1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

Marine Biodiversity in South Africa: An Evaluation of Current States of Knowledge

Continental South Africa has a coastline of some 3,650 km and an Exclusive Economic Zone (EEZ) of just over 1 million km². Waters in the EEZ extend to a depth of 5,700 m, with more than 65% deeper than 2,000 m. Despite its status as a developing nation, South Africa has a relatively strong history of marine taxonomic research and maintains comprehensive and well-curated museum collections totaling over 291,000 records. Over 3 million locality records from more than 23,000 species have been lodged in the regional AfrOBIS (African Ocean Biogeographic Information System) data center (which stores data from a wider African region). A large number of regional guides to the marine fauna and flora are also available and are listed.The currently recorded marine biota of South Africa numbers at least 12,914 species, although many taxa, particularly those of small body size, remain poorly documented. The coastal zone is relatively well sampled with some 2,500 samples of benthic invertebrate communities have been taken by grab, dredge, or trawl. Almost none of these samples, however, were collected after 1980, and over 99% of existing samples are from depths shallower than 1,000 m—indeed 83% are from less than 100 m. The abyssal zone thus remains almost completely unexplored.South Africa has a fairly large industrial fishing industry, of which the largest fisheries are the pelagic (pilchard and anchovy) and demersal (hake) sectors, both focused on the west and south coasts. The east coast has fewer, smaller commercial fisheries, but a high coastal population density, resulting in intense exploitation of inshore resources by recreational and subsistence fishers, and this has resulted in the overexploitation of many coastal fish and invertebrate stocks. South Africa has a small aquaculture industry rearing mussels, oysters, prawns, and abalone—the latter two in land-based facilities.Compared with many other developing countries, South Africa has a well-conserved coastline, 23% of which is under formal protection, however deeper waters are almost entirely excluded from conservation areas. Marine pollution is confined mainly to the densely populated KwaZulu-Natal coast and the urban centers of Cape Town and Port Elizabeth. Over 120 introduced or cryptogenic marine species have been recorded, but most of these are confined to the few harbors and sheltered sites along the coast.     

Bacillus Probiotic Supplementations Improve Laying Performance,Egg Quality,Hatching of Laying Hens,and Sperm Quality of Roosters

Probiotics and Antimicrobial Proteins - The study aims at elucidating the effect of bacilli probiotic preparations on the physiology of laying hens and roosters. Probiotic formulations were...     

Methane emission,digestive characteristics and faecal archaeol in heifers fed diets based on silage from brown midrib maize as compared to conventional maize

The aim of the present experiment was to compare silage prepared from maize having a brown midrib (BMR) mutation with control (CTR) maize to identify their effects on enteric methane emission, digesta mean retention time (MRT), ruminal fermentation and digestibility. In addition, the utility of archaeol present in faecal samples was validated as a proxy for methane production. Seven German Holstein heifers were fed total mixed rations with a maize-silage proportion (either BMR or CTR) of 920 g/kg dry matter (DM) in a change-over design. Heifers were fed boluses with markers to measure MRT; faeces were collected for 7 days and rumen fluid was collected on the penultimate day. Methane emission was measured in respiration chambers on one day. Data were analysed by t-test and regression analysis. DM intake did not differ between the two diets. The apparent digestibility of DM and most nutrients was unaffected by diet type, but apparent digestibility of neutral and acid detergent-fibre was higher in those heifers fed BMR than in those fed CTR. Comparisons between diets revealed no difference in particle or solute MRT in the gastro-intestinal tract and the reticulorumen. Concentrations of short-chain fatty acid and ammonia in rumen fluid and its pH were not affected by silage type. Independent of the mode of expression [l/d, l/kg DM intake, l/kg digested organic matter], methane emissions were not affected by maize-silage type, but with BMR, there was a trend towards lower methane production per unit of digested neutral detergent fibre than there was with CTR silage. Results of the present study show that feeding heifers BMR silage does not increase methane emissions despite a higher fibre digestibility as compared to CTR silage. Therefore, it is assumed that improvements in animal productivity achieved by feeding BMR silage, as some studies have reported, can be obtained without extra environmental cost per unit of milk or meat. Neither faecal archaeol content [µg/g] nor daily amount excreted [mg/d] is suitable to predict methane production in absolute terms [l per day]. However, faecal archaeol content has a certain potential for predicting the methane yield [l per kg DM intake] of individual animals.     

Metabolomic Derangements Are Associated with Mortality in Critically Ill Adult Patients

Objective

To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults.

Rationale

Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible.

Methods

We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study.

Results

We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations).

Conclusion

Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.     

The β1a Subunit of the Skeletal DHPR Binds to Skeletal RyR1 and Activates the Channel via Its 35-Residue C-Terminal Tail

Although it has been suggested that the C-terminal tail of the β_1a subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca²⁺ release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β_1a bound to RyR1 in affinity chromatography. The full-length β_1a subunit and the C-terminal peptide increased [³H]ryanodine binding and RyR1 channel activity with an AC₅₀ of 450–600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca²⁺, ATP, and Mg²⁺ concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg²⁺ inhibition or addition of 100 nM Ca²⁺ (without ATP). Maximum increases were seen with 1–10 μM Ca²⁺, in the absence of Mg²⁺ inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [³H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β_1a subunit and RyR1 may support an in vivo function of β_1a during voltage-activated Ca²⁺ release.     

Chronic hyperhomocysteinemia alters antioxidant defenses and increases DNA damage in brain and blood of rats: protective effect of folic acid

We have previously demonstrated that acute hyperhomocysteinemia induces oxidative stress in rat brain. In the present study, we initially investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative damage, namely total radical-trapping antioxidant potential and activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), as well as on DNA damage in parietal cortex and blood of rats. We also evaluated the effect of folic acid on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of Hcy (0.3-0.6 micromol/g body weight), and/or folic acid (0.011 micromol/g body weight) from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, parietal cortex and total blood was collected. Results showed that chronic homocysteine administration increased DNA damage, evaluated by comet assay, and disrupted antioxidant defenses (enzymatic and non-enzymatic) in parietal cortex and blood/plasma. Folic acid concurrent administration prevented homocysteine effects, possibly by its antioxidant and DNA stability maintenance properties. If confirmed in human beings, our results could propose that the supplementation of folic acid can be used as an adjuvant therapy in disorders that accumulate homocysteine.     

Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO₂ chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide ⁹⁰VSELpSFR⁹⁶ (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1_P216, F2_P216, F3_P216) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1_P216, F2_P216 and F3_P216 adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae . Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.