Examination of association of genes in the serotonin system to autism.

Autism is characterized as one of the pervasive developmental disorders, a spectrum of often severe behavioral and cognitive disturbances of early development. The high heritability of autism has driven multiple efforts to identify genetic variation that increases autism susceptibility. Numerous studies have suggested that variation in peripheral and central metabolism of serotonin (5-hydroxytryptamine) may play a role in the pathophysiology of autism. We screened 403 autism families for 45 single nucleotide polymorphisms in ten serotonin pathway candidate genes. Although genome-wide linkage scans in autism have provided support for linkage to various loci located within the serotonin pathway, our study does not provide strong evidence for linkage to any specific gene within the pathway. The most significant association (p = 0.0002; p = 0.02 after correcting for multiple comparisons) was found at rs1150220 (HTR3A) located on chromosome 11 ( approximately 113 Mb). To test specifically for multilocus effects, multifactor dimensionality reduction was employed, and a significant two-way interaction (p value = 0.01) was found between rs10830962, near MTNR1B (chromosome11; 92,338,075 bp), and rs1007631, near SLC7A5 (chromosome16; 86,413,596 bp). These data suggest that variation within genes on the serotonin pathway, particularly HTR3A, may have modest effects on autism risk.

Kinetic studies of Haemophilus influenzae malate dehydrogenase.

Haemophilus influenzae malate dehydrogenase [S)-malate: NAD+ oxidoreductase EC 1.1.1.37) was purified 109-fold with a 26% recovery through a four-step procedure involving salt fractionation, hydrophobic and dye affinity chromatography. The purified enzyme was demonstrated to be a dimer of Mr 61,000. Initial velocity studies of all four substrates in the forward and reverse reactions indicated a sequential mechanism for the enzyme. Product and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme and NADH, the second product released. Several analogs of NAD structurally altered in either the pyridine or purine moiety were observed to function as coenzymes in the reaction catalyzed by the purified malate dehydrogenase. Alterations in the purine portion of the dinucleotides had a more pronounced effect on the kinetic parameters observed in malate oxidation. The enzyme was inactivated by incubation with diethylpyrocarbonate, whereas no inactivation was observed with sulfhydryl reagents.

Kinetic studies of rat ovarian 20 alpha-hydroxysteroid dehydrogenase.

Rat ovarian 20 alpha-hydroxysteroid dehydrogenase was purified 230-fold with a 48% recovery through a 3-step process involving hydrophobic, gel filtration and green dye affinity chromatography. The purified enzyme was demonstrated to be a single polypeptide chain of Mr 36 000. Initial velocity studies of all four substrates in the forward and reverse reactions indicated a sequential mechanism for the enzyme. Product inhibition and dead-end inhibition studies with substrate analogs were consistent with an ordered bi-bi mechanism in which NADP is the first substrate bound to the enzyme and NADPH, the second product released. Several NADP analogs were demonstrated to function as coenzymes in the reaction catalyzed. The purified enzyme was denatured at moderate temperatures and the binding of NADP protected the enzyme against thermal denaturation.

Simultaneous inactivation of the catalytic activities of yeast glutathione reductase by N-alkylmaleimides.

A series of straight chain N-alkymaleimides was shown to simultaneously inactivate the reductase, transhydrogenase and diaphorase activities of yeast glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2.) at pH 7.5 and 25 degrees C. Apparent second-order rate constants for the inactivation of all enzyme activities exhibited parallel increases with increasing chainlength from C-2 through C-7 of the alkyl substituent of the enhanced binding of maleimides through nonpolar interactions with the enzyme. Reduction of the active site disulfide with NADPH was required prior to addition of maleimide for inactivation to occur. NADP, AcPyADP, SNADP, AADP, and oxidized glutathione all protected the enzyme from inactivation. 2'AMP, 3' AMP, 2'-phospho-5' AMP, 2'-phospho5'-ADP and 2'-phospho-ADP-ribose although all coenzyme-competitive inhibitors failed to protect the enzyme from N-ethylmaleimide inactivation. N-Phenyl and N-alkylmaleimides covalently modified two, of six available sulfhydryl groups per subunit. No other amino acid residues were modified. The reactivity of these sulfhydryl groups was at least two orders of magnitude higher than any reported for the N-ethylmaleimide reaction with many other 'essential sulfhydryl' enzymes. No change in the charge transfer band of the reduced enzyme was observed upon complete inactivation by N-ethyl, N-heptyl or N-phenylmaleimide. The retention of the charge transfer band after selective modification of two sulfhydryl groups suggests the involvement of a third reactive sulfhydryl group in the functioning of the yeast enzyme. No inactivation was observed when coenzymatically reduced enzyme was incubated with the site-specific sulfhydryl reagent, diazotized AADP.

Nonpolar interactions in the maleimide inactivation of Haemophilus influenzae D-lactate dehydrogenase.

A series of N-alkylmaleimides varying in chain length from N-ethyl up to and including N-heptyl, was shown to effectively inactivate Haemophilus influenzae D-lactate dehydrogenase at pH 7.0 and 25 degrees C in processes proposed to involve covalent modification of cysteine residues. The inactivation proceeded through an initial reversible binding of maleimides facilitated by nonpolar interactions with a hydrophobic region of the enzyme. Subsequent irreversible inactivation of the enzyme indicated the modification of a fast-reacting group leading to approx. 80% loss of enzyme activity followed by a second slower-reacting modification process. At saturating concentrations of maleimides, the second inactivation process exhibited a common pseudo-first-order rate constant of 0.6 min-1. The initial reversible binding of N-alkylmaleimides resulted in inhibition of the enzyme that was competitive with respect to NADH. Positive chain length effects were observed in the second-order rate constants for inactivation and in the 6-fold better binding of N-heptylmaleimide as compared to that for N-ethylmaleimide. It is suggested that the nonpolar interactions stabilizing the 1,4-dihydronicotinamide moiety of the reduced coenzyme also facilitate the initial binding of N-alkylmaleimides.

Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase.

Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chromatography. The purified enzyme was demonstrated to be a dimer of Mr 70,000, and to catalyze a sequential reaction process. The enzyme was NADP-specific and kinetic parameters for the oxidation of 6-phosphogluconate were determined for NADP and four structural analogs of NADP. Coenzyme-competitive inhibition by adenosine derivatives was significantly enhanced by the presence of a 2'-phosphoryl group consistent with the observed coenzyme specificity of the enzyme. The purified enzyme was effectively inhibited by 3-aminopyridine adenine dinucleotide phosphate, but at concentrations higher than that observed to inhibit growth of the organism. Rates of inactivation of the enzyme by N-ethylmaleimide were suggestive of sulfhydryl involvement in the reaction catalyzed.

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.

Azotobacter vinelandii glucose 6-phosphate dehydrogenase properties of NAD- and NADP-linked reactions.

Glucose 6-phosphate oxidation, catalyzed by purified Azotobacter vinelandii glucose 6-phosphate dehydrogenase, was studied with respect to the selective utilization of NAD, NADP, thionicotinamide adenine dinucleotide or thionicotinamide adenine dinucleotide phosphate as coenzyme. A sigmoidal relationship was observed for the effect of substrate concentration on initial velocities when either NAD, NADP or thionicotinamide adenine dinucleotide was used as coenzyme, with N values from the Hill equation equalling 2.0, 1.7, and 1.7, respectively. The thionicotinamide analogs of NAD and NADP both functioned as coenzyme-competitive inhibitors of the enzyme-catalyzed NAD- and NADP-linked reactions. A dual wavelength assay, using a combination of NADP and thio-NAD, was established and was used to demonstrate that increasing glucose 6-phosphate concentration did not change the enzyme preference for the coenzyme form used. Sigmoidal relationships were observed for reduction of both dinucleotides, and N values were the same as those observed when each dinucleotide was studied as the only coenzyme form present in reaction mixtures. Using the dual wavelength assay, inhibition by isocitrate, 6-phosphogluconate, ATP, and palmitoyl-CoA was shown to be equally effective in both NAD- and NADP-linked reactions. An enzyme activator, glucosamine 6-phosphate, altered the glucose 6-phosphate sigmoidicity through activation at low substrate concentrations.

Nonpolar interactions in the modification of an essential sulfhydryl of sorbitol dehydrogenase by N-alkylmaleimides.

A series of N-alkylmaleimides varying in chainlength from N-methyl- to N-octylmaleimide inclusive was shown to effectively inactivate sheep liver sorbitol dehydrogenase at pH 7.5 and 25 degrees C. The apparent second-order rate constants for inactivation increased with increasing chainlength of the N-alkylmaleimide used. Positive chainlength effects were also indicated by the Kd values for the N-ethyl and N-heptyl derivatives obtained from studies of the saturation kinetics observed for inactivation of the enzyme at high concentrations of these maleimides. The complete inactivation of sorbitol dehydrogenase was demonstrated to occur through the selective covalent modification of one cysteine residue per subunit of enzyme. The stoichiometry of enzyme inactivation was supported on the one hand by fluorescence titration with fluorescein mercuric acetate of the native and the inactivated enzyme, and, on the other hand, by the simultaneous inactivation of the enzyme with selective modification of one sulfhydryl per subunit by N-[p-(2-benzoxazolyl)phenyl]maleimide. Protection of the enzyme from N-alkylmaleimide inactivation was observed with the binding of NADH, whereas both NAD and sorbitol were ineffective as protecting ligands. Diazotized 3-aminopyridine adenine dinucleotide, in contrast to previous studies of this reagent with yeast alcohol dehydrogenase and rabbit muscle glycerophosphate dehydrogenase, did not function as a site-labeling reagent for sorbitol dehydrogenase.

Effects of dalfampridine on multi-dimensional aspects of gait and dexterity in multiple sclerosis among timed walk responders and non-responders.

BACKGROUND: Dalfampridine extended release 10mg tablets (D-ER) have demonstrated improvement in walking for ambulatory persons with multiple sclerosis (pwMS), termed "responders." OBJECTIVE: This study examined the extent additional aspects of gait and dexterity change for patients prescribed D-ER. METHODS: Over 14-weeks, walking endurance, dynamic gait, self-report walking ability and fine and gross dexterity were examined in pwMS prescribed D-ER as a part of routine clinical care. RESULTS: The final results (n=39) validate that a subset of pwMS improve walking speed (Time 25-Foot Walk Test, p<0.0001). Significant improvements in gait and dexterity were observed even among participants who did not improve walking speed. Improvements were evident in gait and dexterity domains including Six Minute Walk Test, p=0.007, Six-Spot Step Test, p<0.0001, Multiple Sclerosis Walking Scale-12, p<0.0001, Nine Hole Peg Test, p<0.0001 dominant and non-dominant sides, and Box and Blocks Test, p=0.005 and 0.002, dominant and non-dominant sides, respectively. CONCLUSIONS: These findings suggest that D-ER may be a potential treatment for gait impairments, beyond walking speed and dexterity in pwMS. Further investigation regarding D-ER response is warranted.

The iron and zinc status of long-term vegetarian women.

Iron and zinc status of 56 Seventh-Day Adventist Canadian women (mean age 52.9 +/- 15.3 yr) following vegetarian diets for 19 +/- 17 yr were investigated. Energy, protein, iron, available iron, zinc, and total dietary fiber intakes were calculated from 3-day dietary records. Hemoglobin, serum iron, total iron binding capacity, serum and hair zinc concentrations were also determined. Plant products provided 92 and 77% of the total dietary iron and zinc intakes, respectively. Calculated mean daily intakes (+/- SD) for energy, protein, iron, zinc, and total dietary fiber were 1630 +/- 354 kcal, 58 +/- 14 g, 12.5 +/- 3.0 mg, 9.2 +/- 2.5 mg, and 30.9 +/- 11.0 g, respectively. Mean hemoglobin (13.1 +/- 1.0 g/dl), calculated serum transferrin saturation (37.5 +/- 12.9%), mean serum zinc (99 +/- 24 microgram/dl), and hair zinc concentrations (187 +/- 44 ppm) were all within the normal range. The iron and zinc status of these long-term Seventh-Day Adventist vegetarian women appeared adequate despite their low intake of readily absorbed iron and zinc from flesh foods and their high intake of total dietary fiber and phytate.

Regional differences in hair zinc concentrations: a possible effect of water hardness.

Hair Zn, Cu, and Mn concentrations were determined by neutron activation procedures in two groups of healthy, Caucasian children aged 4.5 to 5.5 yr from Halifax, Nova Scotia and Guelph, Ontario and consuming soft water (hardness - 33 ppm) and very hard tap water (hardness = 330 ppm), respectively. Median hair Zn level for the Halifax children (26 males and 26 females) was 140 microgram/g, significantly higher (p less than 0.001) than the Guelph children (median = 82 microgram/g; 25 males and 26 females). No significant differences existed between the two groups for median hair Cu and Mn levels (Halifax hair Cu 12.1 microgram/g. Guelph hair Cu 11.0 microgram/g. Halifax hair Mn 0.17 microgram/g. Guelph hair Mn 0.18 microgram/g). The Halifax children were significantly heavier (p less than 0.001) and slightly taller (p = 0.09) than their Guelph counterparts, although mean daily intakes of energy, protein, Zn, Cu, Mn, and dietary fiber, calculated from 3-day food records were not significantly different in the two groups. The large differences in the hair zinc levels may be due to the high concentration of calcium in the Guelph hard water, which may decrease the absorption of zinc.

The trace metal status of a group of post-menopausal vegetarians.

The copper, manganese, and selenium status of 36 post-menopausal vegetarians was compared with that of a group of post-menopausal omnivores, using dietary trace element intake data, serum and hair copper levels, and hair manganese and hair selenium levels as the principal indexes. Findings of this study suggest that the copper and selenium status of these long-term vegetarian women was comparable to that of non-vegetarians, despite the high intake of dietary fiber by the vegetarian group. In contrast, the manganese status of the vegetarians, as indicated by elevated hair manganese levels, was higher, almost certainly as a result of the significantly higher manganese intake of this group.

Studies of self-inactivation of bovine seminal fluid NAD glycohydrolase.

Bovine seminal fluid NAD glycohydrolase (NADase) was observed to be rapidly inactivated during catalytic hydrolysis of the substrate NAD. The first-order rate constant for the self-inactivation process was independent of enzyme concentration. The enzyme self-inactivation was a turnover-related process and the number of moles of NAD hydrolyzed required for inactivation was proportional to the enzyme concentration. A number of dinucleotides serving as substrates for the enzyme also promoted self-inactivation. The self-inactivation was an irreversible process having a different rate-limiting step from NAD hydrolysis and was not related to the reversible binding of products and substrate-competitive inhibitors. Modification of arginine residues of the enzyme resulted in the loss of NAD hydrolase activity with no differential effect on the self-inactivation process.

Characterization of H. parasuis periplasmic nucleotide pyrophosphatase as a potential target enzyme for inhibition of growth.

The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis was purified 750-fold to electrophoretic homogeneity through salt fractionation and ion-exchange and affinity chromatography. The purified enzyme was monomeric with an apparent M(r) of 70,000 and catalyzed the hydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as products. The enzyme exhibited negative cooperativity in the hydrolysis of a number of pyridine dinucleotides and structurally-related pyrophosphate compounds as indicated by biphasic double-reciprocal plots and Hill coefficients of 0.5. The kinetic parameters, K(m) and Vm, determined titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitrogen bases other than nicotinamide or adenine to that of NAD. Effective substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromolar concentration range. Although less effective, N1-alkylnicotinamide chlorides also inhibited competitively with respect to the substrate, NAD. In addition to being an effective inhibitor of the purified enzyme, adenosine diphosphate also inhibited growth of H. parasuis at a low micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by the fact that adenosine diphosphate does not effectively inhibit growth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the periplasmic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, which also inhibited growth of H. parasuis at a low micromolar concentration, did not effectively inhibit the purified pyrophosphatase and a different target enzyme needs to be considered to explain growth inhibition by this derivative.

Yeast alcohol dehydrogenase catalyzed reduction of p-nitroso-N, N-dimethylaniline by NADH.

The steady-state kinetics, product identification, stoichiometries, and solvent isotope effects of yeast alcohol dehydrogenase catalyzed reduction of p-nitroso-N,N-dimethylaniline (NDMA) by NADH, are reported. NDMA is enzymatically reduced to p-hydroxylamine-N,N-dimethylaniline, which is further enzymatically dehydrated to corresponding quinonediimine cation (QDI+). QDI+ undergoes nonenzymatic transformations. QDI+ is rapidly reduced by NADH to p-amino-N,N-dimethylaniline (ADMA). Also, QDI+ is readily dismutated with ADMA to form N,N-dimethyl-p-phenylenediamine radicals; radicals are stable under steady-state conditions, below pH 7.5. A complete kinetic mechanism for above reactions has been proposed.

Effect of protein source and lasalocid on nitrogen digestibility and growth by ruminants.

Protein supplements having either a high (soybean meal, SBM) or low (escape protein, EP) extent of ruminal N degradability with or without lasalocid (L) were evaluated in digestion and growth trials. The SBM supplement included soybean meal while EP was a combination of dehydrated alfalfa and distillers dried grains. Nitrogen digestibility of SBM supplements was consistently higher than EP supplements when evaluated with two lamb trials. Digestibility of N was improved 8% in trial one (64.9 vs 60.3%) and 27% in trial two (66.3 vs 52.3%) with SBM vs EP. The addition of L to the supplements improved N digestibility by 6% in trial one (64.5 vs 60.6%) and 13% in trial two (62.9 vs 55.7%). No interactions between protein source and L were measured in either trial. Dry matter digestibility was not changed by protein source or L in either trial. Rumen propionate was increased and acetate to propionate ratio decreased when L was fed. Plasma urea N was lower over a 24 h sampling period when lambs were fed EP supplements compared with SBM supplements (11.07 vs 16.44 mg/100 ml); however, L did not appear to consistently alter the values. When steers were supplemented with the same protein sources during a 105-d winter pasture trial daily gains were not affected (P greater than .10) by either protein source or L (.429, .495, .476 and .514 kg/d for SBM, SBM+L, EP and EP+L, respectively) although numerically there did not appear to be main effect improvements due to EP and L.(ABSTRACT TRUNCATED AT 250 WORDS)

Materials trade study for lunar/gateway missions.

The National Aeronautics and Space Administration (NASA) administrator has identified protection from radiation hazards as one of the two biggest problems of the agency with respect to human deep space missions. The intensity and strength of cosmic radiation in deep space makes this a 'must solve' problem for space missions. The Moon and two Earth-Moon Lagrange points near Moon are being proposed as hubs for deep space missions. The focus of this study is to identify approaches to protecting astronauts and habitats from adverse effects from space radiation both for single missions and multiple missions for career astronauts to these destinations. As the great cost of added radiation shielding is a potential limiting factor in deep space missions, reduction of mass, without compromising safety, is of paramount importance. The choice of material and selection of the crew profile play major roles in design and mission operations. Material trade studies in shield design over multi-segmented missions involving multiple work and living areas in the transport and duty phase of space mission's to two Earth-Moon co-linear Lagrange points (L1) between Earth and the Moon and (L2) on back side of the moon as seen from Earth, and to the Moon have been studied. It is found that, for single missions, current state-of-the-art knowledge of material provides adequate shielding. On the other hand, the choice of shield material is absolutely critical for career astronauts and revolutionary materials need to be developed for these missions. This study also provides a guide to the effectiveness of multifunctional materials in preparation for more detailed geometry studies in progress.

Use of pH studies to determine the kinetic and chemical mechanism of yeast alcohol dehydrogenase with primary aliphatic alcohols and aldehydes.

A complete list of all steady-state kinetic constants for the yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzed oxidation of ethanol, propan-1-ol and butan-1-ol, and for the reduction of acetaldehyde and propionaldehyde was collected in the pH range 6-10, and an appropriate pH profile for each constant was constructed. A common minimal mechanism with all these substrates has been postulated and pKa values and the pH independent limiting values have been assigned for the rate constants.