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S Milstien

Publications and source records attributed to S Milstien.

At least 37 records · Page 2Linked to original sources
PubMed2000

Functional characterization of human sphingosine kinase-1.

Sphingosine kinase catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a novel lipid mediator with both intra- and extracellular functions. Based on sequence identity to murine sphingosine kinase (mSPHK1a), we cloned and characterized the first human sphingosine kinase (hSPHK1). The open reading frame of hSPHK1 encodes a 384 amino acid protein with 85% identity and 92% similarity to mSPHK1a at the amino acid level. Similar to mSPHK1a, when HEK293 cells were transfected with hSPHK1, there were marked increases in sphingosine kinase activity resulting in elevated SPP levels. hSPHK1 also specifically phosphorylated D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N, N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Northern analysis revealed that hSPHK1 was widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. Thus, hSPHK1 belongs to a highly conserved unique lipid kinase family that regulates diverse biological functions.

Amino Acid Sequence↗
PubMed2000

Preferential resistance of dopaminergic neurons to the toxicity of glutathione depletion is independent of cellular glutathione peroxidase and is mediated by tetrahydrobiopterin.

Depletion of glutathione in the substantia nigra is one of the earliest changes observed in Parkinson's disease (PD) and could initiate dopaminergic neuronal degeneration. Nevertheless, experimental glutathione depletion does not result in preferential toxicity to dopaminergic neurons either in vivo or in vitro. Moreover, dopaminergic neurons in culture are preferentially resistant to the toxicity of glutathione depletion, possibly owing to differences in cellular glutathione peroxidase (GPx1) function. However, mesencephalic cultures from GPx1-knockout and wild-type mice were equally susceptible to the toxicity of glutathione depletion, indicating that glutathione also has GPx1-independent functions in neuronal survival. In addition, dopaminergic neurons were more resistant to the toxicity of both glutathione depletion and treatment with peroxides than nondopaminergic neurons regardless of their GPx1 status. To explain this enhanced antioxidant capacity, we hypothesized that tetrahydrobiopterin (BH(4)) may function as an antioxidant in dopaminergic neurons. In agreement, inhibition of BH(4) synthesis increased the susceptibility of dopaminergic neurons to the toxicity of glutathione depletion, whereas increasing BH(4) levels completely protected nondopaminergic neurons against it. Our results suggest that BH(4) functions as a complementary antioxidant to the glutathione/glutathione peroxidase system and that changes in BH(4) levels may contribute to the pathogenesis of PD.

Animals↗
PubMed1999

Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis.

Sphingosine 1-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10-100 nM) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sphinganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell G(i)-coupled SPP receptors.

Animals↗
PubMed1999

Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function.

Subsaturating levels of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS), can lead to endothelial dysfunction as a result of decreased production of nitric oxide. Furthermore, insufficient BH(4) can also result in NOS-uncoupled production of reactive oxygen intermediates, such as superoxide anion and hydrogen peroxide. Nitric oxide and superoxide react rapidly to form peroxynitrite, which may be the reactive species responsible for many of the toxic effects of nitric oxide. Here we show that BH(4) is a primary target for peroxynitrite-catalyzed oxidation because at pH 7.4, physiologically relevant concentrations of BH(4) are oxidized rapidly by low concentrations of peroxynitrite. Peroxynitrite oxidizes BH(4) to quinonoid 5,6-dihydrobiopterin and a large proportion of the quinonoid isomer readily loses its side chain to form 7,8-dihydropterin which is not a cofactor for nitric oxide synthase. Thus, abnormally low levels of BH(4) can promote a cycle of its own destruction mediated by nitric oxide synthase-dependent formation of peroxynitrite. This mechanism might contribute to vascular endothelial dysfunction induced by oxidative stress.

Antioxidants↗
PubMed1999

Effects of oral folic acid supplementation on endothelial function in familial hypercholesterolemia. A randomized placebo-controlled trial.

BACKGROUND: Folates have been suggested to be of benefit in reducing cardiovascular risk. The present study was designed to examine whether oral folic acid supplementation could improve endothelial function as an intermediate end point for cardiovascular risk in patients with increased risk of atherosclerosis due to familial hypercholesterolemia (FH). METHODS AND RESULTS: In a prospective, randomized, double-blind, placebo-controlled study with crossover design, we evaluated the effects of 4 weeks of treatment with oral folic acid (5 mg PO) on endothelial function in FH. In 20 FH patients, forearm vascular function was assessed at baseline, after 4 weeks of folic acid treatment, and after 4 weeks of placebo treatment by venous occlusion plethysmography, with serotonin and sodium nitroprusside used as endothelium-dependent and -independent vasodilators. In addition, we examined the vasoconstrictor response to the NO synthase inhibitor N(G)-monomethyl-L-arginine to assess basal NO activity. In FH patients, folic acid supplementation restored the impaired endothelium-dependent vasodilation, whereas it did not significantly influence endothelium-independent vasodilation or basal forearm vasomotion. There was a trend toward improvement in basal NO activity. CONCLUSIONS: These data demonstrate that oral supplementation of folic acid can improve endothelial function in patients with increased risk of atherosclerotic disease due to hypercholesterolemia, without changes in plasma lipids.

Administration, Oral↗
PubMed1999

GTP cyclohydrolase I feedback regulatory protein is expressed in serotonin neurons and regulates tetrahydrobiopterin biosynthesis.

Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by approximately 20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GTPCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP-mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L-Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration-dependent manner, whereas D-phenylalanine and L-tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L-phenylalanine-induced increase in tetrahydrobiopterin levels. 7,8-Dihydroneopterin, a reduced pteridine capable of inhibiting GTPCH in a GFRP-dependent manner, decreased tetrahydrobiopterin levels in cultures of both serotonin and dopamine neurons. This inhibition was reversed by L-phenylalanine in serotonin but not in dopamine neurons. Our data suggest that GTPCH activity within serotonin neurons is under a tonic inhibitory tone mediated by GFRP and that tetrahydrobiopterin levels are maintained by the balance of intracellular concentrations of tetrahydrobiopterin and L-phenylalanine. In contrast, although tetrahydrobiopterin biosynthesis within dopamine neurons is also feedback-regulated, L-phenylalanine plays no role, and therefore tetrahydrobiopterin may have a direct effect on GTPCH activity.

Animals↗
PubMed1999

Bacterial translocation in cirrhotic rats stimulates eNOS-derived NO production and impairs mesenteric vascular contractility.

Nitric oxide (NO) has been implicated in the arterial vasodilation and associated vascular hyporesponsiveness to vasoconstrictors observed in liver cirrhosis. Bacteria, potent activators of NO and TNF-alpha synthesis, are found in the mesenteric lymph nodes (MLNs) of ascitic cirrhotic rats. Here, we investigated the impact of bacterial translocation (BT) to MLNs on TNF-alpha production, vascular NO release, and contractility in the mesenteric vasculature of ascitic cirrhotic rats. Vascular response to the alpha-adrenoagonist methoxamine, which is diminished in the superior mesenteric arterial beds of cirrhotic rats, is further blunted in the presence of BT. BT promoted vascular NO release in cirrhotic rats, an effect that depended on pressure-induced shear stress and was blocked by the NO inhibitor N(omega)-nitro-L-arginine. Removing the endothelium had the same effect. Endothelial NO synthase (eNOS), but not the inducible isoform (iNOS), was present in mesenteric vasculature of cirrhotic rats with and without BT, and its expression was enhanced compared with controls. TNF-alpha was induced in MLNs by BT and accumulated in parallel in the serum. This TNF-alpha production was associated with elevated levels of tetrahydrobiopterin (BH(4)), a TNF-alpha-stimulated cofactor and enhancer of eNOS-derived NO biosynthesis and NOS activity in mesenteric vasculature. These findings establish a link between BT to MLNs and increased TNF-alpha production and elevated BH(4) levels enhancing eNOS-derived NO overproduction, further impairing contractility in the cirrhotic mesenteric vasculature.

Animals↗
PubMed1998

Purification and characterization of rat kidney sphingosine kinase.

Sphingosine kinase catalyzes the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate, which plays important roles in numerous physiological processes, including growth, survival, and motility. We have purified rat kidney sphingosine kinase 6 x 10(5)-fold to apparent homogeneity. The purification procedure involved ammonium sulfate precipitation followed by chromatography on an anion exchange column. Partially purified sphingosine kinase was found to be stabilized by the presence of high salt, and thus, a scheme was developed to purify sphingosine kinase using sequential dye-ligand chromatography steps (since the enzyme bound to these matrices even in the presence of salt) followed by EAH-Sepharose chromatography. This 385-fold purified sphingosine kinase bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of 1 M NaCl. After concentration, the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and Superdex 75 gel filtration columns. Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa under denaturing conditions on SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is a monomer. Sphingosine kinase shows substrate specificity for D-erythro-sphingosine and does not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, DL-threo-dihydrosphingosine, or N,N-dimethylsphingosine. However, the latter two sphingolipids were potent competitive inhibitors. With sphingosine as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed Michaelis-Menten kinetics, with Km values of 5 and 93 microM for sphingosine and ATP, respectively. This study provides the basis for molecular characterization of a key enzyme in sphingolipid signaling.

Animals↗
PubMed1998

Cytokines stimulate GTP cyclohydrolase I gene expression in cultured human umbilical vein endothelial cells.

In vascular endothelial cells, tetrahydrobiopterin serves as an essential cofactor required for enzymatic activity of nitric oxide synthase. GTP cyclohydrolase I is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Previous studies have demonstrated that proinflammatory cytokines stimulate production of tetrahydrobiopterin in endothelial cells. Long-term regulation of GTP cyclohydrolase I gene expression in endothelium has not been studied. The present study was designed to determine whether the cytokines tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma), and interleukin-1beta (IL-1beta) stimulate tetrahydrobiopterin synthesis by increasing expression of GTP cyclohydrolase I mRNA in endothelial cells. The relative reverse transcription polymerase chain reaction was used to quantify expression of GTP cyclohydrolase I mRNA in cultured human umbilical vein endothelial cells. Nuclear run-on assay was performed to determine the transcription rate of GTP cyclohydrolase I gene. GTP cyclohydrolase I enzymatic activity and production of tetrahydrobiopterin were measured in cell extracts. After incubation with TNF-alpha (2 microg/mL), INF-gamma (200 U/mL), and IL-1beta (5 U/mL) for 24 hours, significantly increased expression of GTP cyclohydrolase I mRNA was detected. Cytokines increased the transcription rate of GTP cyclohydrolase I 3.6-fold. This increase was associated with increased GTP cyclohydrolase I enzymatic activity and elevation of intracellular levels of tetrahydrobiopterin. An RNA synthesis inhibitor, actinomycin D (2 microg/mL), inhibited cytokine-induced expression of GTP cyclohydrolase I gene. A protein synthesis inhibitor, cycloheximide (0.5 microg/mL), did not affect expression of GTP cyclohydrolase I mRNA but blocked the increase in enzyme activity, as well as production of tetrahydrobiopterin. Incubation of endothelial cells for 24 hours in the presence of 8-bromoadenosine 3':5'-cyclic monophosphate (10[-3] mol/L) did not affect expression of GTP cyclohydrolase I mRNA. These results demonstrate that in vascular endothelial cells, cytokines increase production of tetrahydrobiopterin by stimulating expression of GTP cyclohydrolase I gene. This effect is apparently due to increased transcription rather than stabilization of mRNA. Regulation of GTP cyclohydrolase I gene expression by cytokines may play an important role in control of endothelial nitric oxide synthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Tetrahydrobiopterin, nitric oxide and regulation of cerebral arterial tone.

Tetrahydrobiopterin is an essential cofactor required for activity of nitric oxide synthases. Existing evidence suggests that, during activation of constitutive and inducible isoforms of nitric oxide synthase, tetrahydrobiopterin is needed for allosteric and redox activation of enzymatic activity. However, precise mechanisms underlying the role of tetrahydrobiopterin in regulation of nitric oxide formation is not fully understood. In cerebral and peripheral arteries, increased availability of tetrahydrobiopterin can augment production of nitric oxide. In contrast, in arteries depleted of tetrahydrobiopterin, production of nitric oxide is impaired. Proinflammatory cytokines enhance mRNA expression of the rate-limiting enzyme of tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I and stimulate production of tetrahydrobiopterin. The ability of vascular tissues to synthesize tetrahydrobiopterin plays an important role in regulation of nitric oxide synthase under physiological conditions as well as during inflammation and sepsis. More recent studies concerning expression and function of recombinant nitric oxide synthases suggest that availability of tetrahydrobiopterin is important for production of nitric oxide in genetically engineered blood vessels. In this review, mechanisms regulating availability of intracellular tetrahydrobiopterin and its role in control of vascular tone under physiological and pathological conditions will be discussed.

Animals↗
PubMed1997

Inhibition of tetrahydrobiopterin biosynthesis impairs endothelium-dependent relaxations in canine basilar artery.

Tetrahydrobiopterin is an essential cofactor in biosynthesis of nitric oxide. The present study was designed to determine the effect of decreased intracellular tetrahydrobiopterin levels on endothelial function of isolated cerebral arteries. Blood vessels were incubated for 6 h in minimum essential medium (MEM) in the presence or absence of a GTP cyclohydrolase I inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP, 10(-2) M). Rings with and without endothelium were suspended for isometric force recording in the presence of a cyclooxygenase inhibitor, indomethacin (10(-5) M). In arteries with endothelium, DAHP significantly reduced intracellular levels of tetrahydrobiopterin. DAHP in combination with a precursor of the salvage pathway of tetrahydrobiopterin biosynthesis, sepiapterin (10(-4) M), not only restored but increased levels of tetrahydrobiopterin above control values. In DAHP-treated arteries, endothelium-dependent relaxations to bradykinin (10(-10)-10(-6) M) or calcium ionophore A23187 (10(-9)-10(-6) M) were significantly reduced, whereas endothelium-independent relaxations to a nitric oxide donor, 3-morpholinosydnonimine (10(-9)-10(-4) M), were not affected. When DAHP-treated arteries with endothelium were incubated with sepiapterin (10(-4) M) or superoxide dismutase (150 U/ml), relaxations to bradykinin and A23187 were restored to control levels. In contrast, superoxide dismutase did not affect endothelium-dependent relaxations in arteries incubated in MEM. A nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (10(-4) M), abolished relaxations to bradykinin or A23187 in control arteries and in DAHP-treated arteries. These studies demonstrate that in cerebral arteries, decreased intracellular levels of tetrahydrobiopterin can reduce endothelium-dependent relaxations. Production of superoxide anions during activation of dysfunctional endothelial nitric oxide synthase appears to be responsible for the impairment of endothelial function.

Animals↗
PubMed1997

Expression and function of recombinant endothelial NO synthase in coronary artery smooth muscle cells.

Smooth muscle cells (SMCs) play a key role in the pathogenesis of vascular diseases. The objectives of this study were to determine whether transfer of recombinant endothelial nitric oxide synthase (eNOS) gene to porcine coronary artery smooth muscle cell (CSMCs) would result in expression of a functional enzyme and to assess the effect of expression of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and beta-galactosidase (Ad beta Gal). In contrast to Ad beta Gal- or sham-transduced cells, CSMCs transduced with AdeNOS stained positive with the NADPH-diaphorase stain, acquired calcium-dependent NOS activity (measured by the conversion of [3H]L-arginine to [3H]L-citrulline), had increasing cyclic 3',5' cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-transduced cells was augmented by increasing intracellular levels of the eNOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine uptake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as assessed by cell counts 3 and 6 days after serum stimulation of quiescent CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional enzyme whose activity can be augmented by increasing intracellular levels of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs results in inhibition of serum-stimulated DNA synthesis and cell proliferation. These findings imply that eNOS gene transfer to SMCs may be a unique mode of increasing local NO production in the arterial wall.

Adenoviridae↗
PubMed1997

Aromatic L-amino acid decarboxylase deficiency: clinical features, diagnosis, and treatment of a second family.

Aromatic L-amino acid decarboxylase deficiency is an inborn error of metabolism that leads to combined serotonin and catecholamine deficiency, first described by Hyland et al in 1990. The clinical features, biochemical findings, and treatment of the second family with this condition are reported. Our male patient presented with developmental delay, extreme hypotonia, oculogyric crises, and irritability. The diagnosis of this inborn error of biogenic amine metabolism was accomplished by determining low concentrations of homovanillic, 5-hydroxyindoleacetic acid, and 3-methoxy-4-hydroxyphenyl-ethyleneglycol in cerebrospinal fluid with normal biopterin metabolism and increased L-dopa, in plasma, cerebrospinal fluid, and urine. Greatly reduced activity of aromatic L-amino acid decarboxylase in plasma confirmed this diagnosis. Combined treatment with pyridoxine, tranylcypromine, and bromocriptine produced some clinical improvement.

5-Hydroxytryptophan↗
PubMed1996

Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP.

The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and -phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern blot analysis indicated the presence of an 0.8-kilobase GFRP mRNA in most rat tissues, the amounts generally correlating with levels of GTP cyclohydrolase I and tetrahydrobiopterin. Thus, mRNA levels were relatively high in liver and kidney and somewhat lower in testis, heart, brain, and lung. These results suggest that GFRP is widely expressed and may play a role in regulating not only phenylalanine metabolism in the liver, but also the production of biogenic amine neurotransmitters as well as nitric oxide synthesis.

Amino Acid Sequence↗
PubMed1996

Double transduction with GTP cyclohydrolase I and tyrosine hydroxylase is necessary for spontaneous synthesis of L-DOPA by primary fibroblasts.

Gene transfer of tyrosine hydroxylase (TH) in animal models of Parkinson's disease (PD), using either genetically modified cells or recombinant virus vectors, has produced partial restoration of behavioral and biochemical deficits. The limited success of this approach may be related to the availability of the cofactor, tetrahydrobiopterin (BH4), because neither the dopamine-depleted striatum nor the cells used for gene transfer possess a sufficient amount of BH4 to support TH activity. To determine the role of BH4 in gene therapy, fibroblast cells transduced with the gene for TH were additionally modified with the gene for GTP cyclohydrolase l; an enzyme critical for BH4 synthesis. In contrast to cells transduced with only TH, doubly transduced fibroblasts spontaneously produced both BH4 and 3, 4-dihydroxy-L-phenylalanine. To examine further the importance of GTP cyclohydrolase I in gene therapy for PD, in vivo micro-dialysis was used to assess the biochemical changes in the dopamine-denervated striatum containing grafts of genetically modified fibroblasts. Only denervated striata grafted with fibro-blasts possessing both TH and GTP cyclohydrolase I genes displayed biochemical restoration. However, no significant differences from controls were observed in apomorphine-induced rotation. This is partly attributable to a limited duration of gene expression in vivo. These differences between fibroblasts transduced with TH alone and those additionally modified with the GTP cyclohydrolase I gene indicate that BH4 is critical for biochemical restoration in a rat model of PD and that GTP cyclohydrolase I is sufficient for production of BH4.

Animals↗