Influence of two functional polymorphisms in NOS1 on baseline cortisol and working memory in healthy subjects.

INTRODUCTION: The neuronal isoform of the nitric oxide synthase (NOS-I) encoded by NOS1 is the main source of nitric oxide (NO) in the brain. Reduced NO signaling in the prefrontal cortex has been linked to schizophrenia and cognitive processes while reduced striatal NOS1 expression has been associated with impulsive behavior. METHODS: To evaluate the effect of two functional polymorphisms in alternative first exons of NOS1, ex1f-VNTR and ex1c-SNP rs41279104, on the HPA stress axis and neurocognitive abilities, 280 healthy subjects were genotyped, had their salivary cortisol levels measured and were assessed in verbal memory, verbal fluency, working memory and verbal IQ by using the California Verbal Learning Test (CVLT), the Regensburger test of verbal fluency (RWT), a n-back task and subscales of the Wechsler Adult Intelligence Scale III (WAIS-III). RESULTS: Schizophrenia risk (A)-allele carriers of NOS1 ex1c-SNP rs41279104 displayed significantly lower baseline cortisol levels (p = 0.004). NOS1 ex1f-VNTR genotype carriers showed differences in working memory performance (p = 0.05) in a gene-dose effect manner, with homozygous carriers of the short impulsivity-risk allele committing most commission errors. Finally, A-allele carriers of the NOS1 ex1c-SNP rs41279104 tended to react faster during the working memory task (p = 0.065). CONCLUSION: For the first time, we demonstrated an influence of the NOS1 ex1c-SNP rs41279104 on salivary cortisol levels and additionally implicate the A-allele in an enhanced reaction time during a working memory task. Regarding the NOS1 ex1f-VNTR our study supports the previously reported influence on impulsivity, lending further support to the hypothesis that this genetic variant underlies impulsive behavior.

Estimating viral titres in solutions with low viral loads.

An important consideration in the manufacture of products derived from animal or human sources is the virus reduction capacity of the manufacturing process as estimated using validated bench-scale models of relevant manufacturing steps. In these studies, manufacturing process intermediates are spiked with virus and processed using the bench-scale model and the resulting viral titres of input and output samples are typically determined using cell-based infectivity assays. In these assays, the Spearman-Kärber (SK) method is commonly used to estimate titres when there is one or more positive observation (i.e., the presence of any viral cytopathic effect). The SK method is most accurate when the proportion of positive observations ranges from <0.1 to >0.9 across dilutions but can be biased otherwise. Maximum likelihood (ML) based on a single-hit Poisson model is an alternative widely used estimation method. We compared SK with ML and found the methods to have similar properties except for situations in which the concentration of virus is low but measurable. In this case, the SK method produces upwardly biased estimates of titres. Based on our results, we recommend the use of either ML or SK at most virus concentrations; however, at low virus concentrations ML is preferred.

His-391 of beta-galactosidase (Escherichia coli) promotes catalyses by strong interactions with the transition state.

His-391 of beta-galactosidase (Escherichia coli) was substituted by Phe, Glu, and Lys. Homogeneous preparations of the substituted enzymes were essentially inactive unless very rapid purifications were performed, and the assays were done immediately. The inactive enzymes were tetrameric, just like wild-type beta-galactosidase and their fluorescence spectra were identical to the fluorescence spectrum of wild-type enzyme. Analyses of two of the substituted enzymes that were very rapidly purified to homogeneity and rapidly assayed while they were still active (at only a few substrate concentrations so that the data could be rapidly obtained), showed that the kinetic values were very similar to the values obtained with the same enzymes that were only partially purified. This showed that the kinetics were not affected by the degree of purity and allowed kinetic analyses with partially purified enzymes so that large numbers of points could be used for accuracy. The data showed that His-391 is a very important residue. It interacts strongly with the transition state and promotes catalysis by stabilizing the transition state. Activation energy differences (deltadelta G(S) double dagger), as determined by differences in the kcat/Km values, indicated that substitutions for His-391 caused very large destabilizations (22.8-35.9 kJ/mol) of the transition state. The importance of His-391 for transition state stabilization was confirmed by studies that showed that transition state analogs are very poor inhibitors of the substituted enzymes, while inhibition by substrate analogs was only affected in a small way by substituting for His-391. The poor stabilities of the transition states caused significant decreases of the rates of the glycolytic cleavage steps (galactosylation, k2). Degalactosylation (k3) was not decreased to the same extent.

Urinary tract infections in men in a primary care population.

OBJECTIVE: There have been few studies on urinary tract infections (UTIs) in adult males. This study aimed to look at the clinical features of males presenting with urinary tract infections in a predominantly gay general practice population. METHOD: A retrospective audit and analysis was carried out of all male patients presenting with symptoms suggestive of a UTI and in whom a pathogen was cultured from urine. The subjects were drawn from two urban general practices in Melbourne. Subjects with known pre-existing urinary tract abnormalities, or in whom recent urinary tract instrumentation had been performed, were excluded, as were subjects in whom Neisseria gonorrhoeae was cultured from urine. RESULTS: Thirty-three subjects were identified, with a total of 47 presentations. Six subjects were excluded on the basis of pre-existing known factors which would predispose to UTIs, leaving 27 subjects, with 37 episodes of UTI. The mean age was 43 (range = 28-62) and 25 of the 27 identified as gay. Nine out of the 27 (33%) were HIV positive with a mean CD4 of 574/mm3 (range = 41-1812). The main presenting symptoms were dysuria/burning on micturition, urinary frequency, fever/sweats and haematuria. Urethral discharge occurred in two episodes. Multiple symptoms on presentation were common. The main organism cultured was E. coli. Fourteen subjects underwent further radiological investigation and two abnormalities were detected. CONCLUSION: Uncomplicated UTIs are uncommon in males presenting to general practice. The symptoms are similar to those in females with UTIs, though fever may be more common. Treatment with oral antibiotics in a primary care setting is generally curative. Further investigation of males with UTIs may be appropriate, but more studies are needed as to the cost-benefit of this.

Zinc and copper status in ewes supplemented with sulfate- and amino acid-complexed forms of zinc and copper.

Thirty 6-yr-old Targhee ewes were randomly allotted to one of five supplemental treatments to evaluate supplementation effects on liver and fecal Zn and Cu concentrations and serum alkaline phosphatase activity: 1) control, 2) Zn complex, 3) Zn and Cu (ZnCu) complex, 4) Zn sulfate, and 5) ZnCu sulfates. Supplements were administered daily in gelatin capsules for 56 d. Liver biopsies and serum samples were collected every 14 d starting on d 0. Supplemental Zn and Cu levels were formulated to provide 90 mg/kg Zn and 10 mg/kg Cu, respectively, on a daily dry matter intake basis. Form (complex vs sulfate) x type (Zn vs ZnCu) interactions were not detected (P > 0.35). Therefore, contrast statements were used to make the following treatment comparisons: 1) control vs supplement, 2) Zn vs ZnCu, and 3) complex vs sulfate. Ewe BW at the end of the study (P = 0.09) and ewe BW change from beginning to end of the study (P = 0.07) were greater for supplemented than control ewes. Body weight and BW change did not differ between sulfate and complex (P > 0.39) or Zn- and ZnCu- (P > 0.40) supplemented ewes. Liver Cu concentrations did not differ (P = 0.41) between control and supplemented ewes. Liver Cu concentrations were higher (P < 0.10) for ewes supplemented with ZnCu than Zn and complex than sulfate forms of supplement. Liver Zn concentration tended (P = 0.13) to be higher in ZnCu than Zn-supplemented ewes. Liver and fecal Zn concentration were higher (P < 0.06) in ewes fed complex than sulfate supplements. However, serum alkaline phosphatase activity tended (P = 0.12) to be greater in ewes fed sulfate than complex supplements. Supplementing mature ewes with complexed minerals resulted in higher concentrations of Zn and Cu in the liver. In addition, supplemental Cu tended to increase concentrations of Zn in the livers of ewes; however, high levels of supplemental Zn did not negatively impact liver Cu concentrations.

Tyr-503 of beta-galactosidase (Escherichia coli) plays an important role in degalactosylation.

Substitutions for Tyr-503 of beta-galactosidase caused large decreases of the activity. Both the galactosylation (k2) and degalactosylation (k3) rates were decreased. Substitutions by residues without transferable protons, caused k3 to decrease much more than k2 while substitutions with residues having transferable protons, caused approximately equal decreases of k2 and k3. Several lines of evidence showed this. The Km values of the substituted enzymes were much smaller than those for the wild type if the substituted amino acid residues did not have transferable protons; this was not the case when the substituted residues had transferable protons. Inhibition studies showed that the Km values were not small because of small Ks values but were small because of relatively small k3 values (compared with the k2 values). The conclusion that the k3 values are small relative to k2 upon substitution with residues without transferable protons is also based upon other studies: studies indicating that the reaction rates were similar with different substrates, studies in the presence of alcohol acceptors, studies showing that the rate of inactivation by 2,4-dinitrophenyl-2-deoxy-2-F-beta-D-galactopyranoside decreased much less than the rate of reactivation; studies on burst kinetics, and pH studies. The data suggest that Tyr-503 may be important for the degalactosylation reaction because of its ability to transfer protons and thereby facilitate cleavage of the transient covalent bond between galactose and Glu-537.

His-357 of beta-galactosidase (Escherichia coli) interacts with the C3 hydroxyl in the transition state and helps to mediate catalysis.

The His at position 357 of beta-galactosidase (Escherichia coli) was substituted by an Asp, an Asn, a Leu, and a Phe, and studies done with the substituted enzymes showed that the main role of His-357 is to stabilize the transition state by interacting with the C3 hydroxyl. The substituted enzymes were less stable to heat than was wild-type enzyme (40-90% of the activity was lost in 10 min at 52 degreesC compared to wild-type beta-galactosidase which lost no activity), but the gross physical properties of the substituted enzymes at normal temperatures were not changed. There were also no differences in the ability to bind or to be activated by Mg2+. The substitutions (except Asp) did not affect the pKa for binding substrate in the ground state, but the pKa of the kcat was altered as would be expected for a residue important for binding the transition state. Substitution by Asp may cause a conformational change at high pH values. Activation energy differences (Delta DeltaGS), as determined by differences in kcat/Km values, indicated that substitutions for His-357 caused significant destabilizations of the first transition state (for the step in which the galactoside bond is cleaved and the covalent reaction intermediate is formed). This resulted in decreases of up to 900-fold in k2 for the mononitrophenyl substrates. In contrast, the k3 values (which depend on the energy level of the second transition state) were not decreased as much (<90-fold). In some cases, the k3 values even increased (when Asn was substituted for His-357). The importance of His-357 for stabilization of the transition state was confirmed by studies with transition state analogue inhibitors that showed that His-357 forms strong specific interactions with the C3 hydroxyl of the galactose moiety of the transition state. Studies with substrate analogue inhibitors indicated that His-357 is probably not important for the binding of the substrates themselves.

Quaternary structure, Mg2+ interactions, and some kinetic properties of the beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1.

The beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks the alpha-peptide and an important alpha-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg2+ bound per monomer and Mg2+ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg2+ in the beta-galactosidase from Escherichia coli) probably accounts for the low level of Mg2+ binding and the consequent lack of response to Mg2+. Both Na+ and K+ also had no effect on the activity. The enzyme activity with o-nitrophenyl-beta-D-galactopyanoside (ONPG) was very similar to that with p-nitrophenyl-beta-D-beta-D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to the E.coli beta-galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by the T. thermosulfurigenes beta-galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of the E. coli beta-galactosidase. Trp-999 is probably of the most importance. Trp-999 of the E. coli beta-galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of the T. thermosulfurigenes beta-galactosidase is different was strengthened by competitive inhibition studies. Compared to E. coli beta-galactosidase, D-galactonolactone was a very good inhibitor of the T. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate that T. thermosulfurigenes beta-galactosidase binds the transition state differently than does E. coli beta-galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature of T. thermosulfurigenes.

The beta-galactosidase (Escherichia coli) reaction is partly facilitated by interactions of His-540 with the C6 hydroxyl of galactose.

beta-Galactosidases with substitutions for His-540 were only poorly reactive with galactosyl substrates. However, the activity with substrates that were like galactose but did not have a C6 hydroxyl group was not decreased much as a result of such substitutions. The loss of transition state stabilization for galactosyl substrates as a result of substitution was between -15.4 and -22.8 kJ/mol but only between +0.34 and -6.5 for substrates that were identical to galactose but lacked the C6 hydroxyl. These findings indicate that an important function of His-540 is to aid in the stabilization of the transition state by forming a stable interaction with the C6 hydroxyl group. This suggestion was strengthened by the results of competitive inhibition studies showing that L-arabinolactone (a transition state analog inhibitor of beta-galactosidase without a C6 hydroxymethyl group) was bound as well by the substituted enzymes as by wild type, whereas transition state analog inhibitors that contain C6 hydroxyls (L-ribose and D-galactonolactone) were bound much more poorly by the substituted enzymes than by the wild type enzyme. Substrate analog inhibitor studies showed that His-540 was also important for binding interactions with the C6 hydroxyl group of the ground (substrate) state. The activation by Mg2+ was the same for the substituted enzymes as for the wild type, and equilibrium dialysis showed that H540F-beta-galactosidase bound Mg2+ as well as did normal beta-galactosidase. The k2 and Ks values seem to have the same pH interactions as wild type enzyme, whereas the k3 interactions are affected differently by pH in the substituted enzymes than in the wild type enzyme. The rate of the "degalactosylation" reaction was affected more by substitutions for His-540 than was the rate of the "galactosylation" reaction. All three substituted beta-galactosidases were less stable to heat than was wild type, but H540N-beta-galactosidase was somewhat more stable than the other two substituted enzymes. There were some differences in activity and inhibitory properties that resulted from the different substitutions.

Glu-416 of beta-galactosidase (Escherichia coli) is a Mg2+ ligand and beta-galactosidases with substitutions for Glu-416 are inactivated, rather than activated, by MG2+.

Glu-416 of beta-galactosidase (E. coli) was replaced with Gln and Val using site-directed mutagenesis. The substituted enzymes displayed a greatly decreased sensitivity to Mg2+. Equilibrium dialysis studies indicated that wild-type beta-galactosidase bound Mg2+ tightly, whereas E416V-beta-galactosidase did not. In addition, the pH profile of E416V-beta-galactosidase was unaffected by the presence or absence of 1 mM Mg2+. Surprisingly, both substituted enzymes were inactivated, rather than activated, by Mg2+ but high amounts of Mg2+ were needed (1 mM). E416Q-beta-galactosidase was unstable when stored in the presence of Mg2+. The substituted enzymes displayed a dramatically lowered affinity for the synthetic substrate, ONPG, and for IPTG (a substrate analog inhibitor) in both the presence and the absence of Mg2+.

A rapid method for the purification of large amounts of an alpha-complementing peptide derived from beta-galactosidase (E. coli).

A DNA segment coding for residues 6-44 of beta-galactosidase was ligated to a vector with the glutathione-S-transferase gene which also contained a sequence coding for a thrombin recognition site. The fused protein, with an additional 9 amino acids coded for by the vector, was purified using a glutathione agarose affinity column. A peptide made up of residues 6-44 of beta-galactosidase and the 9 additional amino acids was then cleaved from the glutathione-S-transferase using thrombin and purified with a gel filtration column. The peptide was about 3-4 times as active for alpha-complementation as the alpha-peptide derived from CNBr digestion of wild type beta-galactosidase.

Site directed substitutions suggest that His-418 of beta-galactosidase (E. coli) is a ligand to Mg2+.

Site directed mutagenesis was used to replace His-418 of beta-galactosidase with Phe (H418F) or Glu (H418E). Kinetic analysis revealed that H418F beta-galactosidase was not significantly affected by the presence of Mg2+ whereas H418E beta-galactosidase retained its sensitivity to Mg2+. H418F had a kcat similar to that of Mg(2+)-free wild type beta-galactosidase. Its pH profile was shifted 1.0 pH unit lower on the alkaline side as compared to wild type beta-galactosidase (with Mg2+). This was similar to the shifting of the wild type beta-galactosidase pH profile when Mg2+ was absent. H418E beta-galactosidase was inactivated (rather than activated) by Mg2+ binding. Equilibrium dialysis studies indicated that H418E and wild type beta-galactosidase bind Mg2+ tightly whereas H418F does not. The results indicate that His-418 is probably a ligand to Mg2+.

Cold water survival suits for aircrew.

Laboratory and sea trials were used to compare the effectiveness of three aircrew exposure garments--the British Mark 10, the United States CWU 21/P, and the Canadian U.VIC. Thermofloat jacket. The first two are waterproof coveralls, whereas the third is a neoprene-lined jacket designed on the basis of the "wet suit" concept. Rectal and skin temperatures, electrocardiograms and other variables were measured while subjects, wearing the suits, were immersed in water at temperatures of 70 degrees C and 10.5 degrees C. The three garments were found to be similar in the degree of thermal protection provided, but the Thermofloat jacket appeared superior in other ways and has the greater potential for development. A previously unreported observation was a marked reduction in core cooling rate after the expected linear fall in core temperature. This has possible implications in the conduct of research in this field.