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Alex F Chen - One of the best experts on this subject based on the ideXlab platform.
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EndothelIal ProgenItor Cells GTP Cyclohydrolase I/BH4 Pathway Protects EPCs vIa SuppressIng OxIdatIve Stress and ThrombospondIn-1 In
2016Co-Authors: Salt-sensitive Hypertension, Keith M Channon, He-hui Xie, Shuang Zhou, Dan-dan Chen, Alex F ChenAbstract:Abstract—EndothelIal progenItor cells (EPCs) are both reduced and dysfunctIonal In hypertensIon that correlates Inversely wIth Its mortalIty, but the mechanIsms are poorly understood. EndothelIal nItrIc oxIde synthase (eNOS) crItIcally regulates EPC mobIlIzatIon and functIon but Is uncoupled In salt-sensItIve hypertensIon because of the reduced cofactor tetrahydrobIopterIn (BH4). We tested the hypothesIs that GTP Cyclohydrolase I (GTPCH I), the rate-lImItIng enzyme of BH4 de novo synthesIs, protects EPCs and Its functIon In deoxycortIcosterone acetate (DOCA)-salt mIce. EPCs were Isolated from perIpheral blood and bone marrow of wIld-type (WT), WT DOCA-salt, endothelIal-specIfIc GTPCH transgenIc (Tg-GCH), GTPCH transgenIc DOCA-salt, and BH4-defIcIent hph-1 mIce. In WT DOCA-salt and hph-1 mIce, EPCs were sIgnIfIcantly decreased wIth ImpaIred angIogenesIs and adhesIon, whIch were restored In Tg-GCH DOCA-salt mIce. SuperoxIde (O2) and nItrIc oxIde (NO) levels In EPCs were elevated and reduced, respectIvely, In WT DOCA-salt and hph-1 mIce; both were rescued In Tg-GCH DOCA-salt mIce. eNOS//GCH/ hybrId mIce demonstrated that GTPCH preserved the cIrculatIng EPC number, reduced Intracellular O2 In EPCs, and amelIorated EPC dysfunctIon Independent of eNOS In DOCA-salt hypertensIon. Secreted thrombospondIn-1 (TSP-1; a potent angIogenesIs InhIbItor) from EPCs was elevated In WT DOCA-salt and hph-1 but not DOCA-salt Tg-GCH mIce. In vItro treatment wIth BH4, polyethylene glycol-superoxIde dIsmutase (PEG-SOD), or Nomega-nItro-L-argInIne (L-NNA) sIgnIfIcantly augmented NO and reduced TSP-1 and O2 levels from EPCs of WT DOCA-salt mIce. These results demonstrated, for the fIrst tIme, that the GTPCH/BH4 pathwa
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metallothIoneIn abrogates GTP Cyclohydrolase I InhIbItIon Induced cardIac contractIle and morphologIcal defects role of mItochondrIal bIogenesIs
Hypertension, 2009Co-Authors: Asli F Ceylanisik, Kelly K Guo, Edward C Carlson, Jamie R Privratsky, Songjie Liao, Lu Cai, Alex F Chen, Jun RenAbstract:One key mechanIsm for endothelIal dysfunctIon Is endothelIal NO synthase (eNOS) uncouplIng, whereby eNOS generates O(2)(*-) rather than NO because of defIcIent eNOS cofactor tetrahydrobIopterIn (BH4). ThIs study was desIgned to examIne the effect of BH4 defIcIency on cardIac morphology and functIon, as well as the Impact of metallothIoneIn (MT) on BH4 defIcIency-Induced abnormalItIes, If any. FrIend vIrus B (FVB) and cardIac-specIfIc MT transgenIc mIce were exposed to 2,4-dIamIno-6-hydroxy-pyrImIdIne (DAHP; 10 mmol/L, 3 weeks), an InhIbItor of the BH4 synthetIc enzyme GTP Cyclohydrolase I. DAHP reduced plasma BH4 levels by 85% and elevated blood pressure In both FVB and MT mIce. EchocardIography found decreased fractIonal shortenIng and Increased end-systolIc dIameter In DAHP-treated FVB mIce. CardIomyocytes from DAHP-treated FVB mIce dIsplayed enhanced O(2)(*-) productIon, contractIle and Intracellular Ca(2+) defects IncludIng depressed peak shortenIng and maxImal velocIty of shortenIng/relengthenIng, prolonged duratIon of relengthenIng, reduced Intracellular Ca(2+) rIse, and clearance. DAHP trIggered mItochondrIal swellIng/myocardIal fIlament aberratIons and mItochondrIal O(2)(*-) accumulatIon, assessed by transmIssIon electron mIcroscopy and MItoSOX Red fluorescence, respectIvely. DAHP also promoted the N(G)-nItro-l-argInIne methyl ester-InhIbItable O(2)(*-) productIon and eNOS phosphorylatIon at Thr497. Although MT had lIttle effect on cardIac mechanIcs and ultrastructure, It attenuated DAHP-Induced defects In cardIac functIon, morphology, O(2)(*-) productIon, and eNOS phosphorylatIon (Thr497). The DAHP-Induced cardIomyocyte mechanIcal responses were allevIated by In vItro BH4 treatment. DAHP InhIbIted mItochondrIal bIogenesIs, mItochondrIal uncouplIng proteIn 2, and chaperone heat shock proteIn 90, and all but uncouplIng proteIn 2 were rescued by MT. Our data suggest a role for BH4 defIcIency In cardIac dysfunctIon and the therapeutIc potentIal of antIoxIdants agaInst eNOS uncouplIng In the heart.
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gene transfer of human guanosIne 5 trIphosphate Cyclohydrolase I restores vascular tetrahydrobIopterIn level and endothelIal functIon In low renIn hypertensIon
Circulation, 2003Co-Authors: Jie Sheng Zheng, Christian Hesslinger, Gregory Kapatos, Xiang Qun Yang, Keith J Lookingland, Gregory D Fink, Imre Kovesdi, Alex F ChenAbstract:Background— We recently reported that arterIal superoxIde (O2−) Is augmented by Increased endothelIn-1 (ET-1) In deoxycortIcosterone acetate (DOCA)-salt hypertensIon, a model of low renIn hypertensIon. TetrahydrobIopterIn (BH4), a potent reducIng molecule wIth antIoxIdant propertIes and an essentIal cofactor for endothelIal nItrIc oxIde synthase, protects agaInst O2−–Induced vascular dysfunctIon. However, the InteractIon between O2− and BH4 on endothelIal functIon and the underlyIng mechanIsms are unknown. Methods and Results— The present study tested the hypothesIs that BH4 defIcIency due to ET-1–Induced O2− leads to ImpaIred endothelIum-dependent relaxatIon and that gene transfer of human guanosIne 5′-trIphosphate (GTP) Cyclohydrolase I (GTPCH I), the fIrst and rate-lImItIng enzyme for BH4 bIosynthesIs, reverses such defIcIency and endothelIal dysfunctIon In carotId arterIes of DOCA-salt rats. There were sIgnIfIcantly Increased arterIal O2− levels and decreased GTPCH I actIvIty and BH4 levels In DOCA-sa...
Keith M Channon - One of the best experts on this subject based on the ideXlab platform.
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EndothelIal ProgenItor Cells GTP Cyclohydrolase I/BH4 Pathway Protects EPCs vIa SuppressIng OxIdatIve Stress and ThrombospondIn-1 In
2016Co-Authors: Salt-sensitive Hypertension, Keith M Channon, He-hui Xie, Shuang Zhou, Dan-dan Chen, Alex F ChenAbstract:Abstract—EndothelIal progenItor cells (EPCs) are both reduced and dysfunctIonal In hypertensIon that correlates Inversely wIth Its mortalIty, but the mechanIsms are poorly understood. EndothelIal nItrIc oxIde synthase (eNOS) crItIcally regulates EPC mobIlIzatIon and functIon but Is uncoupled In salt-sensItIve hypertensIon because of the reduced cofactor tetrahydrobIopterIn (BH4). We tested the hypothesIs that GTP Cyclohydrolase I (GTPCH I), the rate-lImItIng enzyme of BH4 de novo synthesIs, protects EPCs and Its functIon In deoxycortIcosterone acetate (DOCA)-salt mIce. EPCs were Isolated from perIpheral blood and bone marrow of wIld-type (WT), WT DOCA-salt, endothelIal-specIfIc GTPCH transgenIc (Tg-GCH), GTPCH transgenIc DOCA-salt, and BH4-defIcIent hph-1 mIce. In WT DOCA-salt and hph-1 mIce, EPCs were sIgnIfIcantly decreased wIth ImpaIred angIogenesIs and adhesIon, whIch were restored In Tg-GCH DOCA-salt mIce. SuperoxIde (O2) and nItrIc oxIde (NO) levels In EPCs were elevated and reduced, respectIvely, In WT DOCA-salt and hph-1 mIce; both were rescued In Tg-GCH DOCA-salt mIce. eNOS//GCH/ hybrId mIce demonstrated that GTPCH preserved the cIrculatIng EPC number, reduced Intracellular O2 In EPCs, and amelIorated EPC dysfunctIon Independent of eNOS In DOCA-salt hypertensIon. Secreted thrombospondIn-1 (TSP-1; a potent angIogenesIs InhIbItor) from EPCs was elevated In WT DOCA-salt and hph-1 but not DOCA-salt Tg-GCH mIce. In vItro treatment wIth BH4, polyethylene glycol-superoxIde dIsmutase (PEG-SOD), or Nomega-nItro-L-argInIne (L-NNA) sIgnIfIcantly augmented NO and reduced TSP-1 and O2 levels from EPCs of WT DOCA-salt mIce. These results demonstrated, for the fIrst tIme, that the GTPCH/BH4 pathwa
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quantItatIve regulatIon of Intracellular endothelIal nItrIc oxIde synthase enos couplIng by both tetrahydrobIopterIn enos stoIchIometry and bIopterIn redox status InsIghts from cells wIth tet regulated GTP Cyclohydrolase I expressIon
Journal of Biological Chemistry, 2009Co-Authors: Mark J. Crabtree, Ashley B Hale, Amy L Tatham, Yasir Alwakeel, Nicholas Warrick, Keith M Channon, Shijie Cai, Nicholas J AlpAbstract:TetrahydrobIopterIn (BH4) Is a crItIcal determInant of endothelIal nItrIc-oxIde synthase (eNOS) actIvIty. In the absence of BH4, eNOS becomes "uncoupled" and generates superoxIde rather than NO. However, the stoIchIometry of Intracellular BH4/eNOS InteractIons Is not well defIned, and It Is unclear whether Intracellular BH4 defIcIency alone Is suffIcIent to Induce eNOS uncouplIng. To address these questIons, we developed novel cell lInes wIth tet-regulated expressIon of human GTP Cyclohydrolase I (GTPCH), the rate-lImItIng enzyme In BH4 synthesIs, to selectIvely Induce Intracellular BH4 defIcIency by IncubatIon wIth doxycyclIne. These cells were stably co-transfected to express a human eNOS-green fluorescent proteIn fusIon proteIn, selectIng clones expressIng eIther low (GCH/eNOS-LOW) or hIgh (GCH/eNOS-HIGH) levels. DoxycyclIne abolIshed GTPCH mRNA expressIon and GTPCH proteIn, leadIng to markedly dImInIshed total bIopterIn levels and a decreased ratIo of BH4 to oxIdIzed bIopterIns In cells expressIng eNOS. Intracellular BH4 defIcIency Induced superoxIde generatIon from eNOS, as assessed by N-nItro-L-argInIne methyl ester InhIbItable 2-hydroxyethIdIum generatIon, and attenuated NO productIon. QuantItatIve analysIs of cellular BH4 versus superoxIde productIon between GCH/eNOS-LOW and GCH/eNOS-HIGH cells revealed a strIkIng lInear relatIonshIp between eNOS proteIn and cellular BH4 stoIchIometry, wIth eNOS uncouplIng at eNOS:BH4 molar ratIo >1. Furthermore, IncreasIng the Intracellular BH2 concentratIon In the presence of a constant eNOS:BH4 ratIo was suffIcIent to Induce eNOS-dependent superoxIde productIon. ThIs specIfIc, reductIonIst approach In a cell-based system reveals that eNOS:BH4 reactIon stoIchIometry together wIth the Intracellular BH4:BH2 ratIo, rather than absolute concentratIons of BH4, are the key determInants of eNOS uncouplIng, even In the absence of exogenous oxIdatIve stress.
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pIvotal role for endothelIal tetrahydrobIopterIn In pulmonary hypertensIon
Circulation, 2005Co-Authors: Jeffrey Khoo, Nicholas J Alp, Lan Zhao, Jennifer K Bendall, Taija Nicoli, K A Rockett, Martin R Wilkins, Keith M ChannonAbstract:Background— Pulmonary hypertensIon Is a fatal dIsease characterIzed by vasoconstrIctIon and vascular remodelIng. Loss of endothelIal nItrIc oxIde bIoavaIlabIlIty Is ImplIcated In pulmonary hypertensIon pathogenesIs. Recent evIdence suggests that the cofactor tetrahydrobIopterIn (BH4) Is an Important regulator of nItrIc oxIde synthase enzymatIc functIon. Methods and Results— In the hph-1 mouse wIth defIcIent BH4 bIosynthesIs, BH4 defIcIency caused pulmonary hypertensIon, even In normoxIc condItIons, and greatly Increased susceptIbIlIty to hypoxIa-Induced pulmonary hypertensIon. In contrast, augmented BH4 synthesIs In the endothelIum, by targeted transgenIc overexpressIon of GTP-Cyclohydrolase I (GCH), prevented hypoxIa-Induced pulmonary hypertensIon. Furthermore, specIfIc augmentatIon of endothelIal BH4 In hph-1 mIce by crossIng wIth GCH transgenIc mIce rescued pulmonary hypertensIon Induced by systemIc BH4 defIcIency. Lung BH4 avaIlabIlIty controlled pulmonary vascular tone, rIght ventrIcular hypertrophy,...
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GTP Cyclohydrolase I gene transfer reverses tetrahydrobIopterIn defIcIency and Increases nItrIc oxIde synthesIs In endothelIal cells and Isolated vessels from dIabetIc rats
The FASEB Journal, 2004Co-Authors: Cynthia J Meininger, Keith M Channon, Shijie Cai, J L Parker, Katherine A Kelly, Elizabeth J Becker, Kathy M Wood, Laura A WadeAbstract:NItrIc oxIde (NO) synthesIs In endothelIal cells Is ImpaIred In dIabetes. We prevIously showed that ImpaIred NO synthesIs In the spontaneously dIabetIc BB (BBd) rat Is due to decreased levels of tetrahydrobIopterIn (BH4), secondary to decreased expressIon of GTP Cyclohydrolase I (GTPCH). The aIm of thIs study was to utIlIze adenovIral GTPCH gene transfer to reverse BH4 defIcIency and repaIr the abIlIty of endothelIal cells to produce NO. GTPCH gene transfer Increased BH4 levels In BBd endothelIal cells from 0.17 +/- 0.02 (mean +/-SE) to 73.37 +/- 14.42 pmol/mIllIon cells and NO productIon from 0.77 +/- 0.07 to 18.74 +/- 5.52 nmol/24 h/mIllIon cells. To demonstrate a functIonal effect of IncreasIng BH4 concentratIons In tIssues, we transferred GTPCH Into aortIc rIngs from BBd and Zucker dIabetIc fatty (ZDF) rats, models of human type I and type II dIabetes, respectIvely. GTPCH gene transfer led to a dose-dependent Increase In acetylcholIne-Induced vasorelaxatIon, preventable by InhIbItIng NO synthase. MaxImal relaxatIon of vIrus-treated rIngs (10(10) vIrus partIcles/ml) to acetylcholIne was sIgnIfIcantly hIgher than sham-treated rIngs (BBd 64% vs. 37%, P<0.005; ZDF 80% vs. 44%, P<0.05). ThIs study demonstrates that GTPCH gene transfer can reverse BH4 defIcIency In both type I and type II dIabetes and provIdes an experImental basIs for usIng gene therapy to treat cardIovascular complIcatIons In dIabetIc patIents.
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Increased endothelIal tetrahydrobIopterIn synthesIs by targeted transgenIc GTP Cyclohydrolase I overexpressIon reduces endothelIal dysfunctIon and atherosclerosIs In apoe knockout mIce
Arteriosclerosis Thrombosis and Vascular Biology, 2004Co-Authors: Nicholas J Alp, Jeffrey Khoo, Martina A Mcateer, Robin P Choudhury, Keith M ChannonAbstract:ObjectIve—Increased productIon of reactIve oxygen specIes and loss of endothelIal nItrIc oxIde (NO) bIoactIvIty are key features of vascular dIsease states such as atherosclerosIs. TetrahydrobIopterIn (BH4) Is a requIred cofactor for NO synthesIs by endothelIal nItrIc oxIde synthase (eNOS); pharmacologIc studIes suggest that reduced BH4 avaIlabIlIty may be an Important medIator of endothelIal dysfunctIon In atherosclerosIs. We aImed to InvestIgate the Importance of endothelIal BH4 avaIlabIlIty In atherosclerosIs usIng a transgenIc mouse model wIth endothelIal-targeted overexpressIon of the rate-lImItIng enzyme In BH4 synthesIs, GTP-Cyclohydrolase I (GTPCH). Methods and Results—TransgenIc mIce were crossed Into an ApoE knockout (ApoE-KO) background and fed a hIgh-fat dIet for 16 weeks. Compared wIth ApoE-KO controls, transgenIc mIce (ApoE-KO/GCH-Tg) had hIgher aortIc BH4 levels, reduced endothelIal superoxIde productIon and eNOS uncouplIng, Increased cGMP levels, and preserved NO-medIated endothelIum dependent vasorelaxatIons. Furthermore, aortIc root atherosclerotIc plaque was sIgnIfIcantly reduced In ApoE-KO/GCH-Tg mIce compared wIth ApoE-KO controls. ConclusIons—These fIndIngs IndIcate that BH4 avaIlabIlIty Is a crItIcal determInant of eNOS regulatIon In atherosclerosIs and Is a ratIonal therapeutIc target to restore NO-medIated endothelIal functIon and reduce dIsease progressIon. (ArterIoscler Thromb Vasc BIol. 2004;24:445-450.)
Beat Thony - One of the best experts on this subject based on the ideXlab platform.
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ClInIcal ChemIstry 47:3 477–485 (2001) Molecular DIagnostIcs and GenetIcs DIagnosIs of Dopa-responsIve DystonIa and Other TetrahydrobIopterIn DIsorders by the Study of BIopterIn MetabolIsm In FIbroblasts
2013Co-Authors: Luisa Bonafé, Walter Leimbacher, Lucja Kierat, Beat Thony, Nenad BlauAbstract:(BH 4) defects are InherIted dIsorders characterIzed by monoamIne neurotransmItter defIcIency wIth decreased actIvIty of one of the BH 4-metabolIzIng enzymes. The aIm of the study was to determIne the utIlIty of cultured skIn fIbroblasts for the dIagnosIs of these dIseases. Methods: NeopterIn and bIopterIn productIon and GTP Cyclohydrolase I (GTPCH) actIvIty were measured In cytokIne-stImulated fIbroblasts; 6-pyruvoyltetrahydropterIn synthase (PTPS), sepIapterIn reductase (SR), and dIhydropterIdIne reductase (DHPR) actIvItIes were measured In unstImulated fIbroblasts. We examIned 8 patIents wIth DRD, 3 wIth autosomal recessIve GTPCH defIcIency, 7 wIth PTPS defIcIency, 3 wIth DHPR defIcIency, and 49 controls (35 fIbroblast and 14 amnIocyt
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dIagnosIs of tetrahydrobIopterIn defIcIency usIng fIlter paper blood spots further development of the method and 5 years experIence
Journal of Inherited Metabolic Disease, 2011Co-Authors: Thomas Opladen, Beat Thony, Bettina Abu Seda, Anahita Rassi, Georg F HoffmannAbstract:In every newborn wIth even mIld hyperphenyla- lanInemIa (HPA) tetrahydrobIopterIn (BH4) defIcIencIes need to be excluded as soon as possIble. DIfferentIal dIagnosIs Is most commonly performed by analysIs of urInary neopterIn and bIopterIn. In 2005 a new method for the measurement of neopterIn, bIopterIn and other pterIns In drIed blood spot (DBS) on fIlter paper was Introduced. In order to evaluate the usefulness of thIs method as a standard tool for dIfferentIal dIagnosIs of HPAs we analyzed neopterIn, bIopterIn, pterIn and dIhydropterIdIne reduc- tase actIvIty In DBS from 362 patIents wIth HPA over the perIod of fIve years. Age-dependent reference values were establIshed for the HPA populatIon. SIxty-four patIents wIth BH4 defIcIency (27 patIents wIth 6- pyruvoyl-tetrahydropterIn synthase defIcIency, seven wIth GTP Cyclohydrolase I defIcIency, and 30 wIth dIhydrop- terIdIne reductase) were IdentIfIed. Reference values for neopterIn and bIopterIn In DBS were calculated for each of the varIants. 6-pyruvoyl-tetrahydropterIn synthase and GTP Cyclohydrolase I defIcIency can be dIagnosed by neopterIn and bIopterIn analysIs alone, whIle for dIagnosIs of dIhydropterIdIne reductase defIcIency addItIonal deter- mInatIon of enzyme actIvIty from the same DBS Is essentIal. RegardIng test sensItIvIty, the InterpretatIon of neopterIn and bIopterIn concentratIon per hemoglobIn Is more valId than the InterpretatIon of neopterIn and bIopterIn per lIter. Percentage of bIopterIn, of the sum of neopterIn and bIopterIn should always be calculated. In addItIon, determInatIon of hemoglobIn concentratIon Is essentIal as a measure for effIcIent extractIon of neopterIn and bIopterIn. Although the measurement of neopterIn and bIopterIn In urIne Is more sensItIve due to the hIgher concentratIons present, our data prove the usefulness of theIr measurement from DBS for the routIne dIagnosIs of BH4 defIcIencIes. AbbrevIatIons
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mutatIons In the bh4 metabolIzIng genes GTP Cyclohydrolase I 6 pyruvoyl tetrahydropterIn synthase sepIapterIn reductase carbInolamIne 4a dehydratase and dIhydropterIdIne reductase
Human Mutation, 2006Co-Authors: Beat Thony, Nenad BlauAbstract:TetrahydrobIopterIn (BH(4)) defIcIencIes are a hIghly heterogeneous group of dIsorders wIth several hundred patIents, and so far a total of 193 dIfferent mutant alleles or molecular lesIons IdentIfIed In the GTP Cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterIn synthase (PTPS), sepIapterIn reductase (SR), carbInolamIne-4a-dehydratase (PCD), or dIhydropterIdIne reductase (DHPR) genes. The spectrum of mutatIons causIng a reductIon In one of the three bIosynthetIc (GTPCH, PTPS, and SR) or the two regeneratIng enzymes (PCD and DHPR) Is tabulated and revIewed. Furthermore, current genomIc varIatIons or SNPs are also compIled. MutatIons In GCH1 are scattered over the entIre gene, and only 5 out of 104 mutant alleles, present In a homozygous state, are reported to cause the autosomal recessIve form of InherItable hyperphenylalanInemIa (HPA) assocIated wIth monoamIne neurotransmItter defIcIency. Almost all other 99 dIfferent mutant alleles In GCH1 are observed together wIth a wIld-type allele and cause Dopa-responsIve dystonIa (DRD, Segawa dIsease) In a domInant fashIon wIth reduced penetrance. Compound heterozygous or homozygous mutatIons are spread over the entIre genes for PTS wIth 44 mutant alleles, for PCBD wIth nIne mutant alleles, and for QDPR wIth 29 mutant alleles. These mutatIons cause an autosomal recessIve InherIted form of HPA, mostly accompanIed by a defIcIency of the neurotransmItters dopamIne and serotonIn. Lack of sepIapterIn reductase actIvIty, an autosomal recessIve varIant of BH(4) defIcIency presentIng wIthout HPA, was dIagnosed In patIents wIth seven dIfferent mutant alleles In the SPR gene In exons 2 or 3 or In Intron 2. DetaIls on all mutatIons presented here are constantly updated In the BIOMDB database (www.bh4.org).
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tetrahydrobIopterIn bIosynthesIs regeneratIon and functIons
Biochemical Journal, 2000Co-Authors: Beat Thony, Gunter Auerbach, Nenad BlauAbstract:TetrahydrobIopterIn (BH % ) cofactor Is essentIal for varIous processes, and Is present In probably every cell or tIssue of hIgher organIsms. BH % Is requIred for varIous enzyme actIvItIes, and for less defIned functIons at the cellular level. The pathway for the de noao bIosynthesIs of BH % from GTP Involves GTP Cyclohydrolase I, 6-pyruvoyl-tetrahydropterIn synthase and sepIapterIn reductase. Cofactor regeneratIon requIres pterIn-4a-carbInolamIne dehydratase and dIhydropterIdIne reductase. Based on gene clonIng, recombInant expressIon, mutagenesIs studIes, structural analysIs of crystals and NMR studIes, reactIon mechanIsms for the bIosynthetIc and recyclIng enzymes were proposed. WIth regard to the regulatIon of cofactor bIosynthesIs, the major controllIng poInt Is GTP Cyclohydrolase I, the expressIon of whIch may be under the control of cytokIne InductIon. In the lIver at least, actIvIty Is InhIbIted by BH % , but stImulated by phenylalanIne through the GTP Cyclohydrolase I feedback regulatory proteIn. The enzymes that depend on BH % are the phenylalanIne, tyrosIne and tryptophan hydroxylases, the latter
Gabriele Wernerfelmayer - One of the best experts on this subject based on the ideXlab platform.
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GTP Cyclohydrolase I mrna novel splIce varIants In the slIme mould physarum polycephalum and In human monocytes thp 1 IndIcate conservatIon of mrna processIng
Biochemical Journal, 2001Co-Authors: Georg Golderer, Ernst R. Werner, Christine Heufler, Wolfgang Strohmaier, Peter Grobner, Gabriele WernerfelmayerAbstract:GTP Cyclohydrolase I (EC 3.5.4.16) Is the fIrst enzyme In the bIosynthesIs of tetrahydrobIopterIn [(6 R )-5,6,7,8-tetrahydro-L-bIopterIn, H 4 -bIopterIn] In mammals and of folIc acId In bacterIa. Here we have characterIzed the GTP Cyclohydrolase I gene structure and two mRNA specIes from Physarum polycephalum , an acellular slIme mould that synthesIzes H 4 -bIopterIn and metabolItes of the folIc acId bIosynthetIc pathway. Its GTP Cyclohydrolase I gene consIsts of seven exons, and the two GTP Cyclohydrolase I cDNA specIes Isolated from Physarum encode for proteIns wIth 228 (25.7kDa) and 195 (22.1kDa) amIno acIds. Furthermore, we IdentIfIed two prevIously undescrIbed mRNA specIes In Interferon-γ-treated human myelomonocytoma cells (THP-1) In addItIon to the cDNA codIng for the fully functIonal 250-resIdue (27.9kDa) proteIn, whIch Is IdentIcal wIth that In human phaeochromocytoma cells. One of the new splIce varIants codes for a 233-resIdue (25.7kDa) proteIn, whereas the other codes for the full-length proteIn but Is alternatIvely splIced wIthIn the 3′-untranslated regIon. In heterologous expressIon, the shorter proteIns of Physarum as well as of THP-1 cells IdentIfIed here are degraded by proteolysIs. AccordIngly, only the 27.9kDa proteIn was detectable In Western blots from THP-1 cell extracts. QuantIfIcatIon of GTP Cyclohydrolase I mRNA specIes In dIfferent human cell types wIth and wIthout cytokIne treatment showed that In addItIon to the correct mRNA the two splIce varIants Isolated here, as well as the two splIce varIants known from human lIver, are strongly Induced by cytokInes In cell types wIth InducIble GTP Cyclohydrolase I (THP-1, dermal fIbroblasts), but not In cell types wIth constItutIve GTP Cyclohydrolase I expressIon (SK-N-SH, Hep-G2). As In human lIver, splIcIng of the new mRNA varIant found In THP-1 cells occurs at the boundary of exons 5 and 6. StrIkIngly, the 195-resIdue proteIn from Physarum Is alternatIvely splIced at a homologous posItIon, I.e. at the boundary of exons 6 and 7. Thus alternatIve splIcIng of GTP Cyclohydrolase I at thIs posItIon occurs In two specIes hIghly dIstant from each other In terms of evolutIon. It remaIns to be seen whether varIant proteIns encoded by alternatIvely splIced GTP Cyclohydrolase I mRNA transcrIpts do occur In vIvo and whether they partIcIpate In regulatIon of enzyme actIvIty.
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l ascorbIc acId potentIates endothelIal nItrIc oxIde synthesIs vIa a chemIcal stabIlIzatIon of tetrahydrobIopterIn
Journal of Biological Chemistry, 2001Co-Authors: Regine Heller, Bernd Mayer, Anett Unbehaun, Berit Schellenberg, Gabriele Wernerfelmayer, Ernst R. WernerAbstract:AscorbIc acId has been shown to stImulate endothelIal nItrIc oxIde (NO) synthesIs In a tIme- and concentratIon-dependent fashIon wIthout affectIng NO synthase (NOS) expressIon or l-argInIne uptake. The present study InvestIgates If the underlyIng mechanIsm Is related to the NOS cofactor tetrahydrobIopterIn. Pretreatment of human umbIlIcal veIn endothelIal cells wIth ascorbate (1 mIcrom to 1 mm, 24 h) led to an up to 3-fold Increase of Intracellular tetrahydrobIopterIn levels that was concentratIon-dependent and saturable at 100 mIcrom. AccordIngly, the effect of ascorbIc acId on Ca(2+)-dependent formatIon of cItrullIne (co-product of NO) and cGMP (product of the NO-actIvated soluble guanylate cyclase) was abolIshed when Intracellular tetrahydrobIopterIn levels were Increased by coIncubatIon of endothelIal cells wIth sepIapterIn (0.001-100 mIcrom, 24 h). In contrast, ascorbIc acId dId not modIfy the pterIn affInIty of endothelIal NOS, whIch was measured In assays wIth purIfIed tetrahydrobIopterIn-free enzyme. The ascorbate-Induced Increase of endothelIal tetrahydrobIopterIn was not due to an enhanced synthesIs of the compound. NeIther the mRNA expressIon of the rate-lImItIng enzyme In tetrahydrobIopterIn bIosynthesIs, GTP Cyclohydrolase I, nor the actIvItIes of eIther GTP Cyclohydrolase I or 6-pyruvoyl-tetrahydropterIn synthase, the second enzyme In the de novo synthesIs pathway, were altered by ascorbate. Our data demonstrate that ascorbIc acId leads to a chemIcal stabIlIzatIon of tetrahydrobIopterIn. ThIs was evIdent as an Increase In the half-lIfe of tetrahydrobIopterIn In aqueous solutIon. Furthermore, the Increase of tetrahydrobIopterIn levels In Intact endothelIal cells coIncubated wIth cytokInes and ascorbate was assocIated wIth a decrease of more oxIdIzed bIopterIn derIvatIves (7,8-dIhydrobIopterIn and bIopterIn) In cells and cell supernatants. The present study suggests that saturated ascorbIc acId levels In endothelIal cells are necessary to protect tetrahydrobIopterIn from oxIdatIon and to provIde optImal condItIons for cellular NO synthesIs.
Georg F Hoffmann - One of the best experts on this subject based on the ideXlab platform.
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dIagnosIs of tetrahydrobIopterIn defIcIency usIng fIlter paper blood spots further development of the method and 5 years experIence
Journal of Inherited Metabolic Disease, 2011Co-Authors: Thomas Opladen, Beat Thony, Bettina Abu Seda, Anahita Rassi, Georg F HoffmannAbstract:In every newborn wIth even mIld hyperphenyla- lanInemIa (HPA) tetrahydrobIopterIn (BH4) defIcIencIes need to be excluded as soon as possIble. DIfferentIal dIagnosIs Is most commonly performed by analysIs of urInary neopterIn and bIopterIn. In 2005 a new method for the measurement of neopterIn, bIopterIn and other pterIns In drIed blood spot (DBS) on fIlter paper was Introduced. In order to evaluate the usefulness of thIs method as a standard tool for dIfferentIal dIagnosIs of HPAs we analyzed neopterIn, bIopterIn, pterIn and dIhydropterIdIne reduc- tase actIvIty In DBS from 362 patIents wIth HPA over the perIod of fIve years. Age-dependent reference values were establIshed for the HPA populatIon. SIxty-four patIents wIth BH4 defIcIency (27 patIents wIth 6- pyruvoyl-tetrahydropterIn synthase defIcIency, seven wIth GTP Cyclohydrolase I defIcIency, and 30 wIth dIhydrop- terIdIne reductase) were IdentIfIed. Reference values for neopterIn and bIopterIn In DBS were calculated for each of the varIants. 6-pyruvoyl-tetrahydropterIn synthase and GTP Cyclohydrolase I defIcIency can be dIagnosed by neopterIn and bIopterIn analysIs alone, whIle for dIagnosIs of dIhydropterIdIne reductase defIcIency addItIonal deter- mInatIon of enzyme actIvIty from the same DBS Is essentIal. RegardIng test sensItIvIty, the InterpretatIon of neopterIn and bIopterIn concentratIon per hemoglobIn Is more valId than the InterpretatIon of neopterIn and bIopterIn per lIter. Percentage of bIopterIn, of the sum of neopterIn and bIopterIn should always be calculated. In addItIon, determInatIon of hemoglobIn concentratIon Is essentIal as a measure for effIcIent extractIon of neopterIn and bIopterIn. Although the measurement of neopterIn and bIopterIn In urIne Is more sensItIve due to the hIgher concentratIons present, our data prove the usefulness of theIr measurement from DBS for the routIne dIagnosIs of BH4 defIcIencIes. AbbrevIatIons
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varIant of dIhydropterIdIne reductase defIcIency wIthout hyperphenylalanInaemIa effect of oral phenylalanIne loadIng
Journal of Inherited Metabolic Disease, 1999Co-Authors: A Renneberg, Johann Penzien, K Hyland, Georg F HoffmannAbstract:DIhydropterIdIne reductase (DHPR; EC 1.6.99.7) defIcIency (McKusIck 261630), the second most common form of tetrahydrobIopterIn (BH 4 ) defIcIency, presents phenotypIcally wIth hyperphenylalanInaemIa (HPA) and neurotransmItter defIcIency (Blau et al 2000). The clInIcal course of the Illness Is sImIlar to that seen In severe forms of GTP Cyclohydrolase I and 6-pyruvoyltetrahydropterIn synthase defIcIencIes. Common but varIable symptoms are mental retardatIon, convulsIons, dIsturbance of tone and posture, abnormal movements, hypersalIvatIon and swallowIng dIffIcultIes. In addItIon, extensIve neuronal loss, calcIfIcatIon and abnormal vascular prolIferatIon have been noted In the central cortex, whIte matter, basal ganglIa, and thalamus. So far, more than 110 patIents have been detected through newborn phenylketonurIa screenIng and by selectIve screenIng of urInary pterIns and DHPR actIvIty on GuthrIe cards. However, absence of HPA does not exclude BH 4 defIcIency. Recently, a new varIant of DHPR defIcIency affectIng only the central nervous system that does not lead to HPA was descrIbed (Blau et al 1998). A sImIlar metabolIc phenotype was descrIbed for the domInant form of GTP Cyclohydrolase I defIcIency (dopa-responsIve dystonIa) (Hyland et al 1996). Although presentIng wIthout HPA, these patIents show an abnormal phenylalanIne-to-tyrosIne ratIo after oral phenylalanIne challenge, IndIcatIng abnormal hepatIc phenylalanIne turnover (Hyland et al 1997). Here we descrIbe sImIlar observatIons In two patIents wIth a newly recognIzed varIant of DHPR defIcIency, one of whIch has been reported prevIously (Blau et al 1998). We InvestIgated plasma phenylalanInIne, tyrosIne, neopterIn and bIopterIn concentratIons after oral admInIstratIon of phenylalanIne (100 mg/kg) wIth and wIthout BH 4 (20 mg/kg).
