Knowledge on absolute protein concentrations is mandatory for the simulation of biological processes in the context of systems biology. A novel approach for the absolute quantification of proteins at a global scale has been developed and its applicability demonstrated using glucose starvation of the Gram-positive model bacterium Bacillus subtilis and the pathogen Staphylococcus aureus as proof-of-principle examples. Absolute intracellular protein concentrations were initially determined for a preselected set of anchor proteins by employing a targeted mass spectrometric method and isotopically labeled internal standard peptides. Known concentrations of these anchor proteins were then used to calibrate two-dimensional (2-D) gels allowing the calculation of absolute abundance of all detectable proteins on the 2-D gels. Using this approach, concentrations of the majority of metabolic enzymes were determined, and thus a quantification of the players of metabolism was achieved. This new strategy is fast, cost-effective, applicable to any cell type, and thus of value for a broad community of laboratories with experience in 2-D gel-based proteomics and interest in quantitative approaches. Particularly, this approach could also be utilized to quantify existing data sets with the aid of a few standard anchor proteins.

Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.

In this study the protein and mRNA expression of Monocarboxylate transporter 1 (MCT1) was evaluated in rumen epithelial cells (REC) obtained from sheep fed hay ad libitum (control, h diet, n=4) or a mixed hay/concentrate diet (h/c diet, n=4) for two weeks. REC were isolated via fractionated trypsination and three groups consisting of fractions 3 to 5=G1, fractions 6 to 8=G2, and fractions 9 and 10=G3 were formed. Using an anti-basal cytokeratin antibody and flow cytometric analysis, the proportion of REC originating from the stratum basale (SB) was determined for each group. In addition, MCT1 mRNA and protein expression was determined by qRT-PCR and Western blot, respectively. Feeding the h/c diet led to a 299±93 % elevation of the number of SB cells known to express the MCT1 protein. This is accompanied by an increased MCT1 mRNA (1.8 to 2.2-fold) and protein (1.3-fold) expression. Thus, an increased number of MCT1 expressing cells and upregulation of ruminal MCT1 protein seem to be components of rumen epithelium functional adaptation to high energy diet.Keywords: sheep, rumen, epithelial cells, transport protein, high energy diet Zusammenfassung Die mRNA-und Protein-Expression des MCT1 in Pansenepithelzellen wird durch Zufütterung von Konzentrat zu einer ad libitum Heu-Ration erhöhtIn der vorliegenden Studie wurden die mRNA-und Proteinexpression des Monocarboxylattransporters 1 (MCT1) in Pansenepithelzellen (PEZ) von Schafen, die über zwei Wochen entweder Heu ad libitum (h-Diät, n=4) oder eine Heu/Konzentrat-Mischration (h/c-Diät, n=4) erhielten, untersucht. Ovine PEZ wurden mittels fraktionierter Trypsinierung gewonnen und anschließend drei Zellgruppen, bestehend aus den Fraktionen 3-5 (G1), 6-8 (G2) und 9/10 (G3), zugeordnet. Mittels Flowzytometrie und unter Nutzung eines spezifischen Antikörpers gegen basales Cytokeratin wurde der Anteil von PEZ aus dem Stratum basale (SB) für jede Zellgruppe ermittelt. Die Expression von MCT1-mRNA und -Protein wurde mittels qRT-PCR und Western Blot analysiert. Die Anzahl MCT1-exprimierender SB-Zellen, stieg nach Füt-terung der h/c-Diät um 299±93 %. Damit ist eine Erhöhung der MCT1-mRNA-(1,8-2,2-fach) sowie der Proteinexpression (1,3-fach) verbunden. Ein erhöhter Anteil MCT1-expremierender PEZ und die Aufregulation der MCT1 Proteinmenge scheinen somit Komponenten der funktionellen Anpassung des Pansenepithels an energiereiche Fütterung darzustellen.

A protein of~70-kDa was identified as a candidate Na + /Mg 2+ exchanger in rumen epithelial cells (REC). Melastatin-related Transient Receptor Potential 7 (TRPM7) and Magnesium Transporter 1 (MagT1) transcripts and, from them, encoded proteins were also detected. The regulation of these Mg transport pathways by extracellular [Mg] changes was the main focus of this study. Therefore, a 24-h pre-incubation of ovine REC in control (1.2 mM), low (0.12 mM)-Mg, and high (5 mM)-Mg medium was performed. Na + /Mg 2+ exchangers, TRPM7 and MagT1 abundance and activity were investigated by Western blot analysis, flow cytometry, immunocytochemistry and fluorescence spectroscopic measurements of [Mg 2+ ] i changes. Inhibitors were employed to differentiate Na + /Mg 2+ exchanger-mediated (imipramine) and channel-mediated (cobalt(III)hexaammine, nitrendipine) Mg transport. Basal [Mg 2+ ] i (0.40 ± 0.02 mM) was not influenced by pre-incubation in low-or high-Mg medium. However, compared with control REC (4.1 ± 0.7 μM/min), such cells showed reduced (2.8 ± 0.6 μM/min) or elevated (6.4 ± 0.9 μM/min) Mg extrusion rates that correlated with a decreased (25%) and increased (38%) expression of the putative Na + /Mg 2+ exchanger protein, respectively. Low-and high-Mg pre-incubated REC were both characterized by an increased (30-40%) influx capacity. In low-Mg REC, the latter resulted mainly from a strong activation of the TRPM7-related transport component. The data thus clearly demonstrate the intrinsic regulation of REC transmembrane Mg transport.

The aim of this study was to show the abundance of leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) and to determine the direct effects of leptin and adiponectin on the in vitro growth of porcine skeletal muscle cells. ADIPOR1 and ADIPOR2 were abundant at mRNA and protein level in proliferating and differentiating myoblast cultures derived from semimembranosus and semitendinosus muscles of newborn piglets, whereas LEPR expression was close to the detection limit. Adiponectin (10, 20, 40 μg/ml) attenuated the proliferation of porcine myoblasts, measured as [(3)H]-thymidine incorporation and real-time monitoring of the cells in response to 24- and 48-h exposure, in a dose-dependent manner. This effect resulted from suppressed basic fibroblast growth factor (bFGF)-mediated stimulation of DNA synthesis in serum-free medium (SFM) containing bFGF. No effects of leptin (5, 10, 20, 40, 80 ng/ml) on myoblast proliferation in SFM were detectable. Neither leptin nor adiponectin altered protein synthesis and degradation in differentiating porcine myoblasts cultured in SFM. The results on receptor abundance suggest that porcine skeletal muscle cells may be sensitive to adiponectin and leptin. However, except via inhibitory interaction of adiponectin with bFGF, these adipokines appear not to affect in vitro proliferation and protein metabolism of porcine muscle cells directly under serum-free culture conditions.