12. Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

    Energy Technology Data Exchange (ETDEWEB)

    Guldberg, P.; Henriksen, K.F.; Guettler, F. [John F. Kennedy Inst., Glostrup (Denmark)] [and others

    1996-07-01

    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

  13. Effects of tetrahydrobiopterin and phenylalanine on in vivo human phenylalanine hydroxylase by phenylalanine breath test.

    Science.gov (United States)

    Okano, Yoshiyuki; Takatori, Kazuhiko; Kudo, Satoshi; Sakaguchi, Tomoko; Asada, Minoru; Kajiwara, Masahiro; Yamano, Tsunekazu

    2007-12-01

    BH(4) administration results in the reduction of blood phenylalanine level in patients with tetrahydrobiopterin (BH(4))-responsive phenylalanine hydroxylase (PAH) deficiency. The mechanism underlying BH(4) response remains unknown. Here, we studied the effects of BH(4) and phenylalanine on in vivo PAH activity of normal controls using the phenylalanine breath test (PBT) by converting l-[1-(13)C] phenylalanine to (13)CO(2). Phenylalanine oxidation rates were expressed as Delta(13)C ((13)CO(2)/(12+13)CO(2), per thousand) and cumulative recovery rates over 120min (CRR(120), %; total amount of (13)CO(2)/the administered dose of (13)C-phenylalanine). Under physiological conditions of blood phenylalanine, BH(4) administration reduced the Delta(13)C peak from 40.8 per thousand to 21.6 per thousand and CRR(120) from 16.9% to 10.2%. Under high blood phenylalanine conditions, administration of BH(4) increased the Delta(13)C peak from 30.7 per thousand to 46.0 per thousand, while the CRR(120) was similar between phenylalanine (19.9%) and phenylalanine+BH(4) (21.1%) groups. Corrected Delta(13)C and CRR(120) were calculated against serum phenylalanine levels to remove the effects of phenylalanine loading. After BH(4) administration, the corrected Delta(13)C peak increased from 82.7 per thousand to 112.6 per thousand, while the corrected CRR(120) was similar (47.6% and 45.6%). These results indicate that phenylalanine worked as a regulator of in vivo PAH by serving as both a substrate and an activator for the enzyme. Excessive dosages of BH(4) inhibited PAH under normal phenylalanine conditions and activated PAH under conditions of high phenylalanine. The regulation system is therefore designed to maintain phenylalanine levels in the human body. Appropriate BH(4) supplementation must be reviewed in patients with BH(4)-responsive PAH deficiency.

  14. Activation of phenylalanine hydroxylase induces positive cooperativity toward the natural cofactor.

    Science.gov (United States)

    Gersting, Søren W; Staudigl, Michael; Truger, Marietta S; Messing, Dunja D; Danecka, Marta K; Sommerhoff, Christian P; Kemter, Kristina F; Muntau, Ania C

    2010-10-01

    Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate L-Phenylalanine and its natural cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)). Sapropterin dihydrochloride, the synthetic form of BH(4), was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH(4), a previously unknown finding. Analyses of structurally preactivated variants substantiated BH(4)-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient's metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.

  15. In vivo regulation of phenylalanine hydroxylase in the genetic mutant hph-1 mouse model.

    Science.gov (United States)

    Gunasekera, Richard S; Hyland, Keith

    2009-11-01

    The hph-1 mouse has low liver activity of GTP cyclohydrolase 1, the rate limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH(4)). BH(4) is the cofactor for phenylalanine hydroxylase (PAH) and in the early stages of life the hph-1 mouse is hyperphenylalaninemic. At approximately 15 days after birth the blood phenylalanine levels normalize. During this period the animals provide an in vivo model which can be used to study the regulatory effects of phenylalanine on PAH, and for related pediatric metabolic disease in humans; from birth to youth. We therefore, examined; liver PAH activity using BH(4) and 6-methyltetrahydropterin (6MPH(4)) as cofactor; PAH total enzyme concentration by Western blotting using the PH8 antibody, and PAH state of phosphorylation using the PH7 antibody from 4 to 18 days after birth. The findings were compared to the wild type animals that are not hyperphenylalaninemic during this period. PAH (6MPH(4)) activity and total protein (PH8 antibody) rose steadily in the hph-1 mice. In control mice, both activity and total protein fluctuated. The degree of phosphorylation of PAH in the mutants and the state of activation (as measured by the 6MPH(4)/BH(4) activity ratio) increased as phenylalanine levels rose, and decreased when they fell. Similar patterns were not seen in the control animals. These studies provide in vivo evidence that phenylalanine concentration regulates the activity of PAH in the hph-1 mouse and that this acts via a mechanism that includes phosphorylation of the PAH molecule. The kinetic values (K(m) and V(max)) for mouse PAH are also reported.

  16. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

    Science.gov (United States)

    Arturo, Emilia C; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J; Parker, Emily J; Loll, Patrick J; Jaffe, Eileen K

    2016-03-01

    Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.

  17. In vitro residual activity of phenylalanine hydroxylase variants and correlation with metabolic phenotypes in PKU.

    Science.gov (United States)

    Trunzo, Roberta; Santacroce, Rosa; Shen, Nan; Jung-Klawitter, Sabine; Leccese, Angelica; De Girolamo, Giuseppe; Margaglione, Maurizio; Blau, Nenad

    2016-12-05

    Hyperphenylalaninemias (HPAs) are genetic diseases predominantly caused by a wide range of variants in the phenylalanine hydroxylase (PAH) gene. In vitro expression analysis of PAH variants offers the opportunity to elucidate the molecular mechanisms involved in HPAs and to clarify whether a disease-associated variant is genuinely pathogenic, while investigating the severity of a metabolic phenotype, and determining how a variant exerts its deleterious effects on the PAH enzyme. To study the effects of gene variants on PAH activity, we investigated eight variants: c.611A>G (p.Y204C), c.635T>C (p.L212P), c.746T>C (p.L249P), c.745C>T (p.L249F), c.809G>A (p.R270K), c.782G>C (p.R261P), c.587C>A (p.S196Y) and c.1139C>T (p.T380M), associated with different phenotypic groups. Transient expression of mutant full-length cDNAs in COS-7 cells yielded PAH proteins with PAH activity levels between 7% and 51% compared to the wild-type enzyme. With one exception (p.Y204C, which had no significant impact on PAH function), lower PAH activity was associated with a more severe phenotype (e.g. p.L249P with 7% PAH activity, 100% of classic PKU and no BH4 responsiveness), while higher activity correlated with milder phenotypes (e.g. p.T380M with 28% PAH activity, 97% of mild HPA and 83% of BH4 responsiveness). The results of the in vitro residual PAH activity have major implications, both for our understanding of genotype-phenotype correlations, and thereby existing inconsistencies, but also for the elucidation of the molecular basis of tetrahydrobiopterin (BH4) responsiveness.

  18. 'Roid rage in rats? Testosterone effects on aggressive motivation, impulsivity and tyrosine hydroxylase.

    Science.gov (United States)

    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, pmotivation 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, pmotivation 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 brain area important for behavioral inhibition, motor control and habit learning. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Allosteric activation of midazolam CYP3A5 hydroxylase activity by icotinib - Enhancement by ketoconazole.

    Science.gov (United States)

    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 Vmax values in HLM and rhCYP3A5 with no significant changes in Km values. By adding CYP3cide with ICO to the incubation, the Vmax values increased 2-fold over the CYP3cide control. Addition of ketoconazole with ICO alone or ICO plus CYP3cide resulted in an increase in Vmax values and decrease in Km 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.

  20. Domain Characterization of Cyclosporin Regio-Specific Hydroxylases in Rare Actinomycetes.

    Science.gov (United States)

    Woo, Min-Woo; Lee, Bo-Ram; Nah, Hee-Ju; Choi, Si-Sun; Li, Shengying; Kim, Eung-Soo

    2015-10-01

    Cytochrome P450 hydroxylase (CYP) in actinomycetes plays an important role in the biosynthesis and bioconversion of various secondary metabolites. Two unique CYPs named CYP-sb21 and CYP-pa1, which were identified from Sebekia benihana and Pseudonocardia autotrophica, respectively, were proven to transfer a hydroxyl group at the 4(th) or 9(th) N-methyl leucine position of immunosuppressive agent cyclosporin A (CsA). Interestingly, these two homologous CYPs showed different CsA regio-selectivities. CYP-sb21 exhibited preferential hydroxylation activity at the 4(th) position over the 9(th) position, whereas CYP-pa1 showed the opposite preference. To narrow down the CYP domain critical for CsA regio-selectivity, each CYP was divided into four domains, and each domain was swapped with its counterpart from the other CYP. A total of 18 hybrid CYPs were then individually tested for CsA regioselectivity. Although most of the hybrid CYPs failed to exhibit a significant change in regioselectivity in the context of CsA hydroxylation, hybrid CYP-pa1 swapped with the second domain of CYP-sb21 showed a higher preference for the 9th position. Moreover, hybrid CYPsb21 containing seven amino acids from the 2nd domain of CYP-pa1 showed higher preference for the 4(th) position. These results imply that the 2nd domain of CsA-specific CYP plays a critical role in CsA regio-selectivity, thereby setting the stage for biotechnological application of CsA regio-selective hydroxylation.

  1. The presence of hydrocarbons in southeast Norway

    DEFF Research Database (Denmark)

    Hanken, Niels Martin; Hansen, Malene Dolberg; Kresten Nielsen, Jesper

    Hydrocarbons, mostly found as solid pyrobitumen, are known from more than 30 localities in southeast Norway. They occur as inclusions in a wide range of "reservoir rocks" spanning from Permo-Carboniferous breccias to veins (vein quartz and calcite veins) in Precambrian granites, gneisses and amph......Hydrocarbons, mostly found as solid pyrobitumen, are known from more than 30 localities in southeast Norway. They occur as inclusions in a wide range of "reservoir rocks" spanning from Permo-Carboniferous breccias to veins (vein quartz and calcite veins) in Precambrian granites, gneisses...

  2. Mathematics of Periodic Tables for Benzenoid Hydrocarbons.

    Science.gov (United States)

    Dias, Jerry Ray

    2007-01-01

    The upper and lower bounds for invariants of polyhex systems based on the Harary and Harborth inequalities are studied. It is shown that these invariants are uniquely correlated by the Periodic Table for Benzenoid Hydrocarbons. A modified periodic table for total resonant sextet (TRS) benzenoids based on the invariants of Ds and r(empty) is presented; Ds is the number of disconnections among the empty rings for fused TRS benzenoid hydrocarbons. This work represents a contribution toward deciphering the topological information content of benzenoid formulas.

  3. Electrochemical removal of NOx and hydrocarbons

    DEFF Research Database (Denmark)

    Friedberg, Anja Zarah

    on the electrodes during polarisation, probably because of strong adsorption of the hydrocarbon relative to NO. On LSF/CGO electrode the impregnation of ionic conducting material increased the oxidation of NO to NO2 which is an important step before nitrogen formation. The propene inhibited this reaction because....... This could only be done if the electrode was impregnated with BaO. The nitrate formation did not seem to be inhibited by the presence of the hydrocarbon. However, the oxidation of propene was inhibited by the BaO because the active sites for oxidations were partially covered by the BaO nanoparticles...

  4. Transferable Tight-Binding Potential for Hydrocarbons

    CERN Document Server

    Wang, Y; Wang, Yang

    1994-01-01

    A transferable tight-binding potential has been constructed for heteroatomic systems containing carbon and hydrogen. The electronic degree of freedom is treated explicitly in this potential using a small set of transferable parameters which has been fitted to small hydrocarbons and radicals. Transferability to other higher hydrocarbons were tested by comparison with ab initio calculations and experimental data. The potential can correctly reproduce changes in the electronic configuration as a function of the local bonding geometry around each carbon atom. This type of potential is well suited for computer simulations of covalently bonded systems in both gas-phase and condensed-phase systems.

  5. Cuticular hydrocarbons and aggression in the termite Macrotermes subhyalinus.

    Science.gov (United States)

    Kaib, Manfred; Jmhasly, Patrick; Wilfert, Lena; Durka, Walter; Franke, Stephan; Francke, Wittko; Leuthold, Reinhard H; Brandl, Roland

    2004-02-01

    Cuticular hydrocarbons are among the prime candidates for nestmate recognition in social insects. We analyzed the variation of cuticular hydrocarbons in the termite species M. subhyalinus in West Africa (Comoë National Park) on a small spatial scale (recognition.

  6. Kinetic simulating experiment on the secondary hydrocarbon generation of kerogen

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The kinetic parameters of generation have been obtained for different hydrocarbon classes, including methane, C2-C5 gas hydrocarbons, C6-C13 light hydrocarbons and C13+ heavy hydrocarbons, and vitrinite reflectance (Ro) by the kinetic simulating experiment of kerogen cracking. Then, combined with the detailed geology of Sichuan Basin, the effective gas-generating intensity of the Lower Cambrian source rock is approximately estimated by applying these parameters.

  7. 5-HT7 receptors and tryptophan hydroxylase in lymphocytes of rats: mitogen activation, physical restraint or treatment with reserpine.

    Science.gov (United States)

    Urbina, Mary; Arroyo, Rubén; Lima, Lucimey

    2014-01-01

    Serotonin (5-HT)7 receptors in lymphocytes play a relevant role as modulators of T cell functions and might be modified by stress protocols. The aims of this work were to evaluate: (i) the presence of 5-HT7 receptors in specific lymphocyte populations, (ii) the probable modifications of them by inflammatory stress with mitogen and (iii) the effects of physical and pharmacological stress. Blood lymphocytes were isolated by density gradients and differential adhesion to plastic. Concanavalin A (Con A) was systemically administered (500 μg/kg) or added to lymphocyte cultures (2.5 μg/ml, final volume 200 μl). Physical restraint was performed in Plexiglass boxes for 5 h per day for 5 days. Reserpine administration was 2.5 mg/kg for 3 days. Immunocytochemical labeling of CD4+, CD8+ and 5-HT7 receptors, and also tryptophan hydroxylase cells was performed. mRNA of 5-HT7 receptors was evaluated by RT-PCR. Controls were included for each protocol. Con A treatment or culture exposure increased the number of lymphocytes expressing 5-HT7 receptors or tryptophan hydroxylase, as compared to absence of the mitogen. Receptors were present in 12-16% of total rat lymphocytes, in ∼10% of CD4+ and in ∼5% of CD8+ cells from control rats. CD4+ decreased, and CD8+ and 5-HT7 cells increased after physical restraint. Reserpine treatment elevated CD8+ and 5-HT7 cells. Con A and physical restraint, but not reserpine treatment, significantly augmented 5-HT7 receptor mRNA in lymphocytes. Rat lymphocytes, expressing tryptophan hydroxylase, could synthesize 5-HT, functioning as a direct autocrine modulator. The modifications of CD4+, CD8+ and 5-HT7 receptors in lymphocytes by three stress protocols could have an impact on immune responses. In addition, the differential distribution of 5-HT7 receptors indicates potential specific physiopathological roles. © 2014 S. Karger AG, Basel.

  8. A novel NADPH-dependent reductase of Sulfobacillus acidophilus TPY phenol hydroxylase: expression, characterization, and functional analysis.

    Science.gov (United States)

    Li, Meng; Guo, Wenbin; Chen, Xinhua

    2016-12-01

    The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X 4 -Cys-X 2 -Cys-X 29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.

  9. Dietary (+)-catechin and BHT markedly increase alpha-tocopherol concentrations in rats by a tocopherol-omega-hydroxylase-independent mechanism.

    Science.gov (United States)

    Frank, Jan; Lundh, Torbjörn; Parker, Robert S; Swanson, Joy E; Vessby, Bengt; Kamal-Eldin, Afaf

    2003-10-01

    The effects of dietary (+)-catechin (CAT) and BHT on plasma and tissue concentrations of alpha-tocopherol (alpha-T), gamma-tocopherol (gamma-T) and cholesterol (C) were studied in male Sprague-Dawley rats. The rats were fed the compounds during a 4-wk period at concentrations of 2 g/kg in standardized diets, low but adequate in vitamin E, with 2 g/kg cholesterol. The CAT-regimen did not affect weight gain, feed intake or organ weights. BHT did not affect feed intake but lowered the body weight and the amount of liver lipids and increased the weights of livers and lungs relative to the body weight. Rats consuming CAT had 2.5-3.5-fold increased plasma, liver and lung alpha-T concentrations, but C concentrations remained unchanged. BHT-feeding resulted in 2.4- and 1.7-fold elevation in alpha-T but approximately 50% decrease in gamma-T concentrations in blood plasma and liver, respectively. BHT also lowered total C in the liver without affecting the concentration of C in the liver lipids. To investigate whether the alpha-T-sparing action of the studied compounds was due to the inhibition of tocopherol-omega-hydroxylase, HepG2 cells were incubated with CAT or BHT in the presence of delta-tocopherol (delta-T) and the 3'- and 5'-delta-carboxychromanol metabolites in the media were analyzed by GC/MS. Neither CAT nor BHT inhibited tocopherol-omega-hydroxylase activity in hepatocyte cultures; CAT was also inactive in a rat microsomal assay. In conclusion, both dietary CAT and BHT markedly increased alpha-T concentrations in plasma and organs of Sprague-Dawley rats by a mechanism that apparently does not involve inhibition of tocopherol-omega-hydroxylase, a key enzyme in tocopherol catabolism.

  10. Localization of serotonin, tyrosine hydroxylase, and leu-enkephalin immunoreactive cells in the brainstem of the horn shark, Heterodontus francisci.

    Science.gov (United States)

    Stuesse, S L; Cruce, W L; Northcutt, R G

    1991-06-08

    In previous studies on reptiles and elasmobranchs, we determined that some reticular groups are either absent or may be displaced compared to their locations in mammals. For example, nucleus raphe dorsalis, the largest serotoninergic cell group in mammals, is not present in rays, skates, or guitarfish. In the present study, we chose heterodontid sharks, a sister group to these batoids, for an out-group comparison of this and other characters. We identified cells in the brainstem of Heterodontus francisci by use of antibodies against tyrosine hydroxylase, serotonin, or leu-enkephalin and compared the distribution of these nuclei to descriptions in mammals and other elasmobranchs. The majority of tyrosine hydroxylase-positive cells were found in the midbrain tegmentum (A8-A10) and the hypothalamus. In addition, putative A1, A2, A5, A7 (noradrenergic) groups were found in the metencephalon and myelencephalon. Serotonin-positive cells were found in raphe nuclei and scattered lateral to the raphe. We identified probable homologues to raphe pallidus, raphe obscurus, raphe magnus, and raphe centralis superior (B8) cell groups, which have been described in mammals. A cluster of cells dorsomedial to the medial longitudinal fasciculus was identified as raphe dorsalis. The distributions of leu-enkephalin and serotonin immunoreactive cells were similar to each other, but the tyrosine-hydroxylase immunoreactive cells rarely intermingle with the former two immunoreactive cell types. Other reticular groups that contained both serotonin- and leu-enkephalin-positive cells included reticularis (r.) ventralis, r. magnocellularis, r. paragigantocellularis lateralis, r. pontis caudalis, and r. pontis oralis medialis and lateralis. Thus, this shark contains many of the major brainstem raphe and catecholaminergic cell groups described for rats, but the relative distribution of the immunopositive cell groups differs in mammals and cartilaginous fish.

  11. Human Deoxyhypusine Hydroxylase, an Enzyme Involved in Regulating Cell Growth, Activates O2 with a Nonheme Diiron Center

    Energy Technology Data Exchange (ETDEWEB)

    Vu, V.; Emerson, J; Martinho, M; Kim, Y; Munck, E; Park, M; Que, Jr., L

    2009-01-01

    Deoxyhypusine hydroxylase is the key enzyme in the biosynthesis of hypusine containing eukaryotic translation initiation factor 5A (eIF5A), which plays an essential role in the regulation of cell proliferation. Recombinant human deoxyhypusine hydroxylase (hDOHH) has been reported to have oxygen- and iron-dependent activity, an estimated iron/holoprotein stoichiometry of 2, and a visible band at 630 nm responsible for the blue color of the as-isolated protein. EPR, Moessbauer, and XAS spectroscopic results presented herein provide direct spectroscopic evidence that hDOHH has an antiferromagnetically coupled diiron center with histidines and carboxylates as likely ligands, as suggested by mutagenesis experiments. Resonance Raman experiments show that its blue chromophore arises from a (e-1,2-peroxo)diiron(III) center that forms in the reaction of the reduced enzyme with O2, so the peroxo form of hDOHH is unusually stable. Nevertheless we demonstrate that it can carry out the hydroxylation of the deoxyhypusine residue present in the elF5A substrate. Despite a lack of sequence similarity, hDOHH has a nonheme diiron active site that resembles both in structure and function those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein ?9-desaturase from plants, suggesting that the oxygen-activating diiron motif is a solution arrived at by convergent evolution. Notably, hDOHH is the only example thus far of a human hydroxylase with such a diiron active site.

  12. Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation.

    Science.gov (United States)

    Lukacin, R; Gröning, I; Schiltz, E; Britsch, L; Matern, U

    2000-03-15

    Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the course of flavonol/anthocyanin or catechin biosynthesis. The enzyme from Petunia hybrida consists of a 41,655-Da polypeptide that is prone to rapid proteolysis in crude plant extracts as well as on expression in Escherichia coli, and commercial protease inhibitors were inefficient in stopping the degradation. To pinpoint the primary site of proteolysis and to improve the activity yields, two revised schemes of purification were developed for the recombinant polypeptides. Applying a four-step protocol based on extraction and ion-exchange chromatography at pH 7.5, the primary, catalytically inactive proteolytic enzyme fragment (1.1 mg) was isolated and shown to cross-react on Western blotting as one homogeneous band of about 38 kDa. Mass spectrometric analysis assigned a mass of 37,820 +/- 100 Da to this fragment, and partial sequencing revealed an unblocked amino terminus identical to that of the native 3beta-hydroxylase. Thus, the native enzyme had been degraded by proteolysis of a small carboxy-terminal portion, and the primary site of cleavage must be assigned most likely to the Glu 337-Leu 338 bond, accounting for a loss of about 3800 Da. Alternatively, the enzyme degradation was greatly reduced when the extraction of recombinant bacteria was carried out with phosphate buffer at pH 5.5 followed by size exlusion and anion-exchange chromatography. This rapid, two-step purification resulted in a homogeneous 3beta-hydroxylase of high specific acitivity (about 32 mkat/kg) at roughly 5% yield, and the procedure is a major breakthrough in mechanistic investigations of this class of labile dioxygenases.

  13. 40 CFR 86.1221-90 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 19 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 86...-Fueled and Methanol-Fueled Heavy-Duty Vehicles § 86.1221-90 Hydrocarbon analyzer calibration. The FID hydrocarbon analyzer shall receive the following initial and periodic calibrations. (a) Initial and...

  14. 40 CFR 52.987 - Control of hydrocarbon emissions.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 3 2010-07-01 2010-07-01 false Control of hydrocarbon emissions. 52... (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS Louisiana § 52.987 Control of hydrocarbon... compliance date of January 1, 1980. This shall result in an estimated hydrocarbon emission reduction of...

  15. 30 CFR 250.1202 - Liquid hydrocarbon measurement.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 2 2010-07-01 2010-07-01 false Liquid hydrocarbon measurement. 250.1202..., Surface Commingling, and Security § 250.1202 Liquid hydrocarbon measurement. (a) What are the requirements for measuring liquid hydrocarbons? You must: (1) Submit a written application to, and obtain...

  16. 40 CFR 86.331-79 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 18 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 86....331-79 Hydrocarbon analyzer calibration. The following steps are followed in sequence to calibrate the hydrocarbon analyzer. It is suggested, but not required, that efforts be made to minimize relative...

  17. 40 CFR 721.4365 - Substituted ethoxylated hydrocarbon (generic).

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted ethoxylated hydrocarbon... Specific Chemical Substances § 721.4365 Substituted ethoxylated hydrocarbon (generic). (a) Chemical... as Substituted ethoxylated hydrocarbon (PMN P-99-0313) is subject to reporting under this section...

  18. 40 CFR 86.521-90 - Hydrocarbon analyzer calibration.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 18 2010-07-01 2010-07-01 false Hydrocarbon analyzer calibration. 86... Regulations for 1978 and Later New Motorcycles; Test Procedures § 86.521-90 Hydrocarbon analyzer calibration. (a) The FID hydrocarbon analyzer shall receive the following initial and periodic calibration....

  19. Cuticular hydrocarbons from the bed bug Cimex lectularius L.

    Science.gov (United States)

    Pentane extracts of male and female bed bugs were analyzed by gas chromatography and mass spectrometry in an effort to identify cuticular hydrocarbons. Seventeen hydrocarbons accounting for nearly 99% of the compounds eluting in the cuticular hydrocarbon region were identified. The sample contained ...

  20. 21 CFR 573.740 - Odorless light petroleum hydrocarbons.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Odorless light petroleum hydrocarbons. 573.740... ANIMALS Food Additive Listing § 573.740 Odorless light petroleum hydrocarbons. Odorless light petroleum hydrocarbons complying with § 172.884(a) and (b) of this chapter may be safely used in an amount not in...