Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/deltaF508-CFTR to the plasma membrane

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.     

Assay of the concentration and (13)C isotopic enrichment of propionyl-CoA,methylmalonyl-CoA,and succinyl-CoA by gas chromatography-mass spectrometry

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.     

Models for predicting enteric methane emissions from dairy cows in North America,Europe, and Australia and New Zealand

There are several models in the literature for predicting enteric methane (CH₄) emissions. These models were often developed on region or country‐specific data and may not be able to predict the emissions successfully in every region. The majority of extant models require dry matter intake (DMI) of individual animals, which is not routinely measured. The objectives of this study were to (i) evaluate performance of extant models in predicting enteric CH₄ emissions from dairy cows in North America (NA), Europe (EU), and Australia and New Zealand (AUNZ) and (ii) explore the performance using estimated DMI. Forty extant models were challenged on 55, 105, and 52 enteric CH₄ measurements (g per lactating cow per day) from NA, EU, and AUNZ, respectively. The models were ranked using root mean square prediction error as a percentage of the average observed value (RMSPE) and concordance correlation coefficient (CCC). A modified model of Nielsen et al. (Acta Agriculturae Scand Section A, 63 , 2013 and 126) using DMI, and dietary digestible neutral detergent fiber and fatty acid contents as predictor variables, were ranked highest in NA (RMSPE = 13.1% and CCC = 0.78). The gross energy intake‐based model of Yan et al. (Livestock Production Science, 64 , 2000 and 253) and the updated IPCC Tier 2 model were ranked highest in EU (RMSPE = 11.0% and CCC = 0.66) and AUNZ (RMSPE = 15.6% and CCC = 0.75), respectively. DMI of cows in NA and EU was estimated satisfactorily with body weight and fat‐corrected milk yield data (RMSPE < 12.0% and CCC > 0.60). Using estimated DMI, the Nielsen et al. (2013) (RMSPE = 12.7 and CCC = 0.79) and Yan et al. (2000) (RMSPE = 13.7 and CCC = 0.50) models still predicted emissions in respective regions well. Enteric CH₄ emissions from dairy cows can be predicted successfully (i.e., RMSPE < 15%), if DMI can be estimated with reasonable accuracy (i.e., RMSPE < 10%).     

Modelling mammary metabolism in the dairy cow to predict milk constituent yield, with emphasis on amino acid metabolism and milk protein production: model construction

Previous efforts to simulate mammary metabolism have focused on energy, mostly considering amino acids (AA) in aggregate. The main objective of this work was to build a model of mammary metabolism, based on data from arterio-venous difference studies, which considered AA in sufficient detail to predict yields of milk solids. The model contains 19 state variables and considers the removal of 37 metabolites from blood, including 22 AA. It is driven by blood flow and arterial concentrations, and outputs include milk protein, milk lactose, and three classes of milk fat (by chain length). The model was parameterized using a balance version of it and the mean observations from four arterio-venous difference experiments, with a limited number of assumptions, and evaluated against these experiments. In assembling the balance model, milk protein output was not predicted satisfactorily, as some essential AA were not present in quantities great enough to support the rates of milk protein synthesis observed experimentally. Tryptophan showed the greatest deficit, followed by tyrosine plus phenylalanine, methionine, and histidine. In addition, significant quantities of pyruvate were needed to synthesize serine, glycine, and alanine. The supply of alpha-ketoglutarate plus glutamate to synthesize proline and glutamine was provided in part by catabolism of arginine; the remainder was derived from catabolism of other AA and energetic substrates.     

Hydrogen peroxide production and myo‐inositol metabolism as important traits for virulence of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia. In our previous work, we reconstructed the metabolic models of this species along with two other mycoplasmas from the respiratory tract of swine: Mycoplasma hyorhinis, considered less pathogenic but which nonetheless causes disease and Mycoplasma flocculare, a commensal bacterium. We identified metabolic differences that partially explained their different levels of pathogenicity. One important trait was the production of hydrogen peroxide from the glycerol metabolism only in the pathogenic species. Another important feature was a pathway for the metabolism of myo‐inositol in M. hyopneumoniae. Here, we tested these traits to understand their relation to the different levels of pathogenicity, comparing not only the species but also pathogenic and attenuated strains of M. hyopneumoniae. Regarding the myo‐inositol metabolism, we show that only M. hyopneumoniae assimilated this carbohydrate and remained viable when myo‐inositol was the primary energy source. Strikingly, only the two pathogenic strains of M. hyopneumoniae produced hydrogen peroxide in complex medium. We also show that this production was dependent on the presence of glycerol. Although further functional tests are needed, we present in this work two interesting metabolic traits of M. hyopneumoniae that might be directly related to its enhanced virulence.     

PCR-RFLP of ITS rDNA for the rapid identification of Penicillium subgenus Biverticillium species

RFLP of ITS rDNA is proposed as a useful tool for molecular identification of the most common species of biverticillate penicillia. 60 isolates were analysed representing 13 species and 21 unique sequences were produced. The combination of five restriction enzymes was successful in separating 12 species. However, the variety Penicillium purpurogenum var. rubrisclerotium remained indistinguishable from Penicillium funiculosum. P. funiculosum appeared as the most confused species, being mis-identified with Penicillium miniolutum and Penicillium pinophilum, which were originally part of the species, and with P. purpurogenum perhaps because of the common production of red pigment. Penicillium variabile was difficult to investigate as introns were found on half of the isolates. Penicillium piceum, Penicillium rugulosum, Penicillium loliense, Penicillium erythromellis and P. purpurogenum were homogeneous from molecular and morphological positions and corresponded to a well circumscribed taxon. Furthermore, intraspecific variability was evidenced within P. pinophilum and P. funiculosum. The ex-type isolate of P. funiculosum produced a unique pattern. The method is sensitive, rapid and inexpensive and can be used for isolate identification of the biverticillate species. It is recommended particularly when many isolates have to be authentificated prior to analysis for phylogenetic assessment or population genetics.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.