Development and validation of dry reagent time-resolved fluoroimmuno assays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 961 23\EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin a-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TR-FIAs, the results being available within 1 h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.

DNA double-strand break distributions in X-ray and alpha-particle irradiated V79 cells: Evidence for non-random breakage

Many studies have shown that with increasing LET of ionizing radiation the RBE (relative biological effectiveness) for dsb (double strand breaks) induction remains around 1.0 despite the increase in the RBE for cell killing. This has been attributed to an increase in the complexity of lesions, classified as dsb with current techniques, at multiply damaged sites. This study determines the molecular weight distributions of DNA from Chinese hamster V79 cells irradiated with X-rays or 110 keV/mu m alpha-particles. Two running conditions for pulsed-field gel-electrophoresis were chosen to give optimal separation of fragments either in the 225 kbp-5.7 Mbp range or the 0.3 kbp to 225 kbp range. Taking the total fraction of DNA migrating into the gel as a measure of fragmentation, the RBE for dsb induction was less than 1.0 for both molecular weight regions studied. The total yields of dsb were 8.2 x 10(-9) dsb/Gy/bp for X-rays and 7.8 x 10(-9) dsb/Gy/bp for a-particles, measured using a random breakage model. Analysis of the RBE of alpha-particles versus molecular weight gave a different response. In the 0.4 Mbp-57 Mbp region the RBE was less than 1.0; however, below 0.4 Mbp the RBE increased above 1.0. The frequency distributions of fragment sizes were found to differ from those predicted by a model assuming random breakage along the length of the DNA and the differences were greater for alpha-particles than for X-rays. An excess of fragments induced by a single-hit mechanism was found in the 8-300 kbp region and for X-rays and alpha-particles these corresponded to an extra 0.8 x 10(-9) and 3.4 x 10(-9) dsb/bp/Gy, respectively. Thus for every alpha-particle track that induces a dsb there is a 44% probability of inducing a second break within 300 kbp and for electron tracks the probability is 10%. This study shows that the distribution of damage from a high LET alpha-particle track is significantly different from that observed with low LET X-rays. In particular, it suggests that the fragmentation patterns of irradiated DNA may be related to the higher-order chromatin repealing structures found in intact cells.

A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage

An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA, Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA, As well as strand breaks, these sites may contain multiple damaged bases, We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH radicals with either Co-60 gamma-rays or alpha-particles from a Pu-238 source. Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1x10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp, This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For alpha-particles this increased to 9%. Given that endo III sensitive sites may only constitute similar to 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.

The relationship between the RBE of alpha particles and the radiosensitivity of different mutations of Chinese hamster cells

The RBE of alpha -particles in different mutations of Chinese hamster cells was determined with the aim of identifying differences in the sensitivity to x-ray and alpha -particle-induced DNA damage. Two parental lines of Chinese hamster cells and four radiosensitive mutants were irradiated with different single doses of x-rays and alpha -particles and clonogenic cell survival was determined. Radiosensitivity to x-rays varied by a factor of 5 between the cell strains whereas sensitivity to alpha -particle irradiation was almost identical among all strains. The RBE is only determined by the sensitivity of the cells towards x-rays. Since cells with different defects of repair or cell cycle control have different radiosensitivities, we conclude that the effects of x-ray irradiation and the RBE are mostly determined by the activity of repair processes.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal:GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.

Treatment of intestinal Behçet's syndrome with chimeric tumour necrosis factor alpha antibody

Few patients with Behçet's syndrome have gastrointestinal ulceration. Such patients are difficult to treat and have a higher mortality. Faced with refractory symptoms in two patients with intestinal Behçet's, we used the tumour necrosis factor alpha (TNF-alpha) monoclonal antibody infliximab to induce remission. Both women (one aged 27 years, the other 30 years) presented with orogenital ulceration, pustular rash, abdominal pain, bloody diarrhoea due to colonic ulceration, weight loss, and synovitis. One had thrombophlebitis, digital vasculitis, perianal fistula, and paracolic abscess; the other had conjunctivitis and an ulcer in the natal cleft. Treatment with prednisolone, methyl prednisolone, and thalidomide in one and prednisolone, colchicine, and cyclosporin in the other was ineffective. After full discussion, infliximab (3 mg/kg, dose reduced because of recent sepsis in one, and 5 mg/kg in the other) was administered. Within 10 days the ulcers healed, with resolution of bloody diarrhoea and all extraintestinal manifestations. A second infusion of infliximab was necessary eight weeks later in one case, followed by sustained (>15 months) remission on low dose thalidomide. Remission was initially sustained for 12 months in the other but thalidomide had to be stopped due to intolerance, and a good response to retreatment lasted only 12 weeks without immunosuppression, before a third infusion. The cause of Behçet's syndrome is unknown but peripheral blood CD45 gammadelta T cells in Behçet's produce >50-fold more TNF-alpha than controls when stimulated with phorbol myristate acetate and anti-CD3. Infliximab could have a role for inducing remission in Behçet's syndrome.

Effect of B-group vitamins and antioxidant vitamins on hyperhomocysteinemia:a double-blind, randomized, factorial-design, controlled trial

Mild hyperhomocysteinemia is accepted as a risk factor for premature cardiovascular disease. In a population with a high prevalence of cardiovascular disease, we screened a group of clinically healthy working men aged 30-49 y (n = 509) for plasma homocysteine and 5,10-methylene tetrahydrofolate reductase (MTHFR) genotype status. Those with mildly elevated homocysteine concentrations (> or = 8.34 micromol/L) were selected for intervention. In a randomized, factorial-design, controlled trial we assessed the effects of B-group vitamins and antioxidant vitamin supplementation on homocysteine concentrations. The 132 men were randomly assigned to one of four groups: supplementation with B-group vitamins alone (1 mg folic acid, 7.2 mg pyridoxine, and 0.02 mg cyanocobalamin), antioxidant vitamins alone (150 mg ascorbic acid, 67 mg RRR-alpha-tocopherol, and 9 mg beta-carotene), B-group vitamins with antioxidant vitamins, or placebo. Intervention was double-blind. A total of 101 men completed the 8-wk intervention. When homocysteine concentrations were analyzed by group, significant (P <0.001) decreases (32.0% and 30.0%, respectively) were observed in both groups receiving B-group vitamins either with or without antioxidants. The effect of B-group vitamins alone over 8 wk was a reduction in homocysteine concentrations of 27.9% (95% CI: 22.0%, 33.3%; P <0.001) whereas antioxidants alone produced a nonsignificant increase of 5.1% (95% CI: -2.8%, 13.6%; P = 0.21). There was no evidence of any interaction between the two groups of vitamins. The effect of B-group vitamin supplementation seemed to depend on MTHFR genotype. Supplementation with the B-group vitamins with or without antioxidants reduced homocysteine in the men with mildly elevated concentrations, and hence may be effective in reducing cardiovascular risk.

The common 'thermolabile' variant of methylene tetrahydrofolate reductase is a major determinant of mild hyperhomocysteinaemia

Mild hyperhomocysteinaemia is a major risk factor for vascular disease and neural tube defects (NTDs), conferring an approximately three-fold relative risk for each condition. It has several possible causes: heterozygosity for rare loss of function mutations in the genes for 5,10-methylene tetrahydrofolate reductase (MTHFR) or cystathionine-beta-synthase (CBS); dietary insufficiency of vitamin co-factors B6, B12 or folates; or homozygosity for a common 'thermolabile' mutation in the MTHFR gene which has also been associated with vascular disease and NTDs. We quantified the contribution of the thermolabile mutation to the hyperhomocysteinaemic phenotype in a working male population (625 individuals). Serum folate and vitamin B12 concentrations were also measured and their relationship with homocysteine status and MTHFR genotype assessed. The homozygous thermolabile genotype occurred in 48.4, 35.5, and 23.4% of the top 5, 10, and 20% of individuals (respectively) ranked by plasma homocysteine levels, compared with a frequency of 11.5% in the study population as a whole, establishing that the mutation is a major determinant of homocysteine levels at the upper end of the range. Serum folate concentrations also varied with genotype, being lowest in thermolabile homozygotes. The MTHFR thermolabile genotype should be considered when population studies are designed to determine the effective homocysteine-lowering dose of dietary folate supplements, and when prophylactic doses of folate are recommended for individuals.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1) . The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

Selective Hydrogenation of alpha,beta-Unsaturated Aldehydes and Ketones using Novel Manganese Oxide and Platinum Supported on Manganese Oxide Octahedral Molecular Sieves as Catalysts

The selective hydrogenation of ,-unsaturated aldehydes and ketones has been studied using ketoisophorone and cinnamaldehyde as model substrates using manganese oxide octahedral molecular sieve (OMS-2) based catalysts. For the first time, OMS-2 has been shown to be an efficient and selective hydrogenation catalyst. High selectivities for either the CC or CO double bond at approximate to 100% conversion were achieved by using OMS-2 and platinum supported on OMS-2 catalysts. Density functional theory (DFT) calculations showed that the dissociation of H2 on OMS-2 was water assisted and occurred on the surface Mn of OMS-2(001) that had been modified by an adsorbed H2O molecule. The theoretically calculated activation barrier was in good agreement with the experimentally determined value for the hydrogenation reactions, indicating that H2 dissociation on OMS-2 is likely to be the rate-determining step. A significant increase in the rate of reaction was observed in the presence of Pt as a result of the enhancement of H2 dissociative adsorption and subsequent reaction on the Pt or spillover of the hydrogen to the OMS-2 support. The relative adsorption strengths of ketoisophorone and cinnamaldehyde on the OMS-2 support compared with the Pt were found to determine the product selectivity.

Community-living nonagenarians in northern ireland have lower plasma homocysteine but similar methylenetetrahydrofolate reductase thermolabile genotype prevalence compared to 70-89-year-old subjects

This cross-sectional study assessed relationships between plasma homocysteine, 'thermolabile' methylenetetrahydrofolatereductase (MTHFR) genotype, B vitamin status and measures of renal function in elderly (70-89 years) and nonagenarian (90+ years) subjects, with the hypothesis that octo/nonagenarian subjects who remain healthy into old age as defined by 'Senieur' status might show reduced genetic or environmental risk factors usually associated with hyperhomocysteinaemia. Plasma homocysteine was 9.1 micromol/l (geometric mean [GM]) for all elderly subjects. Intriguingly, homocysteine was significantly lower in 90+ (GM; 8.2 micromol/l) compared to 70-89-year-old subjects (GM; 9.8 micromol/l) despite significantly lower glomerular filtration rate (GFR) and serum B12 in nonagenarian subjects and comparable MTHFR thermolabile (TT) genotype frequency, folate and B6 status to 70-89-year-olds. For all elderly subjects, the odds ratio and 95% confidence intervals for plasma homocysteine being in the highest versus lowest quartile was 4.27 (2.04-8.92) for age 90 years, 3.4 (1.5-7.8) for serum folate 10.7nmol/l, 3.0 (0.9-10.2) for creatinine >140 compared

CD69, CD25, and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNF-alpha, IFN-gamma, and sIL-2R levels in aging

Aging is associated with changes in lymphocyte subsets and unexplained HLA-DR upregulation on T-lymphocytes. We further investigated this activation, by measuring early (CD69), middle (CD25), and late (HLA-DR) T-lymphocyte activation markers on CD3+ lymphocytes, across subjects (20-100 years) together with serum tumor necrosis factor (TNF-alpha), interferon-gamma (IFN-gamma), and soluble interleukin-2 receptor (sIL-2R). HLA-DR was present as a CD3+ HLA-DR+ subset that constituted 8% of total lymphocytes, increased twofold with age and included CD4+, CD8+, and CD45RA+ phenotypes. HLA-DR was also expressed on a CD8+ CD57+ subset. The CD3+ CD25+ subset constituted 13% of lymphocytes, fell with age but was weakly associated with the CD3+ HLA-DR+ subset especially in older subjects. A small 3-5% CD3+ CD69+ subsets showed no age effect. Serum sIL-2R, TNF-alpha, but not IFN-gamma, were associated with CD3+ HLA-DR+ lymphocytes, TNF-alpha with CD8+ CD57+ count and sIL-2R and IFN-gamma with the CD3+ CD25+/CD3+ CD4+ ratio. The study confirms age-related upregulation of HLA-DR on CD3+ lymphocytes, shows some evidence for associated upregulation of CD25 on CD3+ cells in older subjects, and links serum TNF-alpha, IFN-gamma, and sIL2-R to T-lymphocyte activation.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-[alpha] 1-antichymotrypsin detection based on surface plasmon resonance

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-a1-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.