Flavanone and flavonoid hydroxylase genes regulate fiber color formation in naturally colored cotton

Using naturally colored cotton(NCC)can eliminate dyeing,printing and industrial processing,and reduce sewage discharge and energy consumption.Proanthocyanidins(PAs),the primary coloration components in brown fibers,are polyphenols formed by oligomers or polymers of flavan-3-ol units derived from anthocyanidins.Three essential structural genes for flavanone and flavonoid hydroxylation encoding flavanone-3-hydroxylase(F3H),flavonoid 3’-hydroxylase(F3’H)and flavonoid 3’5’-hydroxylase(F3’5’H)are initially committed in the flavonoid biosynthesis pathway to produce common precursors.The three genes were all expressed predominantly in developing fibers of NCCs,and their expression patterns varied temporally and spatially among NCC varieties.In GhF3Hi,GhF3’Hi and GhF3’5’Hi silenced lines of NCC varieties XC20 and ZX1,the expression level of the three genes decreased in developing cotton fiber,negatively correlated with anthocyanidin content and fiber color depth.Fiber color depth and type in RNAi lines changed with endogenous gene silencing efficiency and expression pattern,the three hydroxylase genes functioned in fiber color formation.GhF3H showed functional differentiation among NCC varieties and GhF3’H acted in the accumulation of anthocyanin in fiber.Compared with GhF3’H,GhF3’5’H was expressed more highly in brown fiber with a longer duration of expression and caused lighter color of fibers in GhF3’5’H silenced lines.These three genes regulating fiber color depth and type could be used to improve these traits by genetic manipulation.

Effect of α-synuclein on the promoter activity of tyrosine hydroxylase gene

Objective To approach the associated mechanism by which α-synuclein （α-Syn） might regulate the metabolism of dopamine. Methods A DNA fragment, located at --495 to ＋25 of the human tyrosine hydroxylase （TH） gene, was amplified by PCR and inserted into the pGL3-Basic luciferase reporter vector. The recombinant plasmid pGL3-THprom was transfected into a dopammergic cell line MES23.5 or a α-Syn over-expressed MES23.5 （named MES23.5/hα-Syn^＋）. The promoter activity was detected by the Dual Luciferase Assay System. Results The luciferase activities in the MES23.5 cells transfected with pGl.,3-Basic, pGL3-THprom, and pGL3-Control vectors were 5.60±0.67, 26.80±4.11, and 32.90±4.75, respectively. On the other hand, the luciferase activity of pGL3-THprom in the MES23.5 （26.80±4.11） was significantly higher than that in the MES23.5/hα-Syn^＋（14.40±0.61） （P〈0.01）. Conclusion These results indicate that the -495 to ＋25 region in the TH gene possesses promoter activity for controlling the gene expression, and that α-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.

Changes of 5-hydroxytryptamine and tryptophan hydroxylase expression in the ventral horn of spinal cord

Objective To investigate changes of 5-hydroxytryptamine （5-HT） and its synthesis rate-limiting enzyme tryp-tophan hydroxylase （TPH） in the ventral horn of spinal cord after exercise-induced fatigue, and to further discuss the mecha- nism of exercise-induced central fatigue at spinal level. Methods Sixteen healthy adult Wistar rats were randomly divided into 2 groups： exercise-induced fatigue group and control group. Immunohistochemical staining for 5-HT and TPH in the ventral horn were performed and analysized quantitatively. The mean optic densities of 5-HT and TPH positive fibers or terminals were measured by computerized image analyzer. Results Both 5-HT and TPH positive fibers/terminals decreased in the exercise-induced fatigue group. The immunohistochemical staining was weaker and the mean optic densities decreased obviously in the fatigue group compared with those in the control group （P 〈 0.05）. Conclusion 5-HT and TPH in the ventral horn of spinal cord might be involved in exercise-induced fatigue.

In vitro Effect of Dithiocarbamate Pesticides and of CaNa_2EDTA on Human Serum Dopamine-β-hydroxylase

Serum dopamine-β-hydroxylase (DBH) inhibition has been reported in lead workers treated with CaNa2EDTA and in alcoholic patients repeatedly treated with the alcohol aversive drug Disulfiram. The mechanism of inhibition involves Cu-chelation at the active site of DBH. The effect of CaNa2EDTA and Disulfiram on serum DBH has been compared to the effect of dithiocarbamate pesticides in vitro for the possible use of serum DBH determination for the biological monitoring of workers exposed to these pesticides.Most dithiocarbamates inhibit human serum DBH at micromoIar concentrations (range of I50, 0.027-1.6 μmol/L). The inhibitory potency increased from methyl- and dimethyl dithiocarbamates to diethyl dithiocarbamates up to the most potent ethylene bisdithiocarbamates. The I50 of CaNa.EDTA was 3.8 mol/L, higher than those of dithiocarbamates. Copper addition to the test system rcactivated at stoichiometric concentrations dithiocarbamaie-inhibited DBH indicating that both base line values and percent of inhibition can be calculated in a single blood sample. Results suggest that serum DBH determination couId be useful in case of acute poisoning involving high doses of dithiocarbamate pesticides

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

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

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

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

Cloning and Expression Profile of Deoxyhypusine Snyhtase Gene and Deoxyhypusine Hydroxylase Gene in Silkworm, Bombyx mori

Deoxyhypusine snyhtase （DHS） and deoxyhypusine hydroxylase （DOHH） are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A （eIF5A）. Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we described the cloning and expression of two full-length cDNAs, encoding respectively DHS-like protein and DOHH-like protein from Bombyx mori by using the methods of bioinformatics, RACE, and RT-PCR technology, named as BmDHS and BmDOHH. Sequencing results indicate that they are 1 311 and 1 874 bp in length including complete open reading frame （ORF） 1 116 and 915 bp, which encode 371 amino acids （molecular weight is about 41.11 kD and isoelectric point is 5.84） and 304 amino acids （molecular weight is about 34.30 kD and isoelectric point is 4.86）, respectively. BmDHS contains only 1 exon, and BmDOHH contains 4 exons and 3 introns. The deduced amino acid sequence of BmDHS contains a deoxyhypusine synthase domain from 47 to 361 amino acid residues, and the deduced amino acid sequence of BmDOHH contains 6 E-Z type HEAT repeat domains （23-52, 54-83, 87-116, 177-206, 208-237, and 241- 270）. Compared to DHS and DOHH amino acid sequences from other species, such as Homo sapiens and Drosophila melanogaster, both silkworm DHS protein and DOHH protein have more than 55% identity. The conservative regions are very similar with each other. The phylogenetic tree analysis indicated that not only DHS but also DOHH from different species has genus-specific features. The expressions of BmDHS and BmDOHH are no tissue and stage specific in our tested samples.

Altered tryptophan hydroxylase 2 expression in enteric serotonergic nerves in Hirschsprung's-associated enterocolitis

AIM: To determine if expression of colonic tryptophan hydroxylase-2(TPH2), a surrogate marker of neuronal 5-hydroxytryptamine, is altered in Hirschsprung's-associated enterocolitis. METHODS: Entire resected colonic specimens were collected at the time of pull-through operation in children with Hirschsprung's disease(HSCR, n = 12). Five of these patients had a history of pre-operative Hirschsprung's-associated enterocolitis(HAEC). Controls were collected at colostomy closure in children with anorectal malformation(n = 10). The distribution of expression of TPH2 was evaluated using immunofluorescence and confocal microscopy. Protein expression of TPH2 was quantified using western blot analysis in the deep smooth muscle layers. RESULTS: TPH2 was co-expressed in nitrergic and cholinergic ganglia in the myenteric and submucosal plexuses in ganglionic colon in HSCR and healthy controls. Co-expression was also seen in submucosal interstitial cells of Cajal and PDGFRα+ cells. The density of TPH2 immuno-positive fibers decreased incrementally from ganglionic bowel to transition zonebowel to aganglionic bowel in the myenteric plexus. Expression of TPH2 was reduced in ganglionic bowel in those affected by pre-operative HAEC compared to those without HAEC and healthy controls. However, expression of TPH2 was similar or high compared to controls in the colons of children who had undergone diverting colostomy for medically refractory HAEC.CONCLUSION: Altered TPH2 expression in colonic serotonergic nerves of patients with HSCR complicated by HAEC may contribute to intestinal secretory and motor disturbances, including recurrent HAEC.

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

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

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

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

Treatment time influences the effects of a low-frequency pulsed electric field on synthesis of tyrosine hydroxylase and dopamine in PC12 cells

BACKGROUND： Electromagnetic radiation can influence dopamine （DA） synthesis in brain tissues or ceils, but electromagnetic frequencies, intensities, and radiation time can produce different effects. In addition, the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial. OBJECTIVE： To determine tyrosine hydroxylase （TH） expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field （LF-PEF） stimulation, and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors. DESIGN, TIME AND SETTING： A parallel, controlled, cell experiment was performed at the Laboratory of Cell Biology, School of Life Science, East China Normal University, between January and October 2006. MATERIALS： PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. Nerve growth factor was purchased from PeproTech, USA. The protein kinase A inhibitor, H-89, and mitogen-activated protein kinase kinase inhibitor, U0126, were purchased from Sigma, USA. METHODS： （1） Following routine culture in Dulbecco＇s modified eagle medium, primary PC12 cells were stimulated under LF-PEF （pulse frequency 50.Hz, pulse width 20 μs, peak field strength 1 V/m） for 5, 10, 15, 20, and 30 minutes. （2） Inhibitors （H-89 or U0126, 1 μmol/L） were added 30 minutes before LF-PEF stimulation for 10 minutes. MAIN OUTCOME MEASURES： （1） TH expression was determined by Western blot in PC12 cells at 0.5, 1,2, 3, and 4 days after LF-PEF stimulation. Similarly, DA was measured by high-performance liquid chromatography in media at 2, 3, 4, or 5 days after LF-PEE （2） TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEE RESULTS： （1） Short-term LF-PEF stimulation （5 and 10 minutes） increased TH expression and media DA levels after short-term culture （2 days） （P 〈 0.01）, but both parameters decreased with longer culture （3 4 days） （P 〈 0.01）. Long-term LF-PEF stimulation （15, 20, or 30 minutes） decreased TH and DA synthesis, followed by a rapid increase （P 〈 0.01）. （2） H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes, while the inhibition rate of U0126 was 53.2%. CONCLUSION： Short-term LF-PEF first promotes then inhibits, while long-term LF-PEF first inhibits then promotes, TH and DA synthesis. LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.

Molecular Docking of Bacosides with Tryptophan Hydroxylase:A Model to Understand the Bacosides Mechanism

Tryptophan hydroxylase(TPH)catalyses L-tryptophan into 5-hydroxy-L-tryptophan,which is the first and ratelimiting step of serotonin(5-HT)biosynthesis.Earlier,we found that TPH2 up-regulated in the hippocampus of postnatal rats after the oral treatment of Bacopa monniera leaf extract containing the active compound bacosides.However,the knowledge about the interactions between bacosides with TPH is limited.In this study,we take advantage of in silico approach to understand the interaction of bacoside-TPH complex using three different docking algorithms such as HexDock,PatchDock and AutoDock.All these three algorithms showed that bacoside A and A3 well fit into the cavity consists of active sites.Further,our analysis revealed that major active compounds bacoside A3 and A interact with different residues of TPH through hydrogen bond.Interestingly,Tyr235,Thr265 and Glu317 are the key residues among them,but none of them are either at tryptophan or BH4 binding region.However,its note worthy to mention that Tyr 235 is a catalytic sensitive residue,Thr265 is present in the flexible loop region and Glu317 is known to interacts with Fe.Interactions with these residues may critically regulate TPH function and thus serotonin synthesis.Our study suggested that the interaction of bacosides(A3/A)with TPH might up-regulate its activity to elevate the biosynthesis of 5-HT,thereby enhances learning and memory formation.

Expression and effect of proline hydroxylase domain 2 in retina of diabetic rats

AIM： To observe the expression of proline hydroxylase domain 2 （PHD2） in the retina of diabetic rats and investigate the relationship between PHD2 and relevant intraocular vascular proliferation factors, METHODS： Sixty male specific pathogen free （SPF） Sprague-Dawley （SD） rats were randomly divided into two groups： the diabetic group and the control group. The rats in the diabetic group were intraperitoneally injected with 60 mg/kg （0.60 mL/100 g） of streptozotocin to induce a diabetic rat model. The rats in the control group were injected with an equal volume of sodium citrate buffer solution by the same method. Hematoxylin- eosin （HE） staining and immumofiuorescence （IF） method were adopted to observe the pathological changes of retinal tissues and the expression of PHD2, glial fibrillary acidic protein （GFAP）, vascular endothelial growth factor （VEGF） by 8wk. RT-PCR method was applied to detect the expressions of mRNA of PHD2, VEGF and GFAP. The relationship between PHD2 and other vascular proliferation factors was analyzed. RESULTS： HE staining showed that there was the retinal tissue edema in the diabetic group, and the arrangement was in disorder, and proliferation could be seen. IF staining： in the retina of normal rats, PHD2 was not expressed, GFAP and VEGF were mainly expressed in astrocytes; while in the diabetic rats, PHD2, GFAP and VEGF staining showed strong positivity in all retinal layers, mainly in neurogliocytes. PHD2 was co-expressed with VEGF and GFAP. The mRNA expression levels of PHD2, GFAP and VEGF in the diabetic group were obviously higher than that in the control group, respectively 1.83 times, 1.75 times and 2.08 times. The difference had statistical significance （P〈0.01）. CONCLUSION： The high expression of PHD2 in the retina of early-stage diabetic rats might result from secretion of neurogliocytes induced by local high- concentration blood glucose, thus promoting the expression of VEGF and GFAP, PHD2 plays an important role during the occurrence of diabetic retinopathy.

Further Insights on the <i>Datura innoxia</i>Hyoscyamine 6<i>β</i>-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Hyoscyamine, anisodamine and scopolamine are tropane alkaloids present in some Solanaceae species and used in modern medicine. L-Hyoscyamine is hydroxylated to 6β-hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. A natural mutation in the Gly-220 residue to Cys was previously shown to be associated with the loss of function of H6H in Mandragora officinarum, preventing the accumulation of anisodamine and scopolamine in these plants. We show here that a deliberate Gly220Cys mutation in the Datura innoxia DiH6H protein caused a loss of both its enzymatic abilities and rendered it unable to hydroxylate L-hyoscyamine into anisodamine and to epoxidate anisodamine into scopolamine. By using protein modeling based on an available crystal structure of H6H from Datura metel, we show how the Cys220 residue causes a steric interference in the active site cavity impairing the interaction of both substrates, hyoscyamine and anisodamine with the active site of the protein. We also address the enantiomeric preference of DiH6H based on molecular modeling.

Analysis of the Alkane Hydroxylase Gene and Long-Chain Cyclic Alkane Degradation in <i>Rhodococcus</i>

To characterize the long-chain cyclic alkane (c-alkane) degradation of bacteria in Rhodococcus, we analyzed the relationship between the alkane hydroxylase gene (alkB) and long-chain c-alkane degradation in 19 species. Eleven strains which were isolated from nature using long-chain c-alkane as a substrate were identified as R. erythropolisc, and all were shown to carry the alkB [alkB R2 type]. This gene type was also carried by two other species, R. rhodochrous and R. baikonurensis. In total, 17 species of the genus Rhodococcus carried alkB, but the gene types differed from each other. The two species R. rhodnii and R. coprophilus did not carry alkB, and their long-chain c-alkane degradation levels were low.

Tyrosine hydroxylase and Lewy body molecules immunoreactivity in the SNC neurons of an AS/AGU mutantrat

The AS/AGU rat has a recessive single point mutation in the gene coding for the gamma isoform of protein kinase C (PKC-γ) resulting in a failure to release dopamine in the striatum and impaired movement including a staggering gait, difficulty in initiating movement and a slight whole body tremor. This study examined the levels tyrosine hydroxylase, ubiquitin and parkin in individual SNC cell bodies, there was no evidence of a reduction in tyrosine hydroxylase levels although levels of ubiquitin and parkin were elevated in the cytoplasm. The findings support the hypothesis that the initial bar to dopamine availability in the striatum is reduced release, with substantia nigra cell death being a later phenomenon.

TWO NOVEL MUTATIONS IN PHENYLALANINE HYDROXYLASE GENE AND IN VITRO EXPRESSION ANALYSIS ON MUTATION ARG252GLN

We report novel mutations in exon 7 of human phenylalanine hydroxylase (PAH) gene of phenylketonuria (PKU ) in southern Chinese, analysed by using PCR-DGGE (denaturing gradient gel electrophoresis ), solid phase DNA sequencing and Ih vliro expression. One of the 2 novel mutations, IVS6nt1, is an intron-exon Junctional mutation which results a splicing defect in mRNA. Arg252Gln is another novel mutation with residual PAH activity only 24 % compared to wild type in in vitro mutagenesis and expression in Cos-1 cell. Other 3 known mutations and polymorphism including Arg241Cys, Arg243Gln and Val245Val(GTG to GTA) together with these novel mutations composed the mutatlonal profile of exon 7 in the PAH gene of PKUs in this populations.

COEXISTENCE OF FOS-LIKE PROTEIN IN TYROSINE HYDROXYLASE-LIKE IMMUNOREACTIVE NEURONS IN THE HINDBRAIN OF ARTS FOLLOWING PERIPHERAL ELECTRICAL STIMULATION

In the present study we have found that proto-oncogene c-fos protein can expressin the noradrenergic neurons of rat hindbrain following peripheral electrical stimulation. Ratswere given peripheral electrical stimulation via thin stainless steel pins inserted into the pointsnear knee joint (S36) and ankle joint (Sp6) which mimic the manipulation of electroacupuncture(EA) performed in humans. Animals were perfused for double staining immunohistochemistry 2hafter the termination of EA. In rats subjected to EA stimulation Fos-like protein was found in thetyrosine hydroxylase (TH)-like immunoreactive neurons in rat hindbrain. The Fos and TH coex-isting neurons were distributed in the locus coeruleus, solitary tract nucleus, ventrolateral medul-la, periaqeductal gray, as well as superior colliculus. The percentage of the coexisting neuronscompared with the total number of neurons containing Fos-like protein in these nuclei rangedfrom 6% to 32%. The results suggest that the noradrenergic neurons in these regions may be ac-tivted by acupuncture stimulation.

Tyrosine hydroxylase gene transfections to different sites of striatum in the rat model of Parkinson’s disease

The use of gene therapy has been intensively studied as a potential method to treat Parkinson’s disease (PD) and other degenerative brain diseases. However, the effects of experimental measures and approaches on the outcome of gene delivery or on the physiological state of target tissues have not been analyzed as much and systematically. Therefore, we have infused adenovirus vectors expressing either a therapeutic tyrosine hydroxylase (TH) gene or a lacZ reporter gene into striatum in a rat model of PD. The experimental procedures were tested using the Ad lacZ vector in order to optimize concentrations, volumes, infusion speeds and transfection times. The expression of Ad lacZ vector was lower and declined earlier in the lesioned than unlesioned striatum suggesting that the lesion affects on the transfection efficiency and outcome of gene transfection. The effect of three different approaches of Ad TH vector transfection was compared: 1) the delivery of Ad TH gene vector alone into one single site of striatum, 2) the delivery of Ad TH gene vector alone into multiple sites of striatum, and 3) the delivery of Ad TH gene vector into one site of striatum followed by a continuous infusion of tetrahydrobiopterin (BH4) cofactor with a mini pump. There was a small and transient unsignificant decrease in the turning behavior when the Ad TH vector was delivered into one site of the striatum. Simultaneous infusion into several sites or together with BH4 cofactor did not improve more the effect of gene delivery. Thus, although the effects were unsignificant, the Ad TH transfection seemed to decrease the turning behavior in the rat model of PD and the optimal effect was seen at some specific doses and time points. Furthermore, the outcome of gene therapy could depend in addition to the amount and efficacy of gene vectors also on the physiological state and experimental strategies.

MUTATIONS IDENTIFIED IN EXON 7 OFPHENYLALANINE HYDROXYLASE GENE IN CHINESE

Exon 7 of the l’henylalan1ne hydroxylase (PAH) gene was analyzed in 15 chlldren affected wlth classicphenylketonL1rla (PKU) from northern Chlna by uslng PCRxsingle strand conformation polymorphism(PCR-SSCP) technique and DNA direct sequencing. Six missense mutatlons (l. e. R2413Q. R 241H, G247V,1,2 19H, F2541;lnd G257V )and one silent rnutatlon (V245v ) were identified. The latter three missense mu-tations were demonstrated as novel mltations in comparison with the PAH mutation database. one missense mt1tation (R241 H) was flrst dowumeTlted in Chinese. our results showed populatlon ancl reglon tllffer-ences in the PAH mutation clistribution. and suggest that there is more thfln one founding population forPKU in China. The fincling of novel mutations will enhence the molecular diagnosis of PKU.