Urinary metabolomics fingerprinting around parturition identifies metabolites that differentiate lame dairy cows from healthy ones.

Lameness is a very important disorder of periparturient dairy cows with implications on milk production and composition as well as with consequences on reproductive performance. The aetiology of lameness is not clear although there have been various hypotheses suggested over the years. The objective of this study was to metabotype the urine of dairy cows prior to, during and after the onset of lameness by evaluating at weeks -8, -4 pre-calving, the week of lameness diagnosis, and +4 and +8 weeks post-calving. We used a metabolomics approach to analyse urine samples collected from dairy cows around calving (6 cows with lameness v. 20 healthy control cows). A total of 153 metabolites were identified and quantified using an in-house MS library and classified into 6 groups including: 11 amino acids (AAs), 39 acylcarnitines (ACs), 3 biogenic amines (BAs), 84 glycerophospholipids, 15 sphingolipids and hexose. A total of 23, 36, 40, 23 and 49 metabolites were observed to be significantly different between the lame and healthy cows at -8 and -4 weeks pre-calving, week of lameness diagnosis as well as at +4 and +8 weeks post-calving, respectively. It should be noted that most of the identified metabolites were elevated; however, a few of them were also lower in lame cows. Overall, ACs and glycerophospholipids, specifically phosphatidylcholines (PCs), were the metabolite groups displaying the strongest differences in the urine of pre-lame and lame cows. Lysophosphatidylcholines (LysoPCs), although to a lesser extent than PCs, were altered at all time points. Alterations in urinary AA concentrations were also observed during the current study for four time points. During the pre-calving period, there was an observed elevation of arginine (-8 week), tyrosine (-8 week) and aspartate (-4 week), as well as a depression of urinary glutamate (-4 weeks). In the current study, it was additionally observed that concentrations of several sphingomyelins and one BA were altered in pre-lame and lame cows. Symmetric dimethylarginine was elevated at both -8 weeks pre-calving and the week of lameness diagnosis. Data showed that urinary fingerprinting might be a reliable methodology to be used in the future to differentiate lame cows from healthy ones.

An assessment of the Pictorial Test of Intelligence for use with young cerebral-palsied children.

In order to determine the concurrent and predictive validity of the Pictorial Test of Intelligence for use with cerebral-palsied children, this test was administered to 46 cerebral-palsied children aged between four and seven years, together with the Columbia Mental Maturity Scale and Peabody Picture Vocabulary Test. The results, compared with a classroom Achievement Rating Scale, showed the Pictorial Test of Intelligence to be superior to the other two tests as a predictor of classroom achievement of the children in this study.

Purification of glutaryl-CoA dehydrogenase from Pseudomonas sp., an enzyme involved in the anaerobic degradation of benzoate.

Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anaerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of glutaryl-CoA. The green enzymes are homotetramers (m = 170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%).

Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans.

Glutaconate coenzyme A-transferase (Gct) from Acidaminococcus fermentans consists of two subunits (GctA, 35725 Da and GctB, 29168 Da). The N-termini sequences of both subunits were determined. DNA sequencing of a subgenomic fragment of A. fermentans revealed that the genes encoding glutaconate CoA-transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further upstream of gctA, a DNA sequence was detected showing significant similarities to sigma 70-type promoters from Escherichia coli. Primer-extension analysis revealed that this specific DNA sequence was indeed the location of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tentatively been named the hydroxyglutarate operon, since the enzymes encoded by these genes are involved in the conversion of (R)-2-hydroxyglutarate to crotonyl-CoA in the pathway of glutamate fermentation by A. fermentans. The genes gctAB were expressed together in E. coli. Cell-free extracts of a transformant E. coli strain contained glutaconate CoA-transferase at a specific activity of up to 30 U/mg protein. The recombinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisation. The amino acid residue directly involved in catalysis was tentatively identified as E54 of the small subunit of the enzyme (GctB).