Effect of litter size and bacitracin administration on tissue protein synthesis of lactating rabbit does

Bacitracin is an antibiotic used in rabbit husbandry to control microbial digestive pathologies. Collateral effects on absorption and mucosal development have been reported and these may impact on protein metabolism. This study aims to analyse the effect of the antibiotic on protein synthesis in lactating does because mammary gland metabolism and milk output should provide a sensitive index of any undesirable action of bacitracin. Rates of protein synthesis were measured in mammary gland, liver, intestinal mucosa and muscle of lactating rabbits does by injecting a flooding dose of [(2)H(5)]phenylalanine into the auricular artery of two groups (each n = 8) of New Zealand White does fed different experimental diets. The control group (C) received the basal diet and the bacitracin group (B) ingested the same diet but supplemented with bacitracin (100 mg/kg). Animals received the experimental diet from day 28 of pregnancy until day 26 of lactation when they were slaughtered. Just after birth, litter size was adjusted by cross-fostering either to five or nine pups (four does per dietary treatment). The relative weight of the liver tended to be greater in those females receiving the B diet (27 v. 22.5 g/kg BW; P < 0.07), while diet did not affect mammary gland weight (255.7 ± 10.59 g). Fractional protein synthesis rate (FSR) was higher for intestinal mucosa (duodenum; 51.7% ± 2.09%/day) followed by mammary gland and liver (38.29 ± 2.62%/day and 40.2 ± 1.98%/day, respectively), and the lowest value was observed in muscle (2.92 ± 0.26%/day; P < 0.0001). Bacitracin treatment lowered FSR in the mammary gland by 23% (P = 0.024) and this was independent of litter size. Conversely, FSR in the duodenum was not affected by antibiotic treatment but reduced by 15% (P = 0.021) for the larger litter size.     

Real-time detection of common microbial volatile organic compounds from medically important fungi by Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS)

We describe a new method, Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) for the rapid and sensitive real-time detection and quantification of volatile organic compounds from medically important fungi, grown on a range of laboratory media. SIFT-MS utilises the chemical ionisation reactions of mass-selected ions to characterise volatile organic compounds (VOCs) that are produced as metabolites from fungi. This technique has the distinct advantage over others in that it readily detects low molecular weight, reactive volatiles, and allows for real-time, quantitative monitoring. The fungi examined in this study were Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Mucor racemosus, Fusarium solani, and Cryptococcus neoformans grown on or in malt extract agar, Columbia agar, Sabouraud's dextrose agar, blood agar, and brain-heart infusion broth. Common metabolites (ethanol, methanol, acetone, acetaldehyde, methanethiol, and crotonaldehyde) were detected and quantified. We found the fingerprint of volatiles, in terms of presence and quantity of volatiles to be strongly dependent on the culture medium, both in terms of variety and quantity of volatiles produced, but may form the basis for species specific identification of medically important fungi.     

Rumen microbial population dynamics in response to photoperiod

AIMS: This work was carried out to determine if there was a difference in the microbial population of the rumen associated with daylength at which sheep are housed. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) was used to study the ciliate and bacterial diversity in the rumen of Soay rams kept in long day (16 h light) or short day (8 h light) photoperiods. Bacterial diversity varied according to the daylength conditions where the host animal was housed, as did total volatile fatty acids (VFA) concentrations. No differences associated with daylength were detected in ciliate diversity, branched VFA concentrations or the ruminal ammonia concentrations. CONCLUSIONS: As diets had identical composition, yet voluntary intakes levels were higher during long days, it is proposed that the differences in bacterial populations arise because of the differences in amount of food consumed. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this study demonstrated that factors beyond dietary composition must be taken into account when trying to study microbial populations, even in what can be considered a fairly constant environment.     

Phylogenetic Differences in Content and Intensity of Periodic Proteins

Many proteins exhibit sequence periodicity, often correlated with a visible structural periodicity. The statistical significance of such periodicity can be assessed by means of a chi-squared-based test, with significance thresholds being calculated from shuffled sequences. Comparison of the complete proteomes of 45 species reveals striking differences in the proportion of periodic proteins and the intensity of the most significant periodicities. Eukaryotes tend to have a higher proportion of periodic proteins than eubacteria, which in turn tend to have more than archaea. The intensity of periodicity in the most periodic proteins is also greatest in eukaryotes. By contrast, the relatively small group of periodic proteins in archaea also tend to be weakly periodic compared to those of eukaryotes and eubacteria. Exceptions to this general rule are found in those prokaryotes with multicellular life-cycle phases, e.g., Methanosarcina sp., or Anabaena sp., which have more periodicities than prokaryotes in general, and in unicellular eukaryotes, which have fewer than multicellular eukaryotes. The distribution of significantly periodic proteins in eukaryotes is over a wide range of period lengths, whereas prokaryotic proteins typically have a more limited set of period lengths. This is further investigated by repeating the analysis on the NRL-3D database of proteins of solved structure. Some short-range periodicities are explicable in terms of basic secondary structure, e.g., alpha helices, while middle-range periodicities are frequently found to consist of known short Pfam domains, e.g., leucine-rich repeats, tetratricopeptides or armadillo domains. However, not all can be explained in this way.Reviewing Editor: Dr. John Oakeshott     

Cytochrome c551 from Starkeya novella: characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulfidophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: +133 mV (pH 6.0); +104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxX, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.     

Archaeal diversity in two thermophilic chalcopyrite bioleaching reactors

This study used a culture-independent molecular approach to investigate the archaeal community composition of thermophilic bioleaching reactors. Two culture samples, MTC-A and MTC-B, grown with different concentrations of chalcopyrite (CuFeS₂), a copper sulfidic ore, at a temperature of 78°C and pH 1.6 were studied. Phylogenetic analysis of the 16S rRNA genes revealed that both cultures consisted of Archaea belonging to the Sulfolobales . The 16S rRNA gene clone library of MTC-A grown with 4% (w/v) chalcopyrite was dominated by a unique phylotype related to Sulfolobus shibatae (69% of total clones). The remaining clones were affiliated with Stygiolobus azoricus (11%), Metallosphaera sp. J1 (8%), Acidianus infernus (2%), and a novel phylotype related to Sulfurisphaera ohwakuensis (10%). In contrast, the clones from MTC-B grown with 12% (w/v) chalcopyrite did not appear to contain Sulfolobus shibatae -like organisms. Instead the bioleaching consortium was dominated by clones related to Sulfurisphaera ohwakuensis (73.9% of total clones). The remaining microorganisms detected were similar to those found in MTC-A.     

Copper redistribution in murine macrophages in response to Salmonella infection

The movement of key transition metal ions is recognized to be of critical importance in the interaction between macrophages and intracellular pathogens. The present study investigated the role of copper in mouse macrophage responses to Salmonella enterica sv. Typhimurium. The copper chelator BCS (bathocuproinedisulfonic acid, disodium salt) increased intracellular survival of S. Typhimurium within primary mouse BMM (bone-marrow-derived macrophages) at 24 h post-infection, implying that copper contributed to effective host defence against this pathogen. Infection of BMM with S. Typhimurium or treatment with the TLR (Toll-like receptor) 4 ligand LPS (lipopolysaccharide) induced the expression of several genes encoding proteins involved in copper transport [Ctr (copper transporter) 1, Ctr2 and Atp7a (copper-transporting ATPase 1)], as well as the multi-copper oxidase Cp (caeruloplasmin). Both LPS and infection with S. Typhimurium triggered copper accumulation within punctate intracellular vesicles (copper 'hot spots') in BMM as indicated by the fluorescent reporter CS1 (copper sensor 1). These copper hot spots peaked in their accumulation at approximately 18 h post-stimulation and were dependent on copper uptake into cells. Localization studies indicated that the copper hot spots were in discrete vesicles distinct from Salmonella containing vacuoles and lysosomes. We propose that copper hot spot formation contributes to antimicrobial responses against professional intracellular bacterial pathogens.     

Intrinsic disorder in the androgen receptor: identification, characterisation and drugability

The androgen receptor (AR) regulates networks of genes in response to the steroid hormones testosterone and dihydrotestosterone. The receptor protein is made up of both stably folded globular domains, involved in hormone and DNA binding, and regions of intrinsic disorder, including the N-terminal domain (NTD). The AR-NTD has a modular activation function (termed AF1) and is important for gene regulation, participating in multiple protein-protein interactions. Biophysical studies have revealed that AR-NTD/AF1 has limited stable secondary structure and conforms to a 'collapsed disordered' conformation. The AR-NTD/AF1 has the propensity to adopt an α-helical conformation in response to a natural osmolyte or a co-regulatory binding partner. The AR is a key drug target in the management of advanced prostate cancer and recently a small molecule inhibitor was identified that interacts with the NTD/AF1 and impairs protein-protein interactions and recruitment of the receptor to target genes. In this review the role of intrinsic disorder in AR function is discussed along with the potential to develop new drugs that will target the structurally plastic NTD.     

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.