Enrique Viturro

The ABCA subfamily—gene and protein structures, functions and associated hereditary diseases

To date, 12 members of the human ABCA subfamily are identified. They share a high degree of sequence conservation and have been mostly related with lipid trafficking in a wide range of body locations. Mutations in some of these genes have been described to cause severe hereditary diseases related with lipid transport, such as fatal surfactant deficiency or harlequin ichthyosis. In addition, most of them are hypothesized to participate in the subcellular sequestration of drugs, thereby being responsible for the resistance of several carcinoma cell lines against drug treatment. The objective of this review is to summarize the literature for this subfamily of ABC transporter proteins, excluding ABCA1 and ABCA4, which will be discussed in other chapters of this issue.

Cholesterol synthesis in the lactating cow: Induced expression of candidate genes

Despite the extensive knowledge for other species, cholesterol metabolism in ruminants is nowadays still not clear. Huge differences in milk cholesterol concentration are observed between breeds, managing strategies, individuals and moment of the lactating cycle, but the genetic actors working in the process of cholesterol secretion into milk have not been identified. As ruminant diet contains no cholesterol, understanding the mechanisms and regulation of synthesis, transport and secretion into milk is crucial when trying to reduce the amount of this metabolite in dairy products. The present work aims to study the expression of candidate genes for these processes in the liver of Bos taurus during the lactating cycle. Liver biopsies were obtained from 16 adult brown Swiss cows at different time points (2 weeks pre-partum and 0, 2, 4 and 8 weeks post-partum). After RNA extraction and reverse transcription, gene expression of candidate genes was studied using quantitative RT-PCR. Key enzymes of the cholesterol synthesis (3-hydroxy-methyglutaryl-coenzyme-A (HMG-CoA) synthase, HMG-CoA reductase and farnesyldiphosphat-farnesyltransferase (FDFT)) and gene expression feed-back regulators involved in lipid metabolism (sterol regulatory element binding proteins (SREBP1and 2) SREBP-cleavage activating protein (Scap) were selected as candidate genes. HMG-CoA-reductase and FDFT showed a huge expression increase until week 2 post-partum (p<0.01), most probably in response to the new requirements in the mammary gland. As well, and as a possible explanation for such modifications, an increase in the expression of the regulators SREBP1 and Scap was observed (p<0.01 and p<0.05 respectively). Most important, the whole synthesis machinery showed a coordinated regulation, as highly significant positive correlations were found between the expression levels of the above mentioned enzymes (p<0.01). The increase of milk and blood cholesterol levels in B. taurus after parturition might be the result of a coordinated induction in the expression of key liver enzymes and their regulating factors.

Effects of continuous milking during the dry period or once daily milking in the first 4 weeks of lactation on metabolism and productivity of dairy cows.

The objective was to compare the effects of 3 management systems in high-yielding dairy cows on metabolic profiles and milk production. Thirty-six multiparous Brown Swiss cows were randomly assigned to 1 of 3 treatment groups (n=12 cows/group): the control (C) group, in which cows were dried off 56 d before calving and milked twice daily throughout next lactation (305 d); the once daily milking (ODM) group, in which cows were dried off 56 d before calving and milked once daily for the first 4 wk of lactation and twice daily for the remaining lactation; and the continuous milking (CM) group, in which cows were milked twice daily until calving and also during the subsequent lactation. Serum glucose concentrations decreased between wk 1 and 4 exclusively in C cows. Serum concentrations of NEFA and BHBA in the first 4 wk of lactation were highest in C cows compared with ODM and CM cows. Decreased backfat thickness during early lactation and reduction of body condition score were markedly more pronounced in C cows compared with ODM and CM cows. Mean lactational milk yield of C cows [11,310+/-601 kg of energy-corrected milk (ECM)/305 d] was approximately 16% higher compared with ODM cows (9,531+/-477 kg of ECM/305 d) and CM cows (9,447+/-310 kg of ECM/305 d). The lactation curve of CM cows compared with C cows was characterized by a similar time of peak yield (wk 3), a reduced peak yield, and no obvious differences in persistency. Mean percentage of milk protein was significantly higher for CM cows (3.91%) compared with C cows (3.52%). In contrast, once daily milking was accompanied by a reduced and significantly delayed peak yield (wk 8) compared with the control treatment, whereas persistency was better and milk protein (3.79%) was higher in ODM cows than in C cows. In conclusion, continuous milking and once daily milking, targeting the interval before or after calving, respectively, substantially reduced the metabolic challenge of fresh cows and improved milk protein percentage. Continuous milking and once daily milking increased milk protein percentage markedly; furthermore, once daily milking during the first 4 wk of lactation improved the lactation curve.

Rapid method for cholesterol analysis in bovine milk and options for applications.

The adaptation of a colorimetric technique for the analysis of cholesterol in raw milk is presented. Performance quality was satisfying (mean intra-assay coefficient of variation (CV) 4.8, inter-assay CV 9.1%, linearity between 0 and 7 mm, recovery of spiked cholesterol into raw milk 98.1 and 106.3%). However, the milk fat extraction must be carried out within the 48 hours following milk sampling. When performing sampling, the significant variation of milk cholesterol composition during the milking process has to be taken into account.

Temporal variation of milk fat globule diameter, fat and cholesterol content and milk epithelial cell gene expression in dairy cows

It was possible to show a connection between the temporal variation of milk fat globule diameter, fat and cholesterol content in milk and the expression of candidate genes in the mammary gland epithelial cells in milk. The beginning of lactation corresponded with higher levels of fat and cholesterol in the milk as a result of a higher expression of key enzymes in the purified bovine milk epithelial cells, paralleled with an increase in milk fat globule mean size.

Microfluidic high-throughput reverse-transcription quantitative PCR analysis of liver gene expression in lactating animals

AbstractWe have evaluated a microfluidic lab-on-chip quantitative reverse transcription (RT) quantitative PCR (qPCR) method by measuring the expression of key actors of liver metabolism in lactating cattle. Animals in the early and in the late lactation phases were chosen because of the extreme adaptations in gene expression expected to occur. During the lactation cycle, 28 out of 48 genes were significantly regulated, notably in the same direction as previously shown by other techniques. This demonstrates that this high-throughput platform represents an attractive alternative to microarrays due to its ease of application, rapidity and lower costs. A set of 13 genes was identified—in combination with a dynamic PCA algorithm—that allowed the clearest separation between the two physiologically different groups. This paves the way for classification and diagnosis of animals in different metabolic situations by a reliable microfluidic RT-qPCR assay. FigureTo determine the pattern of genes, which visualizes the separation of animals in the two lactation stages best, dynamic PCA was employed. A pattern of 13 genes was identified. Black dots represent the samples obtained from animals in early lactation and white dots represent samples from animals in late lactation.