A chimeric tyrosine/tryptophan hydroxylase

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxylterminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t_1/2 of 84 min at 37°C, TPH was much less stable (t _1/2=28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by at _1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.     

Determination of the ratios of the aromatic amino acid residues by first- or second-derivative UV spectrometry for a simple characterization of peptides

A method for the simultaneous determination of the ratios of the three aromatic amino-acid residues in peptides was set up in acidic conditions. Binary and ternary mixtures of these amino acids were prepared, and first- and second-derivative spectra then calculated from their 0.1 nm resolution spectra between 240 and 320 nm. Certain spectral bands were chosen to differentiate tyrosine from tryptophan on the first-derivative spectra, and phenylalanine from tyrosine and tryptophan on the second-derivative spectra. Variation of the amplitude of the chosen bands was shown to be a linear function of the ratio of the aromatic amino acids in the mixture. This technique was validated with peptides whose sequence was known. The difference between theoretical and experimentally determined ratios was lower than 10%. Since the results are obtained as ratios, neither the concentration nor the nature of the peptide has to be known. The feasibility of application using a photodiode array detector with high resolution in reversed-phase high-performance liquid chromatography is discussed. © Munksgaard 1995.     

Distributive profiles of free and protein-incorporated tryptophan and valine in cerebral cortex slices from adult and 2-day-old rats

The distribution of the specific radioactivity and the incorporation into protein of [³H]-tryptophan and [³H]valine at varying layers from surface to centre were measured in incubated slices of cerebral cortices from infant and adult rats. Specific radioactivity in free amino acids was in both age groups highest in the intact surface layer. Incorporation of tryptophan into protein was even in slices from adult rats but much less than the average in the surface layers in slices from infant rats. Incorporation of valine exhibited similar heterogeneity in both age groups. The results suggest in brain slice preparations a zonal compartmentation of amino acid and protein metabolism which varies for different amino acids.     

Effects of tryptophan depletion on central and peripheral chemoreflexes in man

Klein (Arch. Gen. Psychiatry 50, 306-317, 1993) suggests that panic attacks are the result of a defective 'suffocation alarm' threshold that presents with carbon dioxide (CO(2)) hypersensitivity, exaggerated ventilatory response and panic in panic disorder (PD) patients. Serotonergic deficiencies enhance this ventilatory response in PD patients, as per 'suffocation alarm' theory predictions, suggesting that serotonin (5-HT) normalizes the ventilatory response. Other research supports a serotonin system-mediated stimulation of ventilation. Knowledge of 5-HT's role on ventilatory output and its neurophysiological sources impacts on the 'suffocation alarm' theory validity and predictive value. We used tryptophan depletion (TRP-) in concert with a modified Read rebreathing test to determine the effect of deficient serotonergic modulation on the central and peripheral chemoreflex threshold and sensitivity of response to CO(2) in 11 healthy men. TRP- did not affect central or peripheral chemoreflex threshold or sensitivity of response to CO(2). However, basal ventilation was significantly elevated during TRP-. In contrast to 'suffocation alarm' theory predictions, decreased 5-HT neurotransmission does not significantly affect the respiratory chemoreflex response to CO(2), impacting on non-chemoreflex drives to breathe. Panic associated respiratory abnormalities may be related to defective 5-HT modulation of non-chemoreflex drives to breathe, unrelated to any respiratory chemoreflex abnormality.     

Mechanisms dependent on tryptophan catabolism regulate immune responses in primary Sjögren's syndrome

To investigate the possible role of tryptophan metabolism in immune regulation of primary Sjögren's syndrome (pSS) the serum concentrations of tryptophan and its metabolite kynurenine were measured by reverse‐phase high‐performance liquid chromatography (HPLC) in 103 patients with pSS, 56 patients with sicca symptoms and 309 healthy blood donors. The kynurenine per tryptophan ratio (kyn/trp), which reflects the activity of the indoleamine‐pyrrole 2,3‐dioxygenase (IDO) enzyme involved in tryptophan catabolism, was calculated. Both female and male patients with pSS had significantly higher serum kynurenine concentrations and kyn/trp than subjects with sicca symptoms or healthy blood donors. The median (quartile range) concentration of kynurenine in female patients with pSS was 2·41 µmol/l (1·86–3·26) compared with 1·85 µmol/l (1·58–2·38, P < 0·0001) in subjects with sicca symptoms and 1·96 µmol/l (1·65–2·27, P < 0·0001) in healthy blood donors. Their kyn/trp × 1000 was 34·0 (25·1–44·3) compared with 25·3 (21·1–31·5, P < 0·0001) in subjects with sicca symptoms and 24·3 (21·0–28·9, P < 0·0001) in healthy blood donors. Female pSS patients with high IDO activity (kyn/trp × 1000 ≥ 34·0) had significantly higher ESR, serum C‐reactive protein, serum IgA and serum beta‐2 microglobulin concentrations as well as higher serum creatinine levels, and they had positive antinuclear antibodies more frequently and presented with more American‐European consensus group criteria than those with low IDO activity (kyn/trp × 1000 < 34·0). These data suggest that mechanisms dependent on tryptophan catabolism regulate immune responses in pSS. Tryptophan degradation is enhanced in patients with pSS, and high IDO activity is associated with severity of pSS.     

Tryptophan tolerance and metabolism in endogenous depression

The concentration of free and total tryptophan and kynurenine in plasma from 49 female depressives and 26 female controls was measured following oral loading with l-tryptophan, 100 mg/kg body weight. There was no significant difference between five depressives and six controls in the area under curve for free or total tryptophan or kynurenine in plasma. The peak concentration of kynurenine occured 4 h after loading and it correlated significantly with the area under curve for kynurenine. There was no significant correlation between the l-tryptophan dose (g) and the plasma concentration of kynurenine at 4 h in the 49 depressives or 26 controls. The mean plasma levels of tryptophan and kynurenine at 4 h in the depressives were not significantly different from control levels. There was no clear relationship between the plasma levels of tryptophan or kynurenine at 4 h and the therapeutic response in 13 depressives treated with l-tryptophan for 14 days.It is concluded that the absorption, the plasma clearance, and the degradation to kynurenine of loading doses of l-tryptophan are normal in depressed patients. Results further-more suggest that the plasma levels of tryptophan and kynurenine at 4 h are poor predictors of the response to l-tryptophan treatment in depressives.     

汉黄芩素调控Ywhaz蛋白抑制结肠癌细胞增殖及侵袭转移

目的:探讨汉黄芩素对人结肠癌细胞HCT116和HT29细胞系的增殖、侵袭能力的影响,并研究汉黄芩素对酪氨酸3/色氨酸5-单加氧酶激活蛋白(Ywhaz)蛋白水平的影响。方法:体外培养HCT116和HT29细胞系,设空白组、汉黄芩素不同浓度组(浓度分别为5,10,20,40μmol·L-1)作用不同时间后,采用细胞计数试剂盒(CCK-8)法检测汉黄芩素对结肠癌细胞增殖的影响,并用Annexin V-FITC/PI双标后流式细胞仪检测结肠癌细胞的凋亡率;穿透小室(Transwell)小室法检测处理24 h后细胞侵袭和迁移力的变化;实时荧光定量-聚合酶链式反应(q PCR)检测不同浓度汉黄芩素作用24 h后Ywhaz mRNA的水平,并运用蛋白质免疫印迹(Western blot)检测该蛋白及其磷酸化水平。结果:与空白组比较,汉黄芩素可明显抑制结肠癌细胞系的增殖,具有浓度和时间依赖性,并促进结肠癌细胞系的凋亡率,其中20和40μmol·L-1的汉黄芩素抑制细胞增殖作用显著(P0.01),且不同浓度的汉黄芩素(10,20,40μmol·L-1)作用于结肠癌细胞后,明显降低肿瘤细胞的穿膜数(P0.05,P0.01),汉黄芩素可下调Ywhaz mRNA水平和蛋白水平的表达,并降低Ywhaz的磷酸化水平(P0.05,P0.01)。结论:汉黄芩素可显著抑制HCT116和HT29细胞系的增殖、侵袭和迁移能力,并诱导细胞凋亡,其抗肿瘤机制可能与下调Ywhaz的蛋白水平及其蛋白的磷酸化水平有关。     

诱导吲哚胺2,3-二加氧酶表达的信号通路

吲哚胺2,3-二加氧酶（idoleamine 2,3- dioxygenase,IDO）的高表达是导致肿瘤免疫耐受的一个重要机制.诱导IDO的表达具有两种信号传导途径.一种是干扰素依赖途径,即IDO在Th-1型细胞因子干扰素、CpG ODNs等刺激后优先在巨噬细胞和树突状细胞等中表达,这种途径主要是由信号传导子及转录激活因子1α（STAT1α）和干扰素调节因子1（IRF-1）介导.另一种则是干扰素非依赖途径,即IDO在LPS等刺激后在人外周血单核细胞（PBMC）和人类急性单核白血病细胞（THP-1）等中表达,这种途径可能与p38 MAPK通路及NF-κB通路有关.明确IDO的信号通路且调控其表达,可为肿瘤的免疫治疗提供新策略.     

Increased cerebrospinal fluid corticotropin-releasing factor concentrations during tryptophan depletion in healthy adults.

BACKGROUND: Brain serotonin neurotransmission and hypothalamic-pituitary-adrenal axis function are implicated in the pathophysiology of depression, and these systems interact in a reciprocal modulatory fashion. This study examined the effect of tryptophan depletion, which acutely reduces brain serotonin concentrations, on serial cerebrospinal fluid concentrations of corticotropin-releasing factor in healthy humans. METHODS: Five subjects completed a standard tryptophan depletion protocol, and four subjects participated in a comparison condition. Subjects underwent continuous sampling of cerebrospinal fluid via lumbar peristaltic pump. Concentrations of cerebrospinal fluid corticotropin-releasing factor were measured by radioimmunoassay. RESULTS: No mood changes were observed in either group. Tryptophan-depleted subjects exhibited significantly greater increases in corticotropin-releasing factor concentrations over time than subjects in the comparison condition. CONCLUSIONS: These findings highlight the potential importance of corticotropin-releasing factor and serotonin interactions and suggest that activation of corticotropin-releasing-factor-containing neurons could play a role in the emergence of mood symptoms following tryptophan depletion in vulnerable individuals.