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The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 A, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997) Nat. Struct. Biol. 4, 995-1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, the KM value for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein. 相似文献
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Two tests have been compared for detection of heterozygotes for phenylketonuria, one based on determination of plasma phenylalanine and tyrosine concentrations in fasting individuals and the other on kinetic evaluation of the plasma elimination curve after intravenous loading with L-phenylalanine. The plasma elimination curve was biexponential and the kinetics were evaluated according to the two-compartment model. The constant, beta, expressing the rate of elimination from plasma at pseudo-equilibrium, the rate constant for the elimination from the central compartment, and the total body clearance were determined. Of these three, total body clearance, which on the average was reduced by 32% in the phenylketonuric heterozygotes, showed the best discriminatory ability, but was not better than the information on concentrations of phenylalanine and tyrosine in detecting heterozygotes for phenylketonuria. 相似文献
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S100β, a calcium-binding brain specific protein, may affect both brain development and hippocampal long-term potentiation. S100β levels are elevated in Down's syndrome (DS), and the gene for S100β is located on chromosome 21, which is duplicated in DS. To test the hypothesis that elevated levels of S100β cause behavioral alterations in a mammalian system, 3 transgenic mouse lines with multiple copies of the human gene for S100β were derived and behaviorally tested. The spontaneous alternation behavior of transgenic and normal littermate mice was compared in a T-maze during a 15-trial test. The overall alternation rate was found to be significantly decreased in the transgenic mice compared with their normal littermates. The S100β transgenic mouse model offers one of the first opportunities to investigate the relation between overexpression of a human chromosome 21 gene product and abnormal behavior and brain function. (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
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Well, appropriate-for-gestational age, low-birth-weight infants were divided into three gestational age groups and assigned randomly within each age group to one of five feeding regimens: pooled human milk (BM); formula 1 (F1) = 1.5 gm/dl protein, 60 parts bovine whey proteins: 40 parts bovine caseins; F2 = 3.0 gm/dl, 60:40; F3 = 1.5 gm/dl, 18:82; F4 = 3.0 gm/dl, 18:82. Plasma and urine concentrations of tyrosine and phenylalanine were far higher in the infants fed F1 to F4, especially F2 and F4, than in the infants fed BM. These findings offer further evidence for the limited capacity of the low-birth-weight infant to catabolize tyrosine. Infants fed F3 had significantly higher plasma tyrosine concentrations than infants fed F1, and those fed F4 had higher concentrations than those fed F2. Thus, increased plasma tyrosine concentrations in low-birth-weight infants are related directly both to the quantity and to the quality of the protein in their diets. 相似文献
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We found previously that restriction of tyrosine (Tyr) and phenylalanine (Phe) inhibited growth and metastasis of B16BL6 murine melanoma and arrested these cells in the G0-G1 phase of the cell cycle. Here, we report that deprivation of these two amino acids in vitro induces apoptosis in B16BL6 and in human A375 melanoma cells but not in nontransformed, neonatal murine epidermal cells or human infant foreskin fibroblasts. Four days after deprivation of Tyr and Phe in vitro, 37% of B16BL6 and 51% of A375 melanoma cells were undergoing apoptosis. Apoptosis was not associated with elevation in intracellular calcium or alteration in p53 or c-myc protein expression. Expression and Tyr phosphorylation of focal adhesion kinase (FAK) were inhibited in both melanoma cell lines by deprivation of Tyr and Phe but not by deprivation of glutamine or serum. Tyr phosphorylation of FAK in Tyr- and Phe-deprived melanoma cells was enhanced within 30 min of refeeding with complete DMEM. FAK protein expression recovered within 60 min, and cell viability recovered within 24 h. Genistein, a tyrosine kinase inhibitor that specifically inhibits Tyr phosphorylation of FAK, did not induce apoptosis in A375 melanoma cells at a concentration of 50 microM. Genistein prevented the recovery of cell viability upon refeeding with Tyr and Phe to previously deprived A375 melanoma cells. These data collectively indicate that apoptosis induced by Tyr and Phe deprivation is FAK-dependent. 相似文献
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Treated phenylketonuria (PKU) has been linked to dopaminergic depletion in the dorsolateral prefrontal cortex, potentially leading to selective executive impairment. White matter abnormalities may lead to generalized slowing of information processing. These 2 hypotheses were evaluated in adults with PKU on a lifelong diet. Those with PKU were significantly slower than the control group regardless of working memory load on an n-back task and marginally slower regardless of trial type (inhibitory or noninhibitory) on a flanker task. There were no significant differences in speed on object alternation learning or perceptual judgment tasks. There were no group differences in accuracy on any task. These findings do not appear consistent with the selective executive hypothesis. A cognitive slowing account may prove more informative in adults with PKU, but more evidence is needed. The findings suggest that continuous dietary management is a fairly successful strategy in terms of cognitive outcome for adults. (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
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After an overnight fast, 5 male healthy subjects ingested increasing amounts of a solution containing a fixed proportion of seven essential amino acids (L-isoleucine, 13.3%; L-leucine, 21.0%; L-lysine, 15.2%; L-methionine, 21.0%; L-threonine, 9.5%; L-tryptophan, 4.8% and L-valine, 15.2%) and lacking phenylalanine and tyrosine. The solutions caused a rapid fall in plasma phenylalanine and tyrosine which was proportional to the total amount of amino acids ingested. Following the highest dose administered (31.5 g) plasma phenylalanine and tyrosine fell to a minimum of, respectively, 12.7% and 29.8% the initial levels and remained markedly reduced at 6 hours after treatment. The decrease of tyrosine and phenylalanine levels was associated with a decrease of systolic and diastolic arterial pressure. 相似文献
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Y Wu MJ Nadler LA Brennan GD Gish JF Timms N Fusaki J Jongstra-Bilen N Tada T Pawson J Wither BG Neel N Hozumi 《Canadian Metallurgical Quarterly》1998,8(18):1009-1017
BACKGROUND: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway. RESULTS: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis. CONCLUSIONS: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity. 相似文献
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This study addresses how the timing of a known biological insult affects the developmental progression of executive functions. The sample consisted of children exposed to elevated levels of phenylalanine, either postnatally, as in phenylketonuria (PKU; n = 46), or prenatally, as in maternal PKU (n = 15). Nonhyperphenylalaninemic siblings of children with PKU (n = 18) served as controls. Results indicated that elevated levels of phenylalanine are toxic to the neurological systems that manage executive functions and cognitive tempo. This toxicity is dose dependent, with higher levels of phenylalanine being more detrimental. Executive function difficulties noted in PKU are consistent with attention deficit hyperactivity disorder (ADHD)-inattentive type, whereas maternal PKU offspring had executive function difficulties consistent with ADHD-combined type. (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
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N Kashio W Matsumoto S Parker DM Rothstein 《Canadian Metallurgical Quarterly》1998,273(50):33856-33863
The CD45 protein tyrosine phosphatase (PTPase) has been shown to regulate the activity of Lck and Fyn protein tyrosine kinases in T cells. However, it is not clear that these constitute the only CD45 substrates. Moreover, the manner by which PTPase activity and substrate recruitment are regulated, is poorly understood. Previous in vitro studies suggest that the first cytoplasmic PTPase domain (D1) of CD45 is the active PTPase, which may be regulated by an enzymatically inactive second PTPase domain (D2). However, the function of CD45 D2 in vivo is unknown. In this study, reconstitution of CD45(-) T cells with specific CD45 PTPase mutants allowed demonstration of a critical role for D2 in TCR-mediated interleukin (IL)-2 production. Specifically, replacement of CD45 D2 with that of the LAR PTPase to form a CD45/LAR:D2 chimera, abrogates CD45-dependent IL-2 production. This effect cannot be accounted for by loss of PTPase activity per se. The expression of D1 substrate-trapping mutants reveals an in vivo interaction between CD45 and TCR-zeta that is dependent on CD45 D2. Thus, cells expressing CD45 lacking D2 exhibit abnormal TCR-mediated signaling characterized by hyperphosphorylation of zeta and deficient ZAP-70 phosphorylation. These data suggest an essential role for CD45 D2 in TCR-regulated IL-2 production through substrate recruitment of the zeta chain. 相似文献
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M Deuschle U Schweiger H Standhardt B Weber I Heuser 《Canadian Metallurgical Quarterly》1996,21(8):645-649
We studied corticosteroid-binding globulin (CBG) in 25 drug-free depressed patients and 33 healthy controls over a wide age-range. CBG was measured at 0800, 1400, 2000 and 2400 h in all subjects. Analysis of variance (ANOVA) with repeated measurement design revealed a significant effect of gender and time, but not of diagnosis (depressed patients vs healthy controls) or age group (< 50/> 50 years). In females, regardless of diagnosis, CBG plasma concentrations were significantly increased, when compared with their male counterparts. Although as a group depressed patients had significantly higher plasma cortisol concentrations (108.0 +/- 23.1 vs 70.7 +/- 10.9 micrograms/l), CBG levels did not differ between the two groups. Thus we did not find hypercortisolemia in depression to be paralleled by a decrease in CBG. However, the exaggerated activity of the hypothalamus-pituitary-adrenocortical system in healthy and depressed females is associated with an increase in plasma CBG. 相似文献
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Channon Shelley; German Elaine; Cassina Cristina; Lee Philip 《Canadian Metallurgical Quarterly》2004,18(4):613
The executive deficit hypothesis of treated phenylketonuria (PKU) suggests that dopaminergic depletion in the lateral prefrontal cortex leads to selective executive impairment. This was examined by comparing adults with PKU on a lifelong diet with a matched healthy control group. Those with PKU were impaired on selective and sustained attention, working memory (Self-Ordered Pointing), and letter fluency. However, they failed to show differential sensitivity to increased cognitive load on the attentional and working memory tasks, and they did not differ significantly on the remaining executive tasks (rule finding, inhibition, and multitasking). Nor did they differ significantly on recall or recognition memory. Overall, the findings provided little support for the executive deficit hypothesis. A possible explanation in terms of slowed information processing speed is explored. (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
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The anxiolytics buspirone (BUS), ipsapirone (IPSAP) and gepirone (GEP) were investigated as 5-HT1A receptor-mediated inhibitors of tyrosine hydroxylation (TH) in a synaptosome-rich preparation of rat striatum. BUS, IPSAP and GEP were moderately potent inhibitors of TH with EC50 values of 48.4 microM, 50 microM and 836 microM, respectively. By comparison, 8-OH-DPAT, a 5-HT1A receptor selective agonist, has been previously shown to be more potent with an EC50 value of 7.0 microM. Each of these agents demonstrated full agonist activity at the striatal 5-HT1A receptors regulating TH. The inhibitory effects of each agent were attenuated by prior exposure to the 5-HT1A antagonist NAN-190, (10 microM) (P < 0.05), but not by the dopamine D2 antagonist (-)-sulpiride (10 microM). The potencies of 8-OH-DPAT, BUS, IPSAP and GEP were correlated with their reported affinities for the 5-HT1A receptor (P < 0.01) but not the dopamine D2 receptor. These results support the hypothesis that BUS, IPSAP and GEP inhibit TH through activation of a striatal 5-HT1A heteroreceptor on dopamine nerve terminals. 相似文献
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Phenylalanine at residue 8 in the Aalpha chain of fibrinogen is a highly conserved amino acid that is believed to be critical for binding and catalysis by the serine protease thrombin. We have examined the requirement for Phe at this position by constructing a variant recombinant fibrinogen with a conservative substitution of tyrosine for phenylalanine, Aalpha F8Y fibrinogen. We found that the variant fibrinopeptide A (F8Y 1-16) was cleaved by thrombin, in contrast to the lack of cleavage of an Aalpha 1-23 peptide and an Aalpha 1-50 fusion protein with the same substitution. This result indicates that fibrinogen residues other than Aalpha 1-50 participate in thrombin binding and fibrinogen proteolysis. We found, for the first time, that thrombin-catalyzed lysis of the fibrinogen Bbeta chain preceded lysis of the Aalpha chain, such that fibrinopeptide B (FpB) was released prior to F8Y 1-16. Kinetic analysis demonstrated that F8Y 1-16 was a very poor substrate for thrombin, with a specificity constant 280-fold lower than normal fibrinopeptide A. FpB was also a poor substrate, but the specificity constant for FpB was only 4-fold lower than normal. Consequently, FpB was preferentially released from Aalpha F8Y fibrinogen. This "role reversal" had a dramatic effect on polymerization, such that the rate of Aalpha F8Y fibrinogen polymerization was 13% of the rate of normal recombinant fibrinogen. These results confirm the importance of phenylalanine at Aalpha chain residue 8 for efficient thrombin-catalyzed proteolysis of fibrinogen, and further demonstrate that sequential fibrinopeptide release has an important role in normal polymerization. 相似文献
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The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin. This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E. coli and its purification to approximately 90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity. Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe) in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe261. To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to approximately 90% purity. F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity. The Tyr-->Phe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics. 相似文献
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MA Dudley L Wykes AW Dudley M Fiorotto DG Burrin J Rosenberger F Jahoor PJ Reeds 《Canadian Metallurgical Quarterly》1997,127(5):687-693
Epidermal Langerhans cells (LC) may occur in subsets with different phenotypic and functional characteristics. In this work give further evidence that the CD1a-positive LC population in the normal human epidermis may be heterogeneous. We found that one of our monoclonal antibodies (TE4B) to stratum corneum chymotryptic enzyme (SCCE) stained a population of dendritic cells in the normal epidermis in addition to high suprabasal keratinocytes. The staining of the dendritic cells was seen only when the biopsies had been fixed with formaldehyde and when the sections had been pretreated, either with proteolytic enzymes or with Triton X-100. The binding of the antibody was mediated through its antigen binding site, as it could be inhibited by adsorption with recombinant pro-SCCE. Experiments with double labelling showed that the TE4B-positive dendritic cells were also CD1a-positive. On the other hand, not all CD1a-positive cells were TE4B-positive. By means of confocal microscopy of double-labelled cells, the TE4B binding site could be localized intracellularly. SCCE-mRNA could be detected by in situ hybridization in high suprabasal keratinocytes only. A possible explanation may be that there is a subset of LC which have taken up SCCE secreted by high suprabasal keratinocytes. Alternatively, TE4B may bind to an epitope present in a subgroup of epidermal LC which cross-reacts immunologically with SCCE. It is suggested that the demonstrated heterogeneity of the population of LC in the normal epidermis should be taken into account in studies on the possible role of epidermal autoantigens in the development of immune-mediated skin diseases. 相似文献
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The activity of rat liver phenylalanine hydroxylase (PAH; phenylalanine 4-monooxygenase, EC 1.14.16.1) is regulated by interaction with its substrate, phenylalanine, and its coenzyme, BH4 [tetrahydrobiopterin (6R-dihydroxypropyl-L-erythro-5,6,7,8-tetrahydropterin)]. The structural changes accompanying these interactions have been studied by radiation target analysis. PAH purified from rat liver was incubated with 2 mM phenylalanine to achieve complete activation of the enzyme. Frozen samples were irradiated with various doses of high energy electrons; samples were subsequently thawed, and several surviving properties of the enzyme were determined. Each parameter decreased as a single exponential function of radiation dose. Radiation target analysis of enzymatic activity yielded a dimeric target size. Similar radiation effects on subunit monomers and on tetrameric structure were observed. Together with results from unactivated enzyme, these data show that phenylalanine increases the interactions between the subunits in a dimer and weakens the interactions between dimers in a tetramer. These alterations prevent the natural cofactor, a tetrahydrobiopterin, from exerting a negative effect on activity. 相似文献