Comparison of a novel form of zinc with zinc oxide bolus licensed for prevention of facial eczema, and interaction with copper oxide in sheep.

To evaluate a novel elemental zinc bolus compared with a registered positive control zinc oxide bolus and assess serum zinc concentrations following concomitant treatment with a capsule containing copper oxide needles. Forty Romney-cross ewes were randomly allocated in a 2 × 2 factorial design study. On Day 0, 20 ewes received novel boluses containing elemental zinc (Investigational Veterinary Product, IVP) while 20 received a zinc oxide bolus (control; CP). Half the animals in each zinc treatment group (n = 10) were treated with a copper oxide needle capsule [Copasure® - Ewe]. Weekly, from Day -7 to 56, all ewes were assessed for signs of photosensitization, and for 10 ewes from each zinc treatment groups, samples were collected for analysis of serum GGT activity, serum zinc concentrations, faecal zinc concentrations and on Days -7 and 56, liver copper concentrations. Multivariable random-effects models assessed the effects of zinc treatment, copper treatment, treatment interactions and time on all analytes. Regression models examined associations between serum and faecal zinc concentrations and GGT activity. Low spore numbers indicated low Pithomyces chartarum challenge. Serum zinc levels were significantly higher in the IVP than in the CP group [p < 0.0001] and varied by time [p < 0.001] and positively associated with faecal zinc concentration [p < 0.001]. Copper treatment did not affect serum zinc [p = 0.82] or faecal zinc [p = 0.92] concentrations. Liver copper concentrations did not differ between zinc treatment groups on Day -7 [p = 0.6] or Day 56 [p = 0.95]. Only the CP/no copper group had no increase in liver copper concentrations.

The effect of lipoic acid and vitamin E therapies in individuals with the metabolic syndrome.

The metabolic syndrome is associated with abnormal glucose and lipid metabolism, insulin resistance, increased oxidative stress and pro-inflammatory activity that increase the risk of type 2 diabetes and cardiovascular disease. The aim of this study was to investigate the effect of treatment with the antioxidant α-lipoic acid (ALA) with or without vitamin E supplementation, on markers of insulin resistance and systemic inflammation and plasma nonesterified fatty acid (NEFA) concentrations in individuals with the metabolic syndrome. In a randomized, double-blind, placebo-controlled trial, subjects with the metabolic syndrome received ALA (600 mg/day, n = 34), vitamin E (100 IU/day, n = 36), both ALA and vitamin E (n = 41), or matching placebo (n = 40) for 1 year. Fasting circulating concentrations of glucose and insulin were measure every 3 months and NEFA, markers of inflammation, adiponectin and vitamin E were measured at 6 monthly intervals. Plasma NEFA concentrations decreased [-10 (-18, 0)%] at a marginal level of significance (p = 0.05) in those who received ALA alone compared with placebo and decreased [-8 (-14, -1)% (95% CI)] significantly (P = 0.02) in participants who were randomised to ALA with and without vitamin E compared with those who did not receive ALA. Fasting glucose, insulin, homeostatic model assessment of insulin resistance, adiponectin, and markers of inflammation did not change significantly during the study. These data suggest that prolonged treatment with ALA may modestly reduce plasma NEFA concentrations but does not alter insulin or glucose levels in individuals with the metabolic syndrome.

A descriptive review of the peer and non-peer reviewed literature on the treatment and prevention of foot lameness in cattle published between 2000 and 2011.

The aim of this study was to collate and review the peer and non-peer reviewed English language literature on the treatment and prevention of foot lameness in cattle published since January 2000. The study aimed to identify deficits in knowledge and areas of disparity between what is recommended in the field by veterinarians, foot trimmers and advisors and what has been substantiated experimentally. Peer reviewed literature containing original work was gathered by searching three databases. Papers were categorised and reviewed if they contained material on treatment or prevention. Non-peer reviewed clinical materials were collated from a range of sources. The materials were reviewed and categorised based on whether they recommended a range of possible treatment and prevention strategies. The peer reviewed data base contained 591 papers, of which 286 contained information on treatment or prevention. The vast majority of papers (258) concerned prevention; only a small number covered treatment (31) and of these only three contained information on the treatment of sole ulcers or white line disease. The number of intervention studies and trials was low; most papers on prevention were observational. Generally, lesion specific outcomes were not described making the findings of these papers difficult to use clinically. The non-peer reviewed material contained 46 sources; they varied significantly in regard to the treatments they advocated with some texts directly contradicting each other. Some aspects of prevention recommended in these sources seemed poorly supported by findings from the research literature. Well designed intervention studies are required to address these deficits.

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced Rhodobacter capsulatus cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske iron-sulfur protein.

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 microM) but binds much more weakly to the oxidized form (Kd = 3.1 microM). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 microM. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 microM) and reduced cytochrome c2 binds less strongly (Kd = 0.11 microM) but approximately 30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.

Effect of an intramammary teat seal and dry cow antibiotic in relation to dry period length on postpartum mastitis.

Infusion of either a long-acting antibiotic preparation (cefalonium) or the same antibiotic preparation combined with an internal teat sealant (bismuth subnitrite) were compared for the effect on new intramammary infections at calving and clinical mastitis in the first 100 d of lactation, in relation to dry period length. For all cows, a significant reduction in the incidence of new infections in quarters at calving (3.7 vs. 7.3%) was found for the combination treatment group (150 cows) compared with the antibiotic-alone treatment (133 cows). With a dry period of 10 wk or longer, significantly fewer new quarter infections (3.8 vs. 11.4%) were found in those cows receiving the combination treatment compared with antibiotic treatment alone. When the dry period was less than 10 wk, the incidence of new infections in quarters treated with the combination treatment was lower than for the antibiotic treatment alone (3.7 vs. 6%) but this was not a statistically significant difference. Fewer infections caused by Streptococcus uberis and coagulase-negative staphylococci were found in cows receiving the combination treatment compared with the antibiotic treatment alone (not significant). Coliform isolates were less likely in cows receiving the combination treatment with the longer dry period but the numbers of new intramammary coliform infections were low for both dry period categories. Few infections were caused by coagulase-negative staphylococci. The incidence of clinical mastitis in the first 100 d of lactation in quarters infected at calving was significantly lower (4 vs. 15 cases) for the combination treatment than for the antibiotic treatment alone for both dry period lengths. The clinical incidence in quarters in which a pathogen was not detected in either of the samples taken after calving was comparable between groups. No significant difference was found in the total clinical incidence after calving for both groups irrespective of dry period length.

Errors associated with milk cell counting.

The accuracy of somatic cell counts in milk samples was investigated in four studies. First, the counts recorded by one milk buyer in one supply over six months ranged from 105,000 to 401,000 cells/ml with no apparent changes in the volume of milk consigned or the level of mastitis in the herd that would explain this wide range. Secondly, the counts in daily samples from one bulk milk supply for 28 days ranged from 84,000 to 282,000 cells/ml, again with no apparent changes in the performance of the herd to explain the wide range. Thirdly, the replicated counts recorded for one sample by three separate laboratories agreed closely; however, when a sample with a high cell count was interspersed then two of the three laboratories reported high cell counts suggestive of 'carry-over' in excess of the 2 per cent 'allowable' Finally, cell count data from three separate laboratories on samples from 21 cows for 33 days revealed problems with the misidentification of samples on the farm in 1 per cent of the samples, and misidentification and mishandling of 1 to 2.6 per cent of the samples in the laboratories. All three laboratories differentiated samples from cows with subclinical and clinical mastitis, but the mean cell count of the uninfected cows varied between the laboratories with one of them recording statistically significantly higher counts over the period.

Amphipols: polymeric surfactants for membrane biology research.

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

Prophylactic effects of two selective dry cow strategies accounting for interdependence of quarter.

Infusion of a long-acting antibiotic preparation at drying off in dairy cows as a prophylactic therapy is usually recommended for all quarters where it is in use. Studying the effectiveness of such treatment using quarter as the unit of analysis assumes that each quarter within a cow has a risk of being infected independent of the other quarters of the cow. Failure to account for interdependence of quarters within a cow may lead to inaccurate variance estimates and errors in assessing treatment effects. Data from two trials assessing different dry-cow strategies were examined for interdependence of infection between quarters. Logistic regression with a variance inflation factor or a multilevel analysis was used to assess the effect of antibiotic and internal teat-sealant dry cow strategies. Parity and infection status at drying off were covariates in the analysis. Interdependence of the risk of quarter infections within control-group cows was demonstrated in both dry-cow antibiotic and teat-seal trials. However, cows that received either of these treatments did not demonstrate interdependence. Treated quarters in both trials were 3.0 times less likely to acquire a new infection at calving compared with the untreated controls. Quarters in cows of parity 3 or greater were also at an increased risk in the antibiotic treatment trial. In both trials, quarters with either Corynebacterium spp. or coagulase-negative staphylococci infections at drying off had an increased risk of a new intramammary infection at calving. This study has demonstrated the beneficial and comparable effects of antibiotic and teat seal dry cow strategies; both decreased the risk of intramammary infection at calving. The application of dry-cow strategies at the cow level and not the quarter level is also supported.

The effect of an intramammary teat seal on new intramammary infections.

As concern over the possible overuse of antibacterials increases, attention has focused on reduction of antibiotic usage and on nonantibiotic alternatives. A nonantibiotic intramammary teat sealant, Teat Seal (Cross Vetpharm Group Ltd., Tallaght, Dublin, Ireland), has been available in Ireland, in combination with an intramammary tube of cloxacillin. Teat Seal has been reformulated for use in cows with low cell counts as an alternative to antibiotic dry cow therapy at the end of lactation. The product is now marketed as Orbeseal (Pfizer Animal Health). A comparison between this teat sealant and no treatment was made on new intramammary infections and clinical mastitis, on all cows within four herds, and on low cell count cows in three herds. No cases of clinical mastitis in the dry period were observed in cows treated with Teat Seal (n = 197), whereas a significant number (6 cows) were observed in the untreated cows (n = 204). In all herds, significantly more new infections at calving were found in the untreated group (62 cows in the untreated group compared with 21 cows in the Teat Seal group). In those quarters where infections were first detected at calving, the incidence of clinical mastitis was significantly greater in the untreated group. Quarters in both treatment groups that were infected at drying off with Corynebacterium spp. or coagulase-negative staphylococci were not protected against new infections and had an increased risk of new infection by Streptococcus uberis. The results will inform those restricting their use of antibiotic dry cow therapy in alternative management strategies and the additional risk of new intramammary infection.

The effect of selective dry cow treatment on new intramammary infections.

Dry cow therapy, or antibiotic treatment at end of lactation, is used to eliminate intramammary infections and prevent new infections during the dry period. It is one part of a total management system recommended in controlling intramammary infections in the dairy cow. Public health concerns advise prudent use of antibiotics, as their use may promote bacterial antibiotic resistance and leave antibiotic residues in the food chain. The effects of dry cow treatment and no treatment were compared, on new intramammary infections and clinical mastitis within two low cell count herds and two herds undergoing conversion to organic farming. The results will inform those restricting their use of dry cow therapy on the additional risk of new intramammary infection and aid in development of alternative management strategies. No cases of clinical mastitis in the dry period were observed in treated cows, whereas in the untreated groups a significant number were observed. Significantly more new infections at calving were found in the untreated group in all herds. In those quarters where infections were first detected at calving, the incidence of clinical mastitis was significantly greater in the untreated group in all herds. Clinical mastitis detection was significantly lower in organic herds. Untreated quarters infected at drying with Corynebacterium spp. or coagulase-negative staphylococci were found to have an increased risk of new infection by Streptococcus uberis or coliform bacteria. It can be concluded that dry cow therapy continues to lower significantly the rate of new dry period intramammary infection in herds with elevated somatic cell counts and a high prevalence of infection.

The 2.6 A resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic 'special position'.

Bacterioferritin from Rhodobacter capsulatus was crystallized and its structure was solved at 2.6 A resolution. This first structure of a bacterioferritin from a photosynthetic organism is a spherical particle of 24 subunits displaying 432 point-group symmetry like ferritin and bacterioferritin from Escherichia coli. Crystallized in the I422 space group, its structural analysis reveals for the first time the non-symmetric heme molecule located on a twofold crystallographic symmetry axis. Other hemes of the protomer are situated on twofold noncrystallographic axes. Apparently, both types of sites bind heme in two orientations, leading to an average structure consisting of a symmetric 50:50 mixture, thus satisfying the crystallographic and noncrystallographic symmetry of the crystal. Five water molecules are situated close to the heme, which is bound in a hydrophobic pocket and axially coordinated by two crystallographic or noncrystallographically related methionine residues. Its ferroxidase center, in which Fe(II) is oxidized to Fe(III), is empty or fractionally occupied by a metal ion. Two positions are observed for the coordinating Glu18 side chain instead of one in the E. coli enzyme in which the site is occupied. This result suggests that the orientation of the Glu18 side chain could be constrained by this interaction.

Crystal structure of activated CheY. Comparison with other activated receiver domains.

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.

Crystal structure of an activated response regulator bound to its target.

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

Crystallographic location of two Zn(2+)-binding sites in the avian cytochrome bc(1) complex.

The chicken mitochondrial ubiquinol cytochrome c oxidoreductase (bc(1) complex) is inhibited by Zn(2+) ions, but with higher K(i) ( approximately 3 microM) than the corresponding bovine enzyme. When equilibrated with mother liquor containing 200 microM ZnCl(2) for 7 days, the crystalline chicken bc(1) complex specifically binds Zn(2+) at 4 sites representing two sites on each monomer in the dimer. These two sites are close to the stigmatellin-binding site, taken to be center Q(o) of the Q-cycle mechanism, and are candidates for the inhibitory site. One binding site is actually in the hydrophobic channel between the Q(o) site and the bulk lipid phase, and may interfere with quinone binding. The other is in a hydrophilic area between cytochromes b and c(1), and might interfere with the egress of protons from the Q(o) site to the intermembrane aqueous medium. No zinc was bound near the putative proteolytic active site of subunits 1 and 2 (homologous to mitochondrial processing peptidase) under these conditions.

Structure and function of cytochrome bc complexes.

The cytochrome bc complexes represent a phylogenetically diverse group of complexes of electron-transferring membrane proteins, most familiarly represented by the mitochondrial and bacterial bc1 complexes and the chloroplast and cyanobacterial b6f complex. All these complexes couple electron transfer to proton translocation across a closed lipid bilayer membrane, conserving the free energy released by the oxidation-reduction process in the form of an electrochemical proton gradient across the membrane. Recent exciting developments include the application of site-directed mutagenesis to define the role of conserved residues, and the emergence over the past five years of X-ray structures for several mitochondrial complexes, and for two important domains of the b6f complex.

X-ray structure of a truncated form of cytochrome f from chlamydomonas reinhardtii.

A truncated form of cytochrome f from Chlamydomonas reinhardtii (an important eukaryotic model organism for photosynthetic electron transfer studies) has been crystallized (space group P2(1)2(1)2(1); three molecules/asymmetric unit) and its structure determined to 2.0 A resolution by molecular replacement using the coordinates of a truncated turnip cytochrome f as a model. The structure displays the same folding and detailed features as turnip cytochrome f, including (a) an unusual heme Fe ligation by the alpha-amino group of tyrosine 1, (b) a cluster of lysine residues (proposed docking site of plastocyanin), and (c) the presence of a chain of seven water molecules bound to conserved residues and extending between the heme pocket and K58 and K66 at the lysine cluster. For this array of waters, we propose a structural role. Two cytochrome f molecules are related by a noncrystallographic symmetry operator which is a distorted proper 2-fold rotation. This may represent the dimeric relation of the monomers in situ; however, the heme orientation suggested by this model is not consistent with previous EPR measurements on oriented membranes.

Characterization of cytochrome b from Toxoplasma gondii and Q(o) domain mutations as a mechanism of atovaquone-resistance.

Atovaquone is active in vitro against the tachyzoites of Toxoplasma gondii at nanomolar concentrations and is used clinically to treat acute cases of human toxoplasmosis. In pursuit of the mechanism of action of atovaquone against T. gondii and to understand how resistance might arise, drug-resistant mutants were generated and examined. The previously uncloned cytochrome b gene of T. gondii was cloned and sequenced from wild type and resistant strains as this was a likely candidate for the target of the drug and thus a source of resistance. Mutations are present within the cytochrome b gene of atovaquone-resistant parasites (M129L and I254L) and represent alterations in two different regions of the ubiquinol-binding pocket (Q(o) domain) of cytochrome b, suggesting that atovaquone interferes with electron transport at the cytochrome bc(1) complex in T. gondii. A structural model for how this hydroxynaphthoquinone is binding within the Q(o) domain is presented. Further analysis of the cytochrome b gene suggested that the protein may differ from other homologues by terminating within the mitochondrial membrane. Cytochrome b becomes the first complete mitochondrial gene and cognate protein to be described for T. gondii.