Plasma peroxiredoxin changes and inflammatory cytokines support the involvement of neuro-inflammation and oxidative stress in Autism Spectrum Disorder.

BACKGROUND: It has been established that children with Autism Spectrum Disorders (ASD) are affected by oxidative stress, the origin of which is still under investigation. In the present work, we evaluated inflammatory and pro-oxidant soluble signature in non-syndromic ASD and age-matched typically developing (TD) control children. METHODS: We analyzed leukocyte gene expression of inflammatory cytokines and inflammation/oxidative-stress related molecules in 21 ASD and 20 TD children. Moreover, in another-comparable-group of non-syndromic ASD (N = 22) and TD (N = 21) children, we analyzed for the first time the protein expression of the four members of the antioxidant enzyme family of peroxiredoxins (Prx) in both erythrocyte membranes and in plasma. RESULTS: The gene expression of IL6 and of HSP70i, a stress protein, was increased in ASD children. Moreover, gene expression of many inflammatory cytokines and inflammation/oxidative stress-related proteins correlated with clinical features, and appeared to be linked by a complex network of inter-correlations involving the Aryl Hydrocarbon Receptor signaling pathway. In addition, when the study of inter-correlations within the expression pattern of these molecules was extended to include the healthy subjects, the intrinsic physiological relationships of the inflammatory/oxidative stress network emerged. Plasma levels of Prx2 and Prx5 were remarkably increased in ASD compared to healthy controls, while no significant differences were found in red cell Prx levels. CONCLUSIONS: Previous findings reported elevated inflammatory cytokines in the plasma of ASD children, without clearly pointing to the presence of neuro-inflammation. On the other hand, the finding of microglia activation in autoptic specimens was clearly suggesting the presence of neuro-inflammation in ASD. Given the role of peroxiredoxins in the protection of brain cells against oxidative stress, the whole of our results, using peripheral data collected in living patients, support the involvement of neuro-inflammation in ASD, and generate a rational for neuro-inflammation as a possible therapeutic target and for plasma Prx5 as a novel indicator of ASD severity.

[The Martius flap in stress urinary incontinence treated by suburethral sling]. / Intérêt du lambeau de Martius (LM) dans l'incontinence urinaire d'effort (IUE) traitée par bandelettes sous-urétrales (BSU).

OBJECTIVE: Study of a series of patients treated with suburethral tapes using the Martius flap technique, for complications or in a preventive way, in order to determine its interest and morbidity. PATIENTS: Eleven female patients treated by suburethral tapes for stress urinary incontinence, for which we described the type of tapes used, their complications, the procedure including Martius flap and the outcomes. RESULTS: Indications of the Martius flap were: three tapes through bladder neck, one through urethra, one uro-vaginal fistula, four loss of substance of the vaginal wall, one peri-urethral fibrosis and one preventive use on irradiated pelvis. Complications were transient postoperative pain and one abscess of the greater lip. In our series, the Martius flap never cured stress urinary incontinence. The placement of a suburethral tape on a Martius flap is feasible but the adjustment is more difficult. CONCLUSION: The Martius flap is efficient in the treatment or prevention of suburethral tapes complications with low morbidity. It allows secondary placement of a suburethral tape with difficult adjustment.

How do kinases transfer phosphoryl groups?

Understanding how phosphoryl transfer is accomplished by kinases, a ubiquitous group of enzymes, is central to many biochemical processes. Qualitative analysis of the crystal structures of enzyme-substrate complexes of kinases reveals structural features of these enzymes important to phosphoryl transfer. Recently determined crystal structures which mimic the transition state complex have added new insight into the debate as to whether kinases use associative or dissociative mechanisms of catalysis.

Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold.

The crystal structure of ATP-dependent phosphoenolpyruvate carboxykinase (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1.49; PCK) from Escherichia coli strain K12 has been determined using a combination of multiple isomorphous replacement, density modification, and partial model phase combination, and refined to a conventional R-index of 0.204 (Rfree = 0.244) at 1.9 A resolution. Each PCK molecule consists of a 275 residue N-terminal domain and 265 residue C-terminal or mononucleotide-binding domain, with the active site postulated to be within a cleft between the two domains. PCK is an open-faced, mixed alpha/beta protein, with each domain having an alpha/beta folding topology as found in several other mononucleoside-binding enzymes. The putative phosphate-binding site of ATP adopts the P-loop motif common to many ATP and GTP-binding proteins, and is similar in structure to that found within adenylate kinase. However, the beta-sheet topology within the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphosphate hydrolase superfamily, therefore suggesting it represents the first member in a new family of such proteins. The mononucleotide-binding domain is also different in structure compared to the classical mononucleotide-binding fold (CMBF) common to adenylate kinase, p21ras, and elongation factor-Tu. Several amino acid residues, including R65, K212, K213, H232, K254, D269, K288 and R333 appear to make up the active site of the enzyme, and are found to be absolutely conserved among known members of the ATP-dependent PCK family. A cysteine residue is located near the active-site, as has been suggested for other PCKs, although in the E. coli enzyme C233 is buried and so is most likely not involved in substrate binding or catalysis. Two binding sites of the calcium-analog TB3+ have been determined, one within the active site coordinating to the side-chain of D269, and the other within the C-terminal domain coordinating to the side-chains of E508 and E511.

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate.

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis. Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.

Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution.

Glycosaminoglycans (GAGs) are a family of acidic heteropolysaccharides, including such molecules as chondroitin sulfate, dermatan sulfate, heparin and keratan sulfate. Cleavage of the O-glycosidic bond within GAGs can be accomplished by hydrolases as well as lyases, yielding disaccharide and oligosaccharide products. We have determined the crystal structure of chondroitinase B, a glycosaminoglycan lyase from Flavobacterium heparinum, as well as its complex with a dermatan sulfate disaccharide product, both at 1.7 A resolution. Chondroitinase B adopts the right-handed parallel beta-helix fold, found originally in pectate lyase and subsequently in several polysaccharide lyases and hydrolases. Sequence homology between chondroitinase B and a mannuronate lyase from Pseudomonas sp. suggests this protein also adopts the beta-helix fold. Binding of the disaccharide product occurs within a positively charged cleft formed by loops extending from the surface of the beta-helix. Amino acid residues responsible for recognition of the disaccharide, as well as potential catalytic residues, have been identified. Two arginine residues, Arg318 and Arg364, are found to interact with the sulfate group attached to O-4 of N-acetylgalactosamine. Cleavage of dermatan sulfate likely occurs at the reducing end of the disaccharide, with Glu333 possibly acting as the general base.

The crystal structure of Escherichia coli MoeA, a protein from the molybdopterin synthesis pathway.

MoeA is involved in synthesis of the molybdopterin cofactor, although its function is not yet clearly defined. The three-dimensional structure of the Escherichia coli protein was solved at 2.2 A resolution. The locations of highly conserved residues among the prokaryotic and eukaryotic MoeA homologs identifies a cleft in the dimer interface as the likely functional site. Of the four domains of MoeA, domain 2 displays a novel fold and domains 1 and 4 each have only one known structural homolog. Domain 3, in contrast, is structurally similar to many other proteins. The protein that resembles domain 3 most closely is MogA, another protein required for molybdopterin cofactor synthesis. The overall similarity between MoeA and MogA, and the similarities in a constellation of residues that are strongly conserved in MoeA, suggests that these proteins bind similar ligands or substrates and may have similar functions.

A method of quantitating aggressive behaviour revealing possible dissociation of motor activity and aggression.

A method is described for quantitating isolation-induced aggression in wild male mice by measuring coarse motor activity. It is based on the operational definition that aggression is the form of motor activity in which two (or more) animals are involved in behaviour patterns, such as physical attack and/or defence including pursuit and flight. Both coarse and fine motor activity were measured by means of an electromagnetic activity meter. Four hundred ninety-two fights occurring among mice treated with various hormones, neurotransmitter antagonists, hashish, and cyproterone acetate were recorded; a positive significant correlation (P less than 0.001) between indices of fighting behaviour as well as fighting time and coarse motor activity were found. The simultaneous measurement of motor activity and aggression revealed dissociated effects of some drugs on non-aggressive fine motor activity and aggression.

Growth hormone and isolation-induced aggression in wild male mice.

Aggressive behavior, motor activity and defecation were examined simultaneously in wild male mice following daily growth hormone (GH) administration. GH was found to increase isolation-induced aggression by increasing fighting duration and decreasing latency to fight. There was no influence on non-aggressive motor activity nor on the defecation rate, a presumed parameter of emotionality. The results provide evidence of behavioral properties of GH. The mechanism of this action is discussed in terms of a possible direct central action of GH, not mediated by glucagon, insulin, secretin and gastrin.

Triagem fitoquímica e avaliação da atividade antibacteriana de extratos das flores de Sambucus nigra L. (Caprifoliaceae) / Phytochemical screening and evaluation of the antibacterial activity of Sambucus nigra L. flower extracts (Caprifoliaceae)

RESUMO O presente trabalho teve como objetivo realizar a triagem fitoquímica e avaliar a atividade antibacteriana de extratos das flores de Sambucus nigraL. Os extratos; aquoso (10 %), etanólico (5 %) e Acetato de etila (5 %) foram submetidos a testes colorimétricos para triagem fitoquímica e a avaliação da atividade antibacteriana foi realizada pelo método de disco-difusão em ágar. Os resultados mostraram que nas concentrações de 6 e 12 mg o extrato aquoso apresentou halos significativos de inibição para Staphylococcus aureus, Pseudomonas aeruginosa e Streptococcus pyogenes, porém, quando comparado aos medicamentos usados como referência a atividade não foi satisfatória, e, ainda, evidenciou a ausência de inibição para todas as cepas testadas com o aumento da concentração para 18 e 24 mg. A análise da triagem fitoquímica evidenciou a presença de flavonoides com intensa reação de cor no extrato aquoso e etanólico, e de fraca intensidade no extrato acetato de etila. Nos mesmos extratos, pelos testes realizados, não foram detectados taninos, saponinas, antraquinonas e alcaloides. Concluiu-se que o extrato aquoso apresentou melhor efeito inibitório para Staphylococcus aureus, Pseudomonas aeruginosa e Streptococcus pyogenes, porém insuficiente para promover a inativação eficiente quando comparado aos controles.

ABSTRACT This study aimed to perform a phytochemical screening and to evaluate the antibacterial activity of the extracts of Sambucusnigra L. flowers. The aqueous (10%), ethanolic (5%) and ethyl acetate (5%) extracts were subjected to colorimetric tests for phytochemical screening and the antibacterial activity evaluation was performed by the disk-diffusion method in agar. The results showed that in the 6 and 12 mg concentrations the aqueous extract presented significant inhibition halos for Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes, but when compared with the medicines used as a reference, the activity was not satisfactory and, besides, it indicated the absence of inhibition for all the tested strains with the concentration increase of 18 and 24mg. The phytochemical screening analysis showed the presence of flavonoids with intense color reaction in the aqueous and ethanol extracts, and of low intensity in the ethyl acetate sample. In the same extracts, the tests did not detect tannins, saponins, alkaloids and anthraquinones. It was concluded that the aqueous extract showed better inhibitory effect on the Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes, but not enough to promote an effective inactivation when compared with the medicine tests.

Effect of isolation induced aggression in mice on serum creatine kinase.

It is known that the serum creatine kinase increases as a result of different conditions and that isolation in male mice induces aggressive behaviour after regrouping. The levels of serum creatine kinase were studied in isolated and in regrouped male albino mice. A considerable increase of creatine kinase was found in regrouped male mice fighting for different hours. Further studies are needed to prove whether these results are due to isolation, aggression or motor activity.

Dissociated effects of inhibition of catecholamine synthesis on motor activity, "emotionality," and aggression in mice.

The inhibitor of catecholamine synthesis at the tyrosine hydroxylase level, alpha-methyltyrosine, administered in daily doses of 250 mg/kg IP, altered motor activity and aggressive behavior of isolated male mice in ten-minute paired encounter tests conducted on nine successive days. Motor activity was determined by an electromagnetic activity meter which measures fine and coarse movements separately. Coarse motor activity correlated significantly with aggressive behavior. Nonaggressive fine motor activity was significantly increased, while all the parameters of aggressive behavior showed a significant decrease in aggression. Increased escape behavior, heterogrooming, squeaking, and two cases of stupor were observed, suggesting possible equivalents of anxiousness. The defecation rate did not differ from that of the control group, victors having had a higher rate than losers. Results are discussed in connection with the "construct of emotionality" and the hypothesis of there being several categories of aggression with corresponding biochemical correlations.

The effect of cyproterone acetate on motor activity, aggression, "emotionality", body weight and testes in wild mice.

The literature reports on antiandrogenic and antigonadotropic effects of cyproterone acetate (CA) and on its inhibitory influence on the structure and function of the testis and reduction of the sexual drive in man and animals. No one has as yet been able to confirm a reduction of aggression which could be expected under CA. Isolated male wild mice received daily doses of 0.5 mg (17 mg/kg) CA subcutaneously over 32 days and were studied daily for aggressive behavior, motor activity and emotionality during the last 11 days. Aggression was reduced significantly by CA in the form of an increased latent time and continued to decrease during the test period with a reduction of fighting time and the correlated coarse motor activity. A latent period of approximately 4 weeks was determined for the aggression-inhibiting effect of CA. The detection rate was not changed under CA. It is doubted whether it is an adequate parameter of "emotionality". The overall motor activity was not reduced, so that an aggression-specific effect is assumed rather than a general reduction of drive. The body and testicular weight was reduced, the testicular tissue atrophies. Central points of attack of CA probably play a major role in the reduction of aggression.

Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.

Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo.

Crystallization and preliminary X-ray analysis of chondroitinase B from Flavobacterium heparinum.

Chondroitinase B, a glycosaminoglycan lyase from Flavobacterium heparinum, has been crystallized by hanging-drop vapor diffusion in space group P21 with unit-cell parameters a = 50.6, b = 74.5, c = 58. 7 A, beta = 92.9 degrees and one molecule in the asymmetric unit. This enzyme degrades dermatan sulfate, a glycosaminoglycan primarily made up of a disaccharide repeating unit of iduronic acid and N-acetylgalactosamine. A complete native data set has been collected from a single crystal to 2.2 A resolution using a rotating-anode source.

Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85.

Prevotella (Bacteroides) ruminicola strains B(1)4 and S23 and Selenomonas ruminantium strain D used xylose as the sole source of carbohydrate for growth, whereas Fibrobacter succinogenes was unable to metabolize xylose. Prevotella ruminicola strain B(1)4 exhibited transport activity for xylose. In contrast, F. succinogenes lacked typical xylose uptake activity but did exhibit low binding potential for the sugar. Prevotella ruminicola strains B(1)4 and S23 as well as S. ruminantium D showed low xylose isomerase activities but higher xylulokinase activities, using assays that gave high activities for these enzymes in Escherichia coli. Xylose isomerase appeared to be produced constitutively in these ruminal bacteria, but xylulokinase was induced to varying degrees with xylose as the source of carbohydrate. Fibrobacter succinogenes lacked xylose isomerase and xylulokinase. All three species of ruminal bacteria possessed transketolase, xylulose-5-phosphate epimerase, and ribose-5-phosphate isomerase activities. Neither P. ruminicola B(1)4 nor F. succinogenes S85 showed significant phosphoketolase activity. The data indicate that F. succinogenes is unable to either actively uptake or metabolize xylose as a result of the absence of functional xylose permease, xylose isomerase, and xylulokinase activities, although it and both P. ruminicola and S. ruminantium possess the essential enzymes of the nonoxidative branch of the pentose phosphate cycle.

[Sex role and identity of childless andrology patients]. / Zur Geschlechtsrolle und -identität bei kinderlosen Patienten der Andrologie.

By means of the Bem Sex-Role Inventory (BSRI) 1000 childless married patients of andrology have been examined in order to determine their masculinity, femininity, androgynity and social desire scores. The results have been compared with a control group of 111 students of medicine and dentistry and with data of a study on 444 students at Stanford University. Significant differences have been shown in social desire only, but in no other respect. Thus, the results do not indicate differences of sex role and self image between andrological patients and control groups in Hamburg as well as in Stanford.

Snapshot of an enzyme reaction intermediate in the structure of the ATP-Mg2+-oxalate ternary complex of Escherichia coli PEP carboxykinase.

We report the 1.8 A crystal structure of adenosine triphosphate (ATP)-magnesium-oxalate bound phosphoenolpyruvate carboxykinase (PCK) from Escherichia coli. ATP binding induces a 20 degree hinge-like rotation of the N- and C-terminal domains which closes the active-site cleft. PCK possesses a novel nucleotide-binding fold, particularly in the adenine-binding region, where the formation of a cis backbone torsion angle in a loop glycine residue promotes intimate contacts between the adenine-binding loop and adenine, while stabilizing a syn conformation of the base. This complex represents a reaction intermediate analogue along the pathway of the conversion of oxaloacetate to phosphoenolpyruvate, and provides insight into the mechanistic details of the chemical reaction catalysed by this enzyme.