Possible relationship between amino acids, aggression and psychopathy.

OBJECTIVE: Aggressive behaviour is associated with reduced serotonin metabolism in the brain, but there is not enough knowledge on potential changes of the serotonin precursor levels among violent offenders. In this study, we aimed to evaluate the relationships among the tendency of psychopathy, anger and the basic amino acids. METHODS: Fifty-two young adult male patients with antisocial personality disorder (APD) and 30 healthy men included the study. Serum amino acid levels were measured by HPLC method. Aggression questionnaire and Hare Psychopathology Scale were used for all participants. RESULTS: Blood levels of phosphoserine, aspartic acid, glutamic acid, aminoadipic acid and 1-methylhistidine in group of patients with APD were significantly higher than the control group. Blood levels of TRP, asparagine, citrulline, cystine, isoleucine, tyrosine, histidine, hydroxylysine, lysine, ethanolamine and arginine in the group of patients were found lower than the control group. A significant positive correlation between anger scores and histidine, methionine and GABA was found. GABA and methionine showed a significant correlation with the indirect aggression score. CONCLUSION: Our study showed a relationship between serum amino acid levels and the scores of aggression and psychopathy. We think that this is a productive research area for understanding the relationship among biochemical factors, aggression and psychopathy.

Increased anti-phospholipid antibodies in autism spectrum disorders.

Autism spectrum disorders (ASD) are characterized by impairments in communication, social interactions, and repetitive behaviors. While the etiology of ASD is complex and likely involves the interplay of genetic and environmental factors, growing evidence suggests that immune dysfunction and the presence of autoimmune responses including autoantibodies may play a role in ASD. Anti-phospholipid antibodies are believed to occur from both genetic and environmental factors and have been linked to a number of neuropsychiatric symptoms such as cognitive impairments, anxiety, and repetitive behaviors. In the current study, we investigated whether there were elevated levels of anti-phospholipid antibodies in a cross-sectional analysis of plasma of young children with ASD compared to age-matched typically developing (TD) controls and children with developmental delays (DD) other than ASD. We found that levels of anti-cardiolipin, ß 2-glycoprotein 1, and anti-phosphoserine antibodies were elevated in children with ASD compared with age-matched TD and DD controls. Further, the increase in antibody levels was associated with more impaired behaviors reported by parents. This study provides the first evidence for elevated production of anti-phospholipid antibodies in young children with ASD and provides a unique avenue for future research into determining possible pathogenic mechanisms that may underlie some cases of ASD.

Determination of phosphoserine/threonine by nano ultra-performance liquid chromatography-tandem mass spectrometry coupled with microscale labeling.

Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at microscale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues, such as 2-mercaptoethanol and 3-mercapto-1-propanol, to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl, or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time, NH4HCO3 concentration) and derivatization efficiency parameters (e.g., reaction time, labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra-performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved to be effective for quantifying phosphopeptides in biological samples.

Lipid fingerprinting in women with early-onset preeclampsia: a first look.

OBJECTIVES: The aim of this preliminary study was to characterize the plasma lipid profiling of women with preeclampsia. DESIGN AND METHODS: Plasma samples of 8 pregnant women with early-onset preeclampsia and 8 normal pregnant women were evaluated. Lipids were extracted from plasma using the Bligh-Dyer protocol. The extracts were subjected to MALDI-MS. Data matrix was exported for partial least squares discriminant analysis (PLS-DA) and a parameter VIP was employed to reflect the variable importance in the discriminant analysis. The major discriminant variables were selected and underwent to Mann-Whitney U test. RESULTS: A total of 1290 ions were initially identified and twelve m/z signals were highlighted as the most important lipids for the discrimination of patients with preeclampsia. The identification of these differential lipids was carried out through Lipid Database Search. CONCLUSIONS: The main classes identified were glycerophosphocholines [GP01], glycerophosphoserines [GP03], glycerophosphoglycerols [GP04], glycosyldiradylglycerols [GL05] and glycerophosphates [GP10].

Nitric oxide, erythrocytes and exercise.

Nitric oxide (NO) is accepted to be an important factor affecting the degree of vascular tone in various portions of the circulation. Until recently, research in this area has focused on endothelial cells as a NO source, and there is general agreement that: 1) the level of wall shear stress is the primary determinant of endothelial nitric oxide synthase (eNOS) expression; 2) exercise training induces changes of endothelial cell NO synthesizing activity; 3) phosphorylation patterns of eNOS are altered following exercise episodes. However, there is now a growing body of evidence for the existence of similar nitric oxide synthesizing mechanisms in human red blood cells (RBC). Erythrocyte NOS activity has been demonstrated to be induced by applied shear stress and mechanical deformation of RBC, and there are closely linked increases of intracellular nitric oxide levels and of release of NO into the suspending phase. In brief, the RBC is an enzymatic source of NO that is dependent on flow dynamics and from which NO is released in very close proximity to vessel walls. Although reports regarding the influence of exercise on RBC nitric oxide synthesizing mechanisms are not yet concordant, it seems logical to suggest that this source of NO may play a role in the regulation of local blood flow dynamics during exercise.

Lithium regulates glycogen synthase kinase-3beta in human peripheral blood mononuclear cells: implication in the treatment of bipolar disorder.

BACKGROUND: Bipolar disorder has been linked to alterations in the multifunctional enzyme glycogen synthase kinase-3beta (GSK3beta). The mood stabilizer lithium inhibits GSK3beta in vitro and in mouse brain, and this is currently the strongest known potential therapeutic target of lithium. We tested whether lithium modified GSK3beta in vivo or in vitro in peripheral blood mononuclear cells (PBMCs) from healthy control and bipolar disorder subjects. METHODS: The PBMCs were obtained from 23 healthy control subjects, 9 bipolar subjects currently treated with lithium, and 13 lithium-free bipolar subjects. Immunoblot analyses were used to measure the inhibited, serine9-phosphorylated GSK3beta. RESULTS: The level of phospho-Ser9-GSK3beta in PBMCs was regulated by agents that modified kinases and phosphatases acting on GSK3beta and was increased by in vitro lithium treatment. More important, phospho-Ser9-GSK3beta levels were eightfold higher in PBMCs from lithium-treated bipolar than healthy control subjects. CONCLUSIONS: Signaling pathways regulating serine9-phosphorylation of GSK3beta can be studied in human PBMCs. Both in vitro and in vivo therapeutic lithium treatment is associated with a large increase in phospho-Ser9-GSK3beta in PBMCs. Therefore, the inhibitory serine9-phosphorylation of GSK3beta in human PBMCs may provide a biochemical marker to evaluate the association between GSK3beta inhibition and therapeutic responses to lithium treatment.

Immunoassay of serine-phosphorylated isoform of insulin-like growth factor (IGF) binding protein (IGFBP)-1.

OBJECTIVES: Development of an ELISA for phosphorylated isoform of IGFBP-1. Serine phosphorylation is an important regulator of IGFBP-1 bioactivity, but specific immunoassays for its measurement are currently lacking. DESIGN AND METHODS: Assay design was based on a novel approach of first capturing the phosphorylated and non-phosphorylated IGFBP-1 by an anti-IGFBP-1 antibody and then selectively detecting the phosphorylated form by an anti-phosphoserine antibody. Method development involved pair-wise evaluation of the candidate antibodies and determinations of analytical performance and specificity. Specificity was monitored by reactivity with dephosphorylated IGFBP-1, with antibodies against other phosphorylated residues that are not expressed, and by comparative analysis of sample containing different IGFBP-1 phosphorylation profile. RESULTS: Analytical evaluation demonstrated acceptable performance; detection limit 0.3 microg/L, dynamic range 1.56-100 microg/L; intra- and inter-assay CVs 2.1-8.6%; mean recovery (+/-SD) 97.8+/-9.2%, and mean recovery of sample dilution 93.4+/-6.0%. The phosphorylated and total IGFBP-1 medians in non-pregnant adult serum, which mostly contain the highly phosphorylated isoform, were 11.9 and 18.6 microg/L, respectively, and the sample values were tightly correlated (r=0.99). As expected, the corresponding medians in 1st trimester (17.4 and 63.0 microg/L) and 2nd trimester (30.9 and 75.8) samples with altered IGFBP-1 phosphorylation were significantly different (p<0.001). Similarly, a fraction (1.29%) of total IGFBP-1 (13.3 mg/L) in amniotic fluids was found to be phosphorylated (0.172 mg/L). There was no reactivity with dephosphorylated IGFBP-1. CONCLUSIONS: The present ELISA is highly specific for the phosphorylated isoform of IGFBP-1 and its availability should help expedite further investigations of IGFBP-1 phosphorylation.

Synergism between nitric oxide and hydrogen peroxide in the inhibition of platelet function: the roles of soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein.

In previous studies, a strong synergism between low concentrations of hydrogen peroxide and nitric oxide in the inhibition of agonist-induced platelet aggregation has been established and may be due to enhanced formation of cyclic GMP. In this investigation, hydrogen peroxide and NO had no effect on the activity of pure soluble guanylyl cyclase or its activity in platelet lysates and cytosol. H(2)O(2) was found to increase the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), increasing the amount of the 50-kDa form that results from phosphorylation at serine(157). This occurs both in the presence and in the absence of low concentrations of NO, even at submicromolar concentrations of the peroxide, which alone was not inhibitory to platelets. These actions of H(2)O(2) were inhibited to a large extent by an inhibitor of cyclic AMP-dependent protein kinase, even though H(2)O(2) did not increase cyclic AMP. This inhibitor reversed the inhibition of platelets induced by combinations of NO and H(2)O(2) at low concentrations. The results suggest that the action on VASP may be one site of action of H(2)O(2) but that this event alone does not lead to inhibition of platelets; another unspecified action of NO is required to complete the events required for inhibition.

Serine-578 is a major phosphorylation locus in human plasma plasminogen.

It has been reported that human plasminogen (HPg) exists in plasma in a phosphorylated form. We now document that both major glycoforms of plasma HPg contain a phosphoserine residue in their latent protease chains, as revealed by quantitative protein phosphate determinations and 31P-NMR analysis. The sequence location of the phosphoserine residue was established by time-of-flight matrix-assisted laser desorption ionization with delayed extraction mass spectrometric analysis of peptides resulting from complete tryptic and cyanogen bromide digests of the latent protease chain of HPg. Confirmation of the presence of organic phosphate in the identified peptide was obtained by determination of the resulting mass shift after treatment of the peptide with alkaline phosphatase. The data show that Ser578 is a major phosphorylation site in HPg.

Serine and tyrosine phosphorylation of 28- and 35-kDa proteins of human T lymphocytes stimulated by streptococcal M protein.

Purified polypeptide fragments of certain surface M proteins of group A streptococci stimulate blastogenesis and the differentiation of cytotoxic T lymphocytes of normal human lymphocytes. The biochemical basis of lymphocyte stimulation by a type M5 protein polypeptide fragment (pep M5) was investigated. Optimal blastogenic doses of pep M5 or phytohemagglutinin stimulated the phosphorylation of several cellular proteins. However, pep M5 but not phytohemagglutinin induced the phosphorylation of 28- and 35-kDa proteins. The 28-kDa protein was shown to be phosphorylated only at serine residues, whereas the 35-kDa protein was phosphorylated only at tyrosine residues. Stimulation of peripheral blood lymphocytes with pep M5 caused a two-fold increase in the CD8+ and CD4+ 4B4+ subpopulations of T lymphocytes. The phosphorylation of the 28-kDa protein appeared to be confined to the CD4+ T cell subpopulation.

Erythropoietin rapidly alters phosphorylation of pp43, an erythroid membrane protein.

The phosphorylation of a prominent 43-kDa phosphoprotein (pp43) in the membranes of normal murine erythroid cells was reduced markedly by exposure of the membranes to highly purified erythropoietin. A virtually identical reduction of pp43 phosphorylation was seen when erythropoietin-responsive Rauscher murine erythroleukemia cell membranes were exposed to the hormone. This effect was both time-dependent, occurring within 30 min after erythropoietin exposure, and concentration-dependent. Phosphoamino acid analysis revealed that pp43 is phosphorylated on serine residues. The results provide the first evidence that rapid alterations in membrane protein phosphorylation may serve as a trans-membrane signal for erythropoietin.

Influence of anticoagulant substances on phosphoserine and taurine of human blood.

The influence of heparine (Liquemin) and sodium-EDTA on phosphoserine and taurine of human blood was investigated. Phosphoserine which was not or nearly not present in serum increased after addition of both the anticoagulant substances. The heparine used contained phosphoserine as the only ninhydrinic substance in low concentrations. After addition of heparine phosphoserine in human plasma does not increase proportionally to that change. A mechanism is assumed which leads to the release from cells or which stimulates the synthesis of the substance. A similar effect was found with sodium-EDTA but not with sodium citrate. An opposite effect was detected with taurine in human blood. In serum the highest concentration was measured which decreased slowly under heparine as well as under sodium-EDTA addition.