共查询到20条相似文献,搜索用时 46 毫秒
1.
Abstract: Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic l -amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic l -amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic l -amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. l -DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic l -amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic l -amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic l -amino acid decarboxylase produced less l -DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic l -amino acid decarboxylase near striatal grafts producing l -DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine. 相似文献
2.
3.
Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic l-Amino Acid Decarboxylase: Neurochemical Effects 总被引:1,自引:0,他引:1
Farida G. Kaddis Edward D. Clarkson Michel J. Weber †David J. Vandenbergh †David M. Donovan ‡Jacques Mallet ‡Philippe Horellou †George R. Uhl Curt R. Freed 《Journal of neurochemistry》1997,68(4):1520-1526
Abstract: To study the possibility that increasing striatal activity of aromatic l -amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to l -3,4-dihydroxyphenylalanine ( l -DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos- haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of 14 CO2 from l -[14 C]DOPA. The K m value for l -DOPA in intact and disrupted cells was 0.60 and 0.56 m M , respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 m M . The pH optimum for enzyme activity was 6.8. Grafting Cos- haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of l -DOPA. When measured 2 h after l -DOPA administration, the mean dopamine level in the striata of Cos- haadc -grafted animals was 2 µg/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 µg/g. At 6–8 h after l -DOPA administration, striatal dopamine content in the Cos- haadc -grafted animals was 0.67 µg/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered l -DOPA. 相似文献
4.
Chuanqin Fang Jinbo Yin Zhiqiang Xu Yanjiang Wang Haiwei Xu Huadong Zhou Changyue Gao 《Cell biochemistry and biophysics》2011,59(1):49-56
In order to examine the acute effects of l-DOPA treatment following 6-hydroxydopamine (6-OHDA) injection into rat medial forebrain bundle (MFB). Sprague–Dawley rats
(n = 48) received either 6-OHDA, via intracranial unilateral injection, into the MFB (experimental group) or saline 0.9% (control
group). Administration of l-DOPA or saline 0.9% began 1 month after the 6-OHDA injection for 10 consecutive days. Within 3 days, an increase in the density
of striatal tyrosine hydroxylase (TH) immunoreactive fibers within the striatum, when compared to the control group was observed.
There was no difference in the loss of substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons between. The greater
density of TH fibers in the striatum following l-DOPA may be related to recovery of the DA phenotype and/or sprouting of TH axon terminals. Only animals with severe cell
loss in the SNpc experienced abnormal involuntary movements (AIMs) or “dyskinesias” in response to l-DOPA, which did not correlate with striatal TH fiber density, suggesting that induction of TH-positive fibers does not contribute
to the occurrence of dyskinesia. The relationship between cell loss, fiber density and AIM to the abundance of markers of
microglial activation were also examined. Iba-1, a microglial marker, immunoreactivity was not affected by l-DOPA treatment, was not correlated with the severity of AIM indicating that microglial activation does not contribute to
dyskinetic phenomena. 相似文献
5.
Herpes simplex virus type 1 (HSV-1) plasmid vectors have a number of attractive features for gene transfer into neurons. In particular, the large size of the HSV-1 genome suggests that HSV-1 vectors might be designed to accommodate large inserts. We now report the construction and characterization of a 51 kb HSV-1 plasmid vector. This vector was efficiently packaged into HSV-1 particles using a helper virus-free packaging system. The structure of the packaged vector DNA was verified by both Southern blot and PCR analyses. A vector stock was microinjected into the rat striatum, the rats were sacrificed at 4 days after gene transfer, and numerous X-gal positive striatal cells were observed. This 51 kb vector was constructed using general principles that may support the routine construction of large vectors. Potential applications of such HSV-1 vectors include characterizing large promoter fragments or genomic clones and co-expressing multiple genes. 相似文献
6.
Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme 总被引:4,自引:0,他引:4
P F Fitzpatrick L J Chlumsky S C Daubner K L O'Malley 《The Journal of biological chemistry》1990,265(4):2042-2047
Rat tyrosine hydroxylase has been expressed at high levels in Spodoptera frugiperda cells using a baculovirus expression system. A cDNA containing the coding region for PC12 tyrosine hydroxylase was inserted into the unique EcoRI site of the transfer vector pLJC8 to yield the recombinant vector pLJC9. Spodoptera frugiperda cells were then co-infected with pLJC9 and wild type Autographa californica nuclear polyhedrosis virus. Recombinant virus particles containing the cDNA for tyrosine hydroxylase were selected by hybridization with authentic tyrosine hydroxylase cDNA. Three recombinant viruses were plaque-purified. All expressed a protein of Mr = 55,000 which reacted with antibodies to tyrosine hydroxylase. Forty-eight h after infection of cells with recombinant virus, the specific activity of tyrosine hydroxylase in the cell lysate was 30-100 nmol of dihydroxyphenylalanine produced/min/mg, consistent with 5-10% of the cell protein being tyrosine hydroxylase. Purification from 2.1 g of cells gave 5.8 mg of enzyme with a specific activity of 1.7 mumol of dihydroxyphenylalanine/min/mg. The purified enzyme is a tetramer of identical subunits, containing one covalently bound phosphoryl residue and 0.1 iron atom/subunit. No carbohydrate was detectable. Steady state kinetic results with tetrahydrobiopterin as substrate are consistent with a sequential mechanism for binding of tyrosine and tetrahydrobiopterin. Substrate inhibition occurs at tyrosine concentrations above 50 microM. Steady state kinetic parameters at pH 6.5 are Vmax = 74 min-1, KBH4 = 21 microM, KTyr = 9.4 microM, and Ko2 less than or equal to 6 microM. The Vmax value shows a broad pH optimum around pH 7. The KBH4 value is pH-dependent, increasing from about 20 microM below pH 7 to about 100 microM above pH 8. The KTyr value is independent of pH between pH 6 and pH 8.5. 相似文献
7.
Sukju Gil Changhwan Park Jeongeun Lee Hyunchul Koh 《Cellular and molecular neurobiology》2010,30(6):817-825
The administration of l-DOPA is the standard treatment for Parkinson’s disease (PD). However, the symptomatic relief provided by long-term administration
may be compromised by l-DOPA-induced dyskinesia (LID) that presents as adverse fluctuations in motor responsiveness and progressive loss of motor
control. In the later stages of PD, raphestriatal serotonin neurons compensate for the loss of nigrostriatal dopamine (DA)
neurons by converting and releasing DA derived from exogenous l-DOPA. Since the serotonin system does not have an autoregulatory mechanism for DA, raphe-mediated striatal DA release may
fluctuate dramatically and precede the development of LID. The 6-hydroxydopamine lesioned rats were treated with l-DOPA (6 mg/kg) and benserazide (15 mg/kg) daily for 3 weeks to allow for the development of abnormal involuntary movement
score (AIMs). In rats with LID, chronic treatment with l-DOPA increased striatal DA levels compared with control rats. We also observed a relative increase in the expression of striatal
l-amino-acid decarboxylase (AADC) in LID rats, even though tyrosine hydroxylase (TH) expression did not increase. The administration
of l-DOPA also increased striatal serotonin immunoreactivity in LID rats compared to control rats. Striatal DA and 5-hydroxytryptamine
(5-HT) levels were negatively correlated in l-DOPA-treated rats. These results of this study reveal that 5-HT contributes to LID. Striatal DA positively influences LID,
while 5-HT is negatively associated with LID. Finally, we suggest that by strategic modification of the serotonin system it
may be possible to attenuate the adverse effects of chronic l-DOPA therapy in PD patients. 相似文献
8.
Inactivation of Tyrosine Hydroxylase Activity by Ascorbate In Vitro and in Rat PC12 Cells 总被引:1,自引:1,他引:0
Abstract: Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC50 of 5.9 μM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC50, 11 μM) and dehydroascorbate (EC50, 970 μM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant prote-olysis of the purified enzyme as determined by sodium do-decyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25–50%. The inactivation seen under in vitro conditions appears to have a counterpart under more physiological conditions. 相似文献
9.
Effects of Wild-Type and Mutated Copper/Zinc Superoxide Dismutase on Neuronal Survival and l-DOPA-Induced Toxicity in Postnatal Midbrain Culture 总被引:3,自引:1,他引:2
Maria A. Mena Uzma Khan Daniel M. Togasaki †David Sulzer ‡Charles J. Epstein Serge Przedborski 《Journal of neurochemistry》1997,69(1):21-33
Abstract: Mutations in the free radical-scavenging enzyme copper/zinc superoxide dismutase (Cu/Zn-SOD) are associated with neuronal death in humans and mice. Here, we examine the effects of human wild-type (WT SOD) and mutant (Gly93 → Ala; G93A) Cu/Zn-SOD enzyme on the fate of postnatal midbrain neurons. One-week-old cultures from transgenic mice expressing WT SOD enzyme had significantly more midbrain neurons and fewer necrotic and apoptotic neurons than non-transgenic cultures. In contrast, 1-week-old cultures from transgenic G93A mice expressing mutant SOD enzyme had significantly fewer midbrain neurons and more necrotic and apoptotic neurons than nontransgenic cultures. To subject postnatal midbrain neurons to oxidative stress, cultures were incubated with l -DOPA. l -DOPA at 200 µ M caused ∼50% loss of tyrosine hydroxylase (TH)-positive neurons in nontransgenic cultures and even greater loss in transgenic G93A cultures; no alterations were noted in GABA neuron numbers. In contrast, 200 µ M l -DOPA did not cause any significant reductions in TH-positive or GABA neuron numbers in transgenic WT SOD cultures. l -DOPA at 50 µ M had opposite effects, in that it significantly increased TH-positive, but not GABA neuron numbers in transgenic WT SOD and G93A and in nontransgenic cultures. These results indicate that increased amounts of WT SOD enzyme promote cell survival and protect against l -DOPA-induced dopaminergic neurotoxicity, whereas increased amounts of mutated Cu/Zn-SOD enzyme have inverse effects. As the spontaneous loss and l -DOPA-induced loss of postnatal dopaminergic midbrain neurons appear to be mediated by free radicals, our study supports the view that mutated Cu/Zn-SOD enzyme kills cells by oxidative stress. 相似文献
10.
The newly synthesized pool of dopamine determines the severity of methamphetamine-induced neurotoxicity 总被引:1,自引:0,他引:1
The neurotransmitter dopamine (DA) has long been implicated as a participant in the neurotoxicity caused by methamphetamine (METH), yet, its mechanism of action in this regard is not fully understood. Treatment of mice with the tyrosine hydroxylase (TH) inhibitor α-methyl- p -tyrosine (AMPT) lowers striatal cytoplasmic DA content by 55% and completely protects against METH-induced damage to DA nerve terminals. Reserpine, by disrupting vesicle amine storage, depletes striatal DA by more than 95% and accentuates METH-induced neurotoxicity. l -DOPA reverses the protective effect of AMPT against METH and enhances neurotoxicity in animals with intact TH. Inhibition of MAO-A by clorgyline increases pre-synaptic DA content and enhances METH striatal neurotoxicity. In all conditions of altered pre-synaptic DA homeostasis, increases or decreases in METH neurotoxicity paralleled changes in striatal microglial activation. Mice treated with AMPT, l -DOPA, or clorgyline + METH developed hyperthermia to the same extent as animals treated with METH alone, whereas mice treated with reserpine + METH were hypothermic, suggesting that the effects of alterations in cytoplasmic DA on METH neurotoxicity were not strictly mediated by changes in core body temperature. Taken together, the present data reinforce the notion that METH-induced release of DA from the newly synthesized pool of transmitter into the extracellular space plays an essential role in drug-induced striatal neurotoxicity and microglial activation. Subtle alterations in intracellular DA content can lead to significant enhancement of METH neurotoxicity. Our results also suggest that reactants derived from METH-induced oxidation of released DA may serve as neuronal signals that lead to microglial activation early in the neurotoxic process associated with METH. 相似文献
11.
Tyrosine Hydroxylase Content of Residual Striatal Dopamine Nerve Terminals Following 6-Hydroxydopamine Administration: A Flow Cytometric Study 总被引:2,自引:1,他引:1
Fluorescence-activated cell sorting based on immunolabeling with a monoclonal antibody to tyrosine hydroxylase and a fluorescein-conjugated secondary antibody was used to identify striatal synaptosomes derived from nigrostriatal dopamine nerve terminals. The amount of tyrosine hydroxylase immunoreactivity in dopaminergic striatal synaptosomes prepared from control rats was compared to the amount in dopaminergic synaptosomes prepared from rats that had received intraventricular injections of 6-hydroxydopamine. Although the absolute number of dopaminergic synaptosomes was decreased in lesioned animals, those residual dopamine terminals present contained more tyrosine hydroxylase than did dopamine terminals from control rats. Both the decrease in the absolute number of dopamine terminals and the increase in tyrosine hydroxylase immunoreactivity in residual terminals were proportional to the extent of the lesion, as determined by measurement of striatal dopamine levels. These results suggest that an increase in the amount of tyrosine hydroxylase protein in residual terminals may represent one compensatory mechanism by which residual dopamine neurons maintain normal striatal function after partial destruction of the nigrostriatal dopamine projection. 相似文献
12.
Tyrosine hydroxylase and Parkinson's disease 总被引:7,自引:0,他引:7
A consistent neurochemical abnormality in Parkinson's disease (PD) is degeneration of dopaminergic neurons in substantia nigra,
leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation ofl-DOPA, the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the
striatum. Similarly, some patients with hereditaryl-DOPA-responsive dystonia, a neurological disorder with clinical similarities to PD, have mutations in the TH gene and decreased
TH activity and/or stability. Thus, a logical and efficient treatment strategy for PD is based on correcting or bypassing
the enzyme deficiency by treatment withl-DOPA, DA agonists, inhibitors of DA metabolism, or brain grafts with cells expressing TH. A direct pathogenetic role of TH
has also been suggested, as the enzyme is a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated
oxidative injury. Recently, it has been demonstrated thatl-DOPA is effectively oxidized by mammalian TH in vitro, possibly contributing to the cytotoxic effects of DOPA. This enzyme
may therefore be involved in the pathogenesis of PD at several different levels, in addition to being a promising candidate
for developing new treatments of this disease. 相似文献
13.
H. Päivärinta O. Häppölä T. H. Joh H. Steinbusch T. Watanabe P. Panula 《The Histochemical journal》1987,19(6-7):319-326
Summary The coexistence of histamine, histidine decarboxylase (the enzyme synthesizing histamine), 5-hydroxytryptamine and tyrosine hydroxylase (the rate-limiting enzyme in catecholamine synthesis), was studied in the rat superior cervical ganglion with the indirect immunofluorescence method. Possible colocalization was examined by staining consecutive sections with two different antibodies, or alternatively in the same section by eluting the first antibody with a mild solution containing potassium permanganate and sulphuric acid, and by staining the same section with another antibody. It was shown that tyrosine hydroxylase immunoreactivity was found both in large principal nerve cells and in small cells, which on the basis of their size and high nucleus—cytoplasm ratio corresponded to small intensely fluorescent (SIF) cells. Histamine, histidine decarboxylase and 5-hydroxytryptamine immunoreactivities were observed only in SIF cells. Those SIF cells which were immunoreactive for histamine, histidine decarboxylase or 5-hydroxytryptamine also contained tyrosine hydroxylase immunoreactivity. On the other hand, all tyrosine hydroxylase-immunoreactive SIF cells were also immunoreactive for histidine decarboxylase or 5-hydroxytryptamine. Some of the SIF cells, which were non-reactive for histamine, were immunoreactive for tyrosine hydroxylase. 相似文献
14.
l-3,4-Dihydroxyphenylalanine Increases the Quantal Size of Exocytotic Dopamine Release In Vitro 总被引:3,自引:1,他引:2
Abstract: The catecholamine precursor l -3,4-dihydroxyphenylalanine ( l -DOPA) is used to augment striatal dopamine (DA), although its mechanism of altering neurotransmission is not well understood. We observed the effects of l -DOPA on catecholamine release in ventral midbrain neuron and PC12 pheochromocytoma cell line cultures. In ventral midbrain neuron cultures exposed to 40 m M potassium-containing media, l -DOPA (100 µ M for 1 h) increased DA release by >10-fold. The elevated extracellular DA levels were not significantly blocked by the DA/norepinephrine transport inhibitor nomifensine, demonstrating that reverse transport through catecholamine-uptake carriers plays little role in this release. In PC12 cells, where DA release from individual secretory vesicles can be observed, l -DOPA (50 µ M for 1 h) elevated DA release in high-potassium media by 370%. Amperometric measurements demonstrated that l -DOPA (50 µ M for 40–70 min) did not raise the frequency of vesicular exocytosis but increased the average size of quantal release to at least 250% of control levels. Together, these findings suggest that l -DOPA can increase stimulation-dependent transmitter release from DA cells by augmenting cytosolic neurotransmitter, leading to increased quantal size. 相似文献
15.
These studies were carried out to characterize the activation of rat striatal tyroxine hydroxylase produced by depolarization of the medial forebrain bundle and to evaluate the possible role of cyclic AMP as a mediator of this activation. The enzymatic properties of tyrosine hydroxylase following in vivo depolarization were compared to those produced by treatment of striatal synaptosomes with dibutyryl cyclic AMP (dbcAMP). Similar effects were observed with regard to enzyme distribution, altered sensitivity to dopamine-induced inhibition, and activity as a function of tyrosine concentration. However, differences between the two treatments were also apparent. First, treatment with dbcAMP shifted the pH optimum from 6.2 to 7.0. In contrast, electrical stimulation decreased the rate of decline in activity as the pH was increased above the optimum, but did not shift the pH optimum. Second, plots of tyrosine hydroxylase activity versus cofactor concentration revealed two enzyme forms for both control and electrically stimulated preparations. However, dbcAMP treatment converted the enzyme to a single high affinity form. These results can be explained by one of the following: (1) cyclic AMP is the sole mediator of enzyme activation, but does not produce a maximally activated enzyme following in vivo depolarization (2) cyclic AMP is only one of several mediators involved or (3) cyclic AMP is not involved in depolarization-induced activation, with activation occurring via the mediation of other intracellular messengers, such as calcium. 相似文献
16.
Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems 总被引:2,自引:4,他引:2
Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells. 相似文献
17.
The effect of pseudorabies virus on neuronal functions was investigated in PC12 cells. During the period investigated, choline acetyltransferase was not affected, while the acetylcholinesterase activity declined steadily starting at 12 h post infection (p.i.), reaching its minimal level of 40% of the control value at 24 h p.i. In contrast, the activity of tyrosine hydroxylase, the key enzyme in catecholamine synthesis, increased to 150% of the control level by 15 h p.i., dropping off slowly with the appearance of viral cytopathology. In parallel, the infection induced, by a process independent of the extracellular Ca2+, an increased release of dopamine at 11 h p.i., followed by noradrenaline at 20 h p.i. In the infected cells, the intracellular content of catecholamine was maintained only in the presence of a high amount of catecholamine precursors in the culture medium. Three plaque-forming units per cell was the minimal multiplicity of infection required to obtain the maximal changes in enzyme activities; higher multiplicities induced more rapidly the maximal effects on tyrosine hydroxylase and acetylcholinesterase. Inhibition of DNA synthesis did not prevent the increase in tyrosine hydroxylase activity; however, protein synthesis was required. In conclusion, infection of the PC12 cells with pseudorabies virus induced significant changes in catecholaminergic and cholinergic metabolism, indicating the ability of this virus to interfere selectively with specialized neuronal functions. 相似文献
18.
F Rodríguez-Pascual R Ferrero M T Miras-Portugal M Torres 《Archives of biochemistry and biophysics》1999,366(2):207-214
The phosphorylation of the enzyme tyrosine hydroxylase by the cGMP pathway was investigated in chromaffin cells from the bovine adrenal medulla. The nitric oxide donor, sodium nitroprusside, and the natriuretic peptide, C-type natriuretic peptide, which are able to increase cGMP levels and cGMP-dependent protein kinase activity, produced significant increases in the phosphorylation level of tyrosine hydroxylase in a time- and concentration-dependent manner. The pretreatment of the cells with the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one blocked the effect of sodium nitroprusside. This result indicates that cGMP production by this enzyme mediated this effect. Experiments performed with a cGMP-dependent protein kinase inhibitor, the Rp-isomer of 8-(4-chlorophenylthio)-cyclic guanosine monophosphorothioate, which blocked the effects of both sodium nitroprusside and C-type natriuretic peptide, demonstrated that the phosphorylation increases evoked by both compounds were mediated by the activation of cGMP-dependent protein kinase. In cells incubated with the adenylyl cyclase activator, forskolin, an increase in the phosphorylation level of the tyrosine hydroxylase was also found. When cells were treated simultaneously with forskolin and sodium nitroprusside or C-type natriuretic peptide, an additive effect on tyrosine hydroxylase phosphorylation was not observed. This suggests that cAMP- and cGMP-dependent protein kinases may phosphorylate the same amino acid residues in the enzyme. Western blot analysis of soluble extracts from chromaffin cells detected specific immunoreactivity for two different commercial antibodies raised against cGMP-dependent protein kinase (both Ialpha and Ibeta isoforms). Electrophoretic mobility correlates with that of purified PKG Ialpha. Because the phosphorylation of the tyrosine hydroxylase correlates with increases in its enzymatic activity and thus with augmentation in the cell capacity to synthesize catecholamines, our results indicate that a cGMP-based second messenger pathway participates in catecholamine biosynthesis regulation in chromaffin cells, a mechanism which may be widespread in other catecholamine-synthesizing cells. 相似文献
19.
Expression of human tyrosine hydroxylase cDNA in invertebrate cells using a baculovirus vector 总被引:1,自引:0,他引:1
E I Ginns M Rehavi B M Martin M Weller K L O'Malley M E LaMarca C G McAllister S M Paul 《The Journal of biological chemistry》1988,263(15):7406-7410
A human cDNA containing the complete coding sequence for a human tyrosine hydroxylase (EC 1.14.16.2, form 2) was introduced into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) downstream to the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus resulted in the expression of human tyrosine hydroxylase in these invertebrate cells. Characterization of tyrosine hydroxylase activity in infected SF9 cells demonstrated both substrate and cofactor kinetics that were characteristic of those previously reported for the native human enzyme. Both 3-iodotyrosine and alpha-methyl-p-tyrosine competitively inhibited the recombinantly produced tyrosine hydroxylase with Ki values of 1.2 and 16 microM, respectively, similar to those previously reported for the rat and human enzymes. Western blot analysis of extracts of SF9 cells infected with the recombinant baculovirus containing human tyrosine hydroxylase cDNA revealed a major immunoreactive band with an apparent Mr of 60 kDa, identical to the size of the immunoreactive protein from rat adrenal and caudate nucleus. The use of the baculovirus expression system to produce abundant quantities of each of the multiple forms of active human tyrosine hydroxylase in eukaryotic cells should facilitate structural analysis and help clarify the physiological significance of each of the isoenzymes. 相似文献
20.
Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates. 总被引:16,自引:9,他引:7
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M L Haffey J T Stevens B J Terry D I Dorsky C S Crumpacker S M Wietstock W T Ruyechan A K Field 《Journal of virology》1988,62(12):4493-4498
The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae. 相似文献