HTR1A, TPH2, and 5-HTTLPR Polymorphisms and Their Impact on the Severity of Depressive Symptoms and on the Concentration of Tryptophan Catabolites during Hepatitis C Treatment with Pegylated Interferon-α2a and Oral Ribavirin (PEG-IFN-α2a/RBV).

BACKGROUND: Seeing that there are no data about associations between serotonin gene polymorphism and tryptophan catabolite concentration during PEG-IFN-α2a treatment, the aim of the current study is to examine (a) the associations between polymorphisms within the HTR1A, TPH2, and 5-HTT genes and the severity of depression symptoms and (b) the relationships among rs6295, rs4570625, and 5-HTTLPR rs25531polymorphisms and indoleamine 2,3-dioxygenase (IDO) activity, as well as kynurenine (KYN), tryptophan (TRP), kynurenic acid (KA), and anthranilic acid (AA) concentrations. MATERIALS AND METHODS: The study followed a prospective, longitudinal, single-center cohort design. The severity of the depressive symptoms of 101 adult patients with chronic HCV infections was measured during PEG-IFN-α2a/RBV treatment. We used the Montgomery-Åsberg Depression Rating Scale (MADRS) to assess the severity of depressive symptoms. The subjects were evaluated six times-at baseline and at weeks 2, 4, 8, 12, and 24. At all the time points, MADRS score, as well as KYN, TRP, KA, and AA concentrations, and IDO activity were measured. At baseline, rs6295, rs4570625, and 5-HTTLPR rs25531polymorphisms were assessed. RESULTS: Subjects with C/C genotypes of 5-HT1A and lower-expressing alleles (S/S, LG/LG, and S/LG) of 5-HTTLPR scored the highest total MADRS scores and recorded the highest increase in MADRS scores during treatment. We found associations between TRP concentrations and the TPH-2 and 5-HTTLPR rs25531 genotypes. CONCLUSIONS: Our findings provide new data that we believe can help better understand infection-induced depression as a distinct type of depression.

Cytoguardin: A Tryptophan Metabolite against Cancer Growth and Metastasis.

Cytoguardin was identified in the conditioned medium of fibroblasts as a tryptophan metabolite, 5-methoxytryptophan (5-MTP). It is synthesized via two enzymatic steps: tryptophan hydroxylase (TPH) and hydroxyindole O-methyltransferase (HIOMT). A truncated HIOMT isoform, HIOMT298, catalyzes 5-MTP synthesis. Cancer cells produce scarce 5-MTP due to defective HIOMT298 expression. 5-MTP inhibits cancer cell COX-2 expression and thereby reduces COX-2-mediated cell proliferation and migration. 5-MTP also inhibits MMP-9 expression and thereby reduces cancer cell invasion. 5-MTP exerts its anti-cancer effect by blocking p38 MAPK and p38-mediated NF-κB and p300 HAT activation. The stable transfection of A549 cells with HIOMT298 restores 5-MTP production which renders cancer cells less aggressive. The implantation of HIOMT-transfected A549 into subcutaneous tissues of a murine xenograft tumor model shows that HIOMT-transduced A549 cells form smaller tumors and generate fewer metastatic lung nodules than control A549 cells. HIOMT298 transfection suppresses aromatic amino acid decarboxylase (AADC) expression and serotonin production. Serotonin is a cancer-promoting factor. By restoring 5-MTP and suppressing serotonin production, HIOMT298 overexpression converts cancer cells into less malignant phenotypes. The analysis of HIOMT expression in a human cancer tissue array showed reduced HIOMT levels in a majority of colorectal, pancreatic, and breast cancer. HIOMT298 may be a biomarker of human cancer progression. Furthermore, 5-MTP has the potential to be a lead compound in the development of new therapy for the chemoprevention of certain cancers such as hepatocellular cancer.

The role of anthranilic acid in the increase of depressive symptoms and major depressive disorder during treatment for hepatitis C with pegylated interferon-α2a and oral ribavirin.

Background: Tryptophan metabolism via the kynurenine pathway is considered the link between the immune and endocrine systems. Dysregulation of serotonergic transmission can stem from the direct influence of interferon-α on the activity of serotonergic receptors 5-HT1A and 5-HT2A, and from its indirect effect on tryptophan metabolism. Induction of the kynurenine pathway increases the concentration of neurotoxic kynurenine metabolites, and the activity of kynurenine derivatives is linked to the onset of depression. The aim of our study was to evaluate the relationships between depressive symptoms and kynurenine, tryptophan, anthranilic acid and kynurenic acid concentrations, indolamine 2,3-dioxygenase (IDO) activity and tryptophan availability to the brain. Methods: The study followed a prospective longitudinal cohort design. We evaluated 101 patients with chronic hepatitis C who were treated with pegylated interferon-α2a, and 40 controls who were awaiting treatment. We evaluated the relationships between total score on the Montgomery-Åsberg Depression Rating Scale and kynurenine, tryptophan, anthranilic acid and kynurenic acid concentrations, IDO activity and tryptophan availability to the brain. A logistic regression model was adapted for the diagnosis of major depressive disorder at each time point, taking into account changes in parameters of the kynurenine pathway between a given time point and the baseline measurement. Results: Of the treated patients, 44% fulfilled the criteria for major depressive disorder at least once during the 24 weeks of treatment. Anthranilic acid concentrations were significantly increased compared to baseline for all time points except week 2. Tryptophan availability showed a significant decrease (ß = -0.09, p = 0.01) only in week 12 of treatment. Over time, kynurenine, tryptophan and anthranilic acid concentrations, as well as IDO activity and tryptophan availability to the brain, were significantly associated with total score on the Montgomery-Åsberg Depression Rating Scale. A logistic regression model revealed that participants with decreased tryptophan availability to the brain at 12 weeks of treatment and participants with increased anthranilic acid concentrations at week 24 of treatment were at increased risk for diagnosis of major depressive disorder (odds ratios 2.92 and 3.59, respectively). Limitations: This study had an open-label design in a population receiving naturalistic treatment. Conclusion: The present study provides the first direct evidence of the role of anthranilic acid in the pathogenesis of inflammation-induced major depressive disorder during treatment for hepatitis C with pegylated interferon-α2a.

Fluorogenic glycopolymers available for determining the affinity of lectins by intermolecular FRET.

A convenient assembly of fluorogenic glycopolymers having various polymer compositions was accomplished from the corresponding glycomonomer and dansyl monomer by means of radical polymerization, and the water-soluble glycopolymers gave typical fluorescence spectroscopic profiles due to the dansyl moieties on the glycopolymer in aqueous media. Biological evaluation of the polymer against wheat germ agglutinin (WGA) was accomplished on the basis of fluorescence changes due to tryptophan residues on WGA, and the affinities between the glycopolymers and WGA were estimated to be 4.7 × 105 to 9.3 × 105 M-1. In order to apply the fluorogenic glycopolymers for further biological measurements, efficient resonance energy transfer from tryptophan moieties on WGA to dansyl moieties on the fluorogenic glycopolymers was examined. FRET profiles of both fluorophores were similar compared to the binding profiles on the basis of fluorescence changes of tryptophan residues. This approach is applicable for the determination of an affinity constant between a carbohydrate and a lectin in which no fluorophore exists near the binding site.

Major depression model induced by repeated and intermittent lipopolysaccharide administration: Long-lasting behavioral, neuroimmune and neuroprogressive alterations.

Major depressed patients show increased bacterial translocation with elevated plasma levels of lipopolysaccharide (LPS), which may trigger immune-inflammatory and neuro-oxidative responses. Recently, an animal model based on chronic LPS administration was developed which was associated with long-lasting depressive-like and neuro-oxidative alterations in female mice. The aim of the current study was to investigate behavioral, neuroimmune and neuroprogressive alterations in female mice 6 weeks after LPS chronic exposure. Female mice received increasing doses of LPS during 5 days at one-month intervals repeated for 4 consecutive months. Six weeks after the last LPS-exposure, we assessed behavioral despair and anhedonia, microglial activation, alterations in tryptophan, 5-HT, kynurenine, quinolinic acid (QUIN) levels and spermidine/spermine N1-acetyltransferase (SAT1) expression in the hippocampus, both with and without fluoxetine administration. Our results show that six weeks post-LPS, mice present behavioral despair and anhedonia in association with increased IBA1 expression (a microglia activation marker), NF-kB p65 and IL-1ß levels, indoleamine 2,3-dioxygenase (IDO1) mRNA expression, kynurenine, QUIN levels and QUIN/tryptophan ratio, and lowered tryptophan, 5-HT levels and SAT1 mRNA expression. Fluoxetine reversed the behavioral and neuroimmune alterations but had no effect in the reversal of IDO1 increased expression, QUIN levels and QUIN/tryptophan ratio. In conclusion, our results support the validity of the chronic LPS model of major depression and additionally shows its translational relevance with respect to neuroimmune and neuroprogressive pathways.

Alterations in the metabolism of tryptophan in patients with chronic hepatitis C six months after pegylated interferon-α 2a treatment.

BACKGROUND: Risk of depression and suicide in patients on interferon remains also after the treatment, the pathogenesis of which is still unclear. We aimed to determine the influence of the PEG-IFN-α2a on tryptophan metabolism along the kynurenine pathway during treatment and up to 6 months after the end of treatment. METHODS: We evaluated 101 patients with chronic hepatitis C treated with PEG-IFN-α2a, and 40 controls, so as to determine the activation of indolamine 2,3-dioxygenase (IDO) and tryptophan (TRP) and their metabolites' concentrations/levels: kynurenine (KYN), kynurenic acid (KYNA) and anthranilic acid (AA). The subjects were evaluated before and after weeks 2, 4, 8, 12, 24, 48, as well as 6 months after the end of the treatment. RESULTS: In the group of patients treated 24 weeks, six months after the end of treatment IDO activity was significantly higher compared to baseline (69.5 vs 57.2 ß = 0.21 P = 0.000); TRP concentration was significantly lower compared to baseline (30.0 vs 35.6 ß=-0.21 P = 0.001); KYNA concentration was significantly higher compared to baseline (37.2 nmol/L vs 29.4 nmol/L ß = 0.22 P = 0.02), and AA concentration was significantly higher compered to baseline (51.0 nmol/L vs 38.4 nmol/L ß = 0.22 P = 0.05) In the group of patients treated 48 weeks six months, after the end of treatment both the IDO activity and KYNA concentration were significantly higher compared to baseline (respective values - IDO: 78.8 vs 56.2 ß = 0.14 P = 0.02; KYNA: 39.2 nmol/L vs 27.0 nmol/L ß = 0.26 P = 0.000). CONCLUSIONS: This is the first report of a prolonged activation of IDO six months after the end of PEG-IFN-α2a treatment. The clinical significance of the finding can be implicated in the pathophysiology of depressive episodes.

A Preclinical Study of Casein Glycomacropeptide as a Dietary Intervention for Acute Mania.

Background: Casein glycomacropeptide is a peptide that lacks phenylalanine, tyrosine, and tryptophan. This profile may enable it to deplete phenylalanine, tyrosine, and tryptophan, and subsequently the synthesis of dopamine and serotonin in the brain. Dopamine- and serotonin-depleting amino acid mixtures have shown promise as acute antimanic treatments. In this study, we explore the depleting effects on amino acids, dopamine and serotonin as well as its actions on manic-like and other behavior in rats. Methods: Casein glycomacropeptide and a selection of amino acid mixtures were administered orally at 2, 4, or 8 h or for 1 week chronically. Amino acid and monoamine levels were measured in plasma and brain and behavior was assessed in the amphetamine-hyperlocomotion, forced swim, prepulse inhibition, and elevated plus maze tests. Results: Casein glycomacropeptide induced a time-dependent reduction in tyrosine, tryptophan, and phenylalanine in brain and plasma which was augmented by supplementing with leucine. Casein glycomacropeptide +leucine reduced dopamine in the frontal cortex and serotonin in the hippocampus, frontal cortex, and striatum after 2 and 4 h. Casein glycomacropeptide+leucine also had antimanic activity in the amphetamine-induced hyperlocomotion test at 2 h after a single acute treatment and after 1 week of chronic treatment. Conclusions: Casein glycomacropeptide-based treatments and a branched-chain amino acid mixture affected total tissue levels of dopamine in the frontal cortex and striatum and serotonin in the frontal cortex, striatum, and hippocampus of rats in a time-dependent fashion and displayed antimanic efficacy in a behavioral assay of mania.

Inhibiting tryptophan metabolism enhances interferon therapy in kidney cancer.

Renal cell carcinoma (RCC) is increasing in incidence, and a complete cure remains elusive. While immune-checkpoint antibodies are promising, interferon-based immunotherapy has been disappointing. Tryptophan metabolism, which produces immunosuppressive metabolites, is enhanced in RCC. Here we show indolamine-2,3-dioxygenase-1 (IDO1) expression, a kynurenine pathway enzyme, is increased not only in tumor cells but also in the microenvironment of human RCC compared to normal kidney tissues. Neither kynurenine metabolites nor IDO inhibitors affected the survival or proliferation of human RCC or murine renal cell adenocarcinoma (RENCA) cells in vitro. However, interferon-gamma (IFNγ) induced high levels of IDO1 in both RCC and RENCA cells, concomitant with enhanced kynurenine levels in conditioned media. Induction of IDO1 by IFNα was weaker than by IFNγ. Neither the IDO1 inhibitor methyl-thiohydantoin-DL-tryptophan (MTH-trp) nor IFNα alone inhibited RENCA tumor growth, however the combination of MTH-trp and IFNα reduced tumor growth compared to IFNα. Thus, the failure of IFNα therapy for human RCC is likely due to its inability to overcome the immunosuppressive environment created by increased IDO1. Based on our data, and given that IDO inhibitors are already in clinical trials for other malignancies, IFNα therapy with an IDO inhibitor should be revisited for RCC.

Indoleamine-2,3-dioxygenase mediates neurobehavioral alterations induced by an intracerebroventricular injection of amyloid-ß1-42 peptide in mice.

Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by a progressive cognitive decline along with various neuropsychiatric symptoms, including depression and anxiety. Increasing evidence has been proposed the activation of the tryptophan-degrading indoleamine-2,3-dyoxigenase (IDO), the rate-limiting enzyme of kynurerine pathway (KP), as a pathogenic factor of amyloid-beta (Aß)-related inflammation in AD. In the current study, the effects of an intracerebroventricular (i.c.v.) injection of Aß1-42 peptide (400pmol/mice; 3µl/site) on the regulation of KP biomarkers (IDO activity, tryptophan and kynurerine levels) and the impact of Aß1-42 on neurotrophic factors levels were investigated as potential mechanisms linking neuroinflammation to cognitive/emotional disturbances in mice. Our results demonstrated that Aß1-42 induced memory impairment in the object recognition test. Aß1-42 also induced emotional alterations, such as depressive and anxiety-like behaviors, as evaluated in the tail suspension and elevated-plus maze tests, respectively. We observed an increase in levels of proinflammatory cytokines in the Aß1-42-treated mice, which led to an increase in IDO activity in the prefrontal cortex (PFC) and the hippocampus (HC). The IDO activation subsequently increased kynurerine production and the kynurenine/tryptophan ratio and decreased the levels of neurotrophic factors in the PFC and HC, which contributed to Aß-associated behavioral disturbances. The inhibition of IDO activation by IDO inhibitor 1-methyltryptophan (1-MT), prevented the development of behavioral and neurochemical alterations. These data demonstrate that brain IDO activation plays a key role in mediating the memory and emotional disturbances in an experimental model based on Aß-induced neuroinflammation.

Elevation of Kynurenine Metabolites in Rat Liver and Serum: A Potential Additional Mechanism of the Alcohol Aversive and Anti-cancer Effects of Disulfiram?

AIMS: The tryptophan metabolites 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) inhibit the liver mitochondrial low Km aldehyde dehydrogenase and possess alcohol-aversive and immunosuppressant properties. As the disulfiram (DS) metabolite carbon disulphide activates enzymes forming 3-HK and 3-HAA, we investigated if repeated disulfiram treatment increases the hepatic and serum levels of these 2 metabolites. METHODS: Livers and sera of male Wistar rats were analysed for tryptophan and kynurenine metabolites after repeated DS treatment for 7 days. RESULTS: DS increased liver and serum [3-HK] and [3-HAA] possibly by increasing the flux of tryptophan down the hepatic kynurenine pathway and activation of kynurenine hydroxylase and kynureninase. CONCLUSIONS: We provisionally suggest that elevation of some kynurenine metabolites may be an additional mechanism of the alcohol-aversive and anticancer effects of disulfiram.

Impact of different antipsychotics on cytokines and tryptophan metabolites in stimulated cultures from patients with schizophrenia.

BACKGROUND: An imbalance of tryptophan metabolites plays a role in the pathophysiology of schizophrenia. Also cytokines seem to be involved and are able to enhance the tryptophan metabolism. In this study the impact of cytokines, tryptophan metabolites and antipsychotics was evaluated in schizophrenic patients/ healthy controls and correlated with the psychopathology of schizophrenia. SUBJECTS AND METHODS: This study investigated 12 patients with schizophrenia and 24 matched controls. Peripheral blood mononuclear cell cultures were stimulated in vitro with lipopolysaccharide (LPS) or polyinosine-polycytidylic acid (poly I:C) and different antipsychotics (quetiapine, risperidone, haloperidole and clozapine) were added. The cytokines IL-4, IL-10, IFN-γ and tryptophan metabolites were analysed. Symptom severity was assessed using the positive and negative syndrome scale (PANSS). RESULTS: Peripheral mononuclear cells of schizophrenia patients showed a reduced IFN-γ response to LPS (p=0.008). When quetiapine and risperidone were added this imbalance between patients and controls disappeared. Tryptophan levels were significantly lower in patients' cells cultures when the cells were stimulated with LPS (p=0.029). A group effect for lower levels in the patients' cell culture was evaluated for tryptophan and kynurenine (p=0.043; p=0.05). In addition, high tryptophan levels correlated with low PANSS negative scores in patients and higher kynurenine levels resulted in higher PANSS positive scores. CONCLUSIONS: Only two atypical antipsychotics were identified to reverse the imbalanced cytokine levels in schizophrenia. The low concentrations of tryptophan and kynurenine in these patients could be a sign of a fast degradation of tryptophan - yet tryptophan metabolites could not be changed by any of the investigated antipsychotics.

Lignans from Carthamus tinctorius suppress tryptophan breakdown via indoleamine 2,3-dioxygenase.

Seed extracts of Carthamus tinctorius L. (Asteraceae), safflower, have been traditionally used to treat coronary disease, thrombotic disorders, and menstrual problems but also against cancer and depression. A possible effect of C. tinctorius compounds on tryptophan-degrading activity of enzyme indoleamine 2,3-dioxygenase (IDO) could explain many of its activities. To test for an effect of C. tinctorius extracts and isolated compounds on cytokine-induced IDO activity in immunocompetent cells in vitro methanol and ethylacetate seed extracts were prepared from cold pressed seed cakes of C. tinctorius and three lignan derivatives, trachelogenin, arctigenin and matairesinol were isolated. The influence on tryptophan breakdown was investigated in peripheral blood mononuclear cells (PBMCs). Effects were compared to neopterin production in the same cellular assay. Both seed extracts suppressed tryptophan breakdown in stimulated PBMC. The three structurally closely related isolates exerted differing suppressive activity on PBMC: arctigenin (IC50 26.5µM) and trachelogenin (IC50 of 57.4µM) showed higher activity than matairesinol (IC50 >200µM) to inhibit tryptophan breakdown. Effects on neopterin production were similar albeit generally less strong. Data show an immunosuppressive property of compounds which slows down IDO activity. The in vitro results support the view that some of the anti-inflammatory, anticancer and antidepressant properties of C. tinctorius lignans might relate to their suppressive influence on tryptophan breakdown.

Tryptophan oxidation photosensitized by pterin.

Pterins are normal components of cells and they have been previously identified as good photosensitizers under UV-A irradiation, inducing DNA damage and oxidation of nucleotides. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to photosensitize the oxidation of another class of biomolecules, amino acids, using tryptophan (Trp) as a model compound. Irradiation of Ptr in the UV-A spectral range (350 nm) in aerated aqueous solutions containing Trp led to the consumption of the latter, whereas the Ptr concentration remained unchanged. Concomitantly, hydrogen peroxide (H2O2) was produced. Although Ptr is a singlet oxygen ((1)O2) sensitizer, the degradation of Trp was inhibited in O2-saturated solutions, indicating that a (1)O2-mediated process (type II oxidation) was not an important pathway leading to Trp oxidation. By combining different analytical techniques, we could establish that a type I photooxidation was the prevailing mechanism, initiated by an electron transfer from the Trp molecule to the Ptr triplet excited state, yielding the corresponding radical ions (Trp(·+)/Trp(-H)· and Ptr(·-)). The Trp reaction products that could be identified by UPLC-mass spectrometry are in agreement with this conclusion.

An investigation of the antidepressant action of xiaoyaosan in rats using ultra performance liquid chromatography-mass spectrometry combined with metabonomics.

A rapid, highly sensitive, and selective method was applied in a non-invasive way to investigate the antidepressant action of Xiaoyaosan (XYS) using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and chemometrics. Many significantly altered metabolites were used to explain the mechanism. Venlafaxine HCl and fluoxetine HCl were used as chemical positive control drugs with a relatively clear mechanism of action to evaluate the efficiency and to predict the mechanism of action of XYS. Urine obtained from rats subjected to chronic unpredictable mild stress (CUMS) was analyzed by UPLC-MS. Distinct changes in the pattern of metabolites in the rat urine after CUMS production and drug intervention were observed using partial least squares-discriminant analysis. The results of behavioral tests and multivariate analysis showed that CUMS was successfully reproduced, and a moderate-dose XYS produced significant therapeutic effects in the rodent model, equivalent to those of the positive control drugs, venlafaxine HCl and fluoxetine HCl. Metabolites with significant changes induced by CUMS were identified, and 17 biomarker candidates for stress and drug intervention were identified. The therapeutic effect of XYS on depression may involve regulation of the dysfunctions of energy metabolism, amino acid metabolism, and gut microflora changes. Metabonomic methods are valuable tools for measuring efficacy and mechanisms of action in the study of traditional Chinese medicines.

Photochemical properties and phototoxicity of Pazufloxacin: a stable and transient study.

Photochemical properties and phototoxicity of Pazufloxacin (PAX) were systematically investigated in aqueous solutions using UV-Vis, fluorescence, laser flash photolysis, pulse radiolysis and SDS-PAGE gel electrophoresis techniques. PAX triplet-state ((3)PAX(*)) absorption spectra (λ(max)=570 nm) was determined. (3)PAX(*) was quenched by PAX and O(2), with rate constants of 6.9×10(8) and 3.2×10(8) dm(3) mol(-1) s(-1), respectively. The pK(a) values (5.7 and 8.6) for the protonation equilibrium were determined by UV-Vis and fluorescence techniques. The PAX triplet energy (E(T)=260.3 kJ/mol) was obtained using energy transfer method. The reaction of electron transfer from tryptophan (TrpH) and dGMP to (3)PAX(*) was found with rate constants of 8.8×10(7) and 8.7×10(6) dm(3) mol(-1) s(-1), respectively. The rate constants for reactions of ()OH, SO(4)(-) and hydrated electron with PAX were found to be 5.8×10(8), 2.1×10(9) and 9×10(9)d m(3) mol(-1) s(-1), respectively. Based on the results obtained, a rational scheme for dGMP, TrpH and lysozyme photodamage induced by PAX was proposed.

Optimal salt bridge for Trp-cage stabilization.

Gai and co-workers [Bunagan, M. R., et al. (2006) J. Phys. Chem. B 110, 3759-3763] reported computational design studies suggesting that a D9E mutation would stabilize the Trp-cage. Experimental studies for this mutation were reported in 2008 [Hudaky, P., et al. (2008) Biochemistry 47, 1007-1016]; the authors suggested that [D9E]-TC5b presented a more compact and melting resistant structure because of the "optimal distance between the two sides of the molecule". Nonetheless, the authors reported essentially the same circular dichroism (CD) melting temperature, 38 ± 0.3 °C, for TC5b and its [D9E] mutant. In this study, a more stable Trp-cage, DAYAQ WLKDG GPSSG RPPPS, was examined by nuclear magnetic resonance and CD with the following mutations: [D9E], [D9R,R16E], [R16O], [D9E,R16O], [R16K], and [D9E,R16K]. Of these, the [D9E] mutant displayed the smallest acidification-induced change in the apparent T(m). In analogy to the prior study, the CD melts of TC10b and its [D9E] mutant were, however, very similar; all of the other mutations were significantly fold destabilizing by all measures. A detailed analysis indicates that the original D9-R16 salt bridge is optimal with regard to fold cooperativity and fold stabilization. Evidence of salt bridge formation is also provided for a swapped pair, the [D9R,R16E] mutant. Model systems reveal that an ionized aspartate at the C-terminus of a helix significantly decreases intrinsic helicity, a requirement for Trp-cage fold stability. The CD evidence that was cited as supporting increased fold stability for [D9E]-TC5b at higher temperatures appears to be a reflection of increased helix stability in both the folded and unfolded states rather than a more favorable salt bridge. Our study also provides evidence of other Trp-cage stabilizing roles of the R16 side chain.

Safety and adequacy of an optimized formula for pediatric patients with cow's milk-sensitive enteropathy.

AIM: Infants and children with cow's milk-sensitive enteropathy are treated with extensively hydrolyzed formulas. A formula (New Alfaré) was developed by a protein hydrolysis method that yields an amino acid profile that more closely resembles human milk compared to previous formulas, and contains nucleotides. METHODS: The current study was a prospective, open trial aimed at evaluating the safety and nutritional adequacy of this formula for pediatric patients with clinical indications for the enteral use of semi-elemental diet. Safety was measured as normal growth based on Euro-growth standards for body mass index (BMI)-for-age z-scores, and nutritional adequacy was evaluated based on measurements of blood parameters. Forty-seven patients <32 months old, having a gestational age of ≥ 26 weeks, and weighing ≥ 1,500 g were enrolled, and fed with New Alfaré for four weeks. Weight, length and blood parameters were measured at the beginning and end of the study. Signs of tolerance to the formula (amount of formula intake, gastrointestinal symptoms and stool characteristics) were recorded daily by the parents. Twenty-five patients completed the study with all measurements. RESULTS: There was a significant increase in the mean BMI-for-age z-score (P<0.05) and albumin concentration (P<0.01) after four weeks. Mean plasma threonine concentration decreased significantly (P=0.01) and the mean tryptophan concentration tended to increase by the end of the study (P=0.06). No adverse events related to the study formula were reported. CONCLUSION: These results show that New Alfaré is safe and nutritionally adequate for pediatric patients with cow's milk-sensitive enteropathy.

Melatonin and tryptophan circadian profiles in patients with advanced non-small cell lung cancer.

INTRODUCTION: Accumulating studies indicate that melatonin is a natural oncostatic agent capable of mediating the influence of the psychoneuroendocrine system on cancer growth. Although there is increasing evidence to show that the pineal gland may play a role in human non-small cell lung cancer (NSCLC), there is uncertainty about circadian profiles of melatonin, its precursor tryptophan, and its major metabolite, 6-sulfatoxymelatonin (6-OH-MLT) in NSCLC patients before and after treatment with standard chemotherapy (cisplatin plus vinorelbine). The aim of this study was to investigate the concentration changes of melatonin, tryptophan, and 6-OH-MLT in NSCLC patients treated with standard chemotherapy. METHODS: We examined the circadian melatonin, tryptophan, and 6-OH-MLT rhythms in 30 patients suffering from advanced-stage NSCLC and compared them with those of 63 healthy volunteers free from neoplastic disease. Blood samples were collected at 12 noon and 12 midnight. Urine samples were collected at 7 AM: and 4 PM: . The levels of melatonin in serum and of 6-OH-MLT in urine were measured by high-performance liquid chromatography. The concentration of amino acids including tryptophan in serum was measured by amino acid analyzer. RESULTS: Melatonin, tryptophan, and 6-OH-MLT concentrations were significantly lower in cancer patients, in comparison with healthy subjects. A significant inverse correlation between melatonin and tryptophan was observed. Additionally, after three cycles of standard chemotherapy, there was a tendency of melatonin, tryptophan, and 6-OH-MLT concentrations to progressively decrease in NSCLC patients. CONCLUSION: The results of the present study indicate that the presence of NSCLC influences the metabolism of melatonin, and chemotherapy in NSCLC patients may progressively decrease the production of melatonin.

No effect of covalently linked poly(ethylene glycol) chains on protein internal dynamics.

Poly(ethylene glycol) or PEG is a hydrophilic polymer that covalently linked to therapeutical proteins may significantly increase their pharmacological properties. Despite the extensive production of PEG-conjugated proteins the effects of the polymer on the protein structure and dynamics is poorly understood, making the production of active biomaterials a largely unpredictable process. The present investigation examines the effects of 5 k and 20 k PEG on the internal flexibility of Ribonuclease T1, the mutant C112S of azurin from Pseudomonas aeruginosa, alcohol dehydrogenase and alkaline phosphatase, native and Zn-depleted. These systems encompass structural domains that range from rather superficial, flexible sites to deeply buried, rigid cores. The approach is based on three sensitive parameters related to the phosphorescence emission of internal Trp residues, namely, the intrinsic room-temperature phosphorescence lifetime (tau(0)) that reports on the local flexibility of the protein matrix around the chromophore and the bimolecular rate constant (k(q)) for the quenching of phosphorescence by O(2) and by acrylamide in solution, which are related to the diffusion of these solutes through the protein fold. The results obtained by these three independent, intrinsic probes of protein structure-dynamics concur that mono-PEGylation does not detectably perturb the conformation and dynamics of the protein native fold, over a wide temperature range. The implication is that protein motions are essentially not coupled to the polymer and that adverse effects of chemical modification on biological function are presumably owed to steric hindrance by PEG units blocking the access to sites critical for molecular recognition.

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of L-tryptophan by L-tryptophan indole-lyase.

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2'-methoxyethyl)]-alpha-cyclodextrin; heksakis(2,3-di-O-methyl)-alpha-cyclodextrin; heptakis(2,3-di-O-methyl)-beta-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2'-methoxyethyl)]-alpha-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2'-methoxyethyl)]-beta-cyclodextrin; heksakis[2,3-di-O-(2'-methoxyethyl)]-alpha-cyclodextrin and heptakis[2,3-di-O-(2'-methoxyethyl)]-beta-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-alpha-cyclodextrin and (2-hydroxypropyl)-beta-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of L-tryptophan by L-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and L-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.