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Catecholamine and Cortisol Concentrations in Plasma from Cows at Term: Cows with spontaneous parturition (n = 8) and cows with flumethasone (n = 3) or prostaglandin (n = 3) induced parturition were used in this study. Catecholamine concentrations from cows in the periparturient period were measured in order to evaluate the stress of birth on the dam and the calf. The plasma content of both norepinephrine and epinephrine in late pregnancy was not different from control values in the 3rd to 6th month of pregnancy (norepinephrine 3.27 ± 0.99 nmol/l plasma and epinephrine 0.98 ± 0.45 nmol/l plasma), though both parameters appear elevated in the pregnant group. Norepinephrine concentrations rose to a value of 4.50 nmol/l immediately after birth, whereas epinephrine levels did not show an increase post partum. In two neonates norepinephrine and epinephrine values were 2-3 times higher than in the dams and decreased to the values of the dams within 48 hours. Synthetic glucocorticoids can mimic the increase of Cortisol, which leads to birth. Cortisol concentrations in the cows remained unaffected by induction of parturition by prostaglandins. Newborn calves showed values of about 330 nmol/l, which also declined within 48 hours to values similar to those measured in the dam (9-28 nmol/l). As stress and exogenous glucocorticoids may affect antibody production, the IgG-content of the colostrum was examined. Prostaglandin induction of birth did not alter the IgG-content of colostrum, but flumethasone produced a decrease of about 14.5 %. It is therefore concluded that pre-term birth induction does not produce negative effects on the supply of IgG. Zusammenfassung In dieser Studie wurden Kühe mit physiologischer Geburt (n = 8) sowie Tiere mit Prostaglandin- (n = 3) und Flumethason-induzierter Geburt (n = 3) verwendet. Katecholaminkonzentrationen im Plasma von Kühen um die Geburt wurden gemessen, um anhand dieser Substanzen das Streßgeschehen zu untersuchen, dem Kuh und Kalb während des Geburtsvorganges ausgesetzt sind. Der Plasmagehalt von Noradrenalin und Adrenalin im Endstadium der Trächtigkeit war von Vergleichswerten von Muttertieren im 3. bis 6. Graviditätsmonat nicht signifikant verschieden (Noradrenalin 3,27 ± 0,99 nmol/l und Adrenalin 0,98 ± 0,45 nmol/l Plasma). Die Noradrenlinkonzentrationen des Plasmas stiegen unmittelbar nach der Geburt auf Werte von ca. 4,50 nmol/l an, die Adrenalinwerte blieben unverändert. Bei zwei neugeborenen Kälbern waren die Noradrenalin- und Adrenalinwerte gegenüber den Muttertieren etwa 2-3 fach erhöht. Innerhalb von 48 Stunden nach der Geburt sanken die Hormonkonzentrationen auf die Werte der Muttertiere ab. Synthetische Glukokortikoide können den Cortisolanstieg, der zur Geburt führt, nachahmen. Die Cortisolwerte waren bei Muttertieren mit Prostaglandin-induzierter Geburt, verglichen mit spontan gebärenden Tieren, nicht unterschiedlich. Neugeborene Kälber zeigten Werte um 330 nmol/l. Diese Werte sanken innerhalb von 48 Stunden nach der Geburt gleichfalls in den Bereich der Konzentrationen der Muttertiere (9-28 nmol/l) ab. Da Streß und exogene Glukokortikoide die Antikörperproduktion hemmen können, wurde der IgG-Gehalt des Kolostrums untersucht. Die Prostaglandininduktion hatte keinen Einfluß auf den IgG-Gehalt und auch nach Flumethasonverabreichung war die Antikörperkonzentration des Kolostrums nur um 14,5 % niedriger als in der Kontrollgruppe. Es ist daher anzunehmen, daß sich auch eine frühzeitige Gebunsinduktion nicht nachteilig auf die Immunglobulinversorgung der Kälber auswirkt. [ABSTRACT FROM AUTHOR]

Saliva contains a number of proteins that may be useful as biomarkers of health and disease and can be easily obtained from large numbers of animals in a non-invasive, stress-free way. The objective of this study was to explore the protein composition of porcine saliva from 10 specific pathogen free pigs using first one-dimensional SDS-PAGE and then two-dimensional electrophoresis and mass spectrometry. A reference proteome pattern for porcine saliva was established with the identification of 13 different, mainly saliva-specific, proteins. These reference data will facilitate the investigation of salivary proteins potentially altered in disease and could serve as novel diagnostic biomarkers. [ABSTRACT FROM AUTHOR]

Polyclonal immunoglobulin (Ig) concentrates are important biological medicinal products and the assurance of their quality and safety is crucial. In our present approach we used proteomic methods to check the purity of commercial Ig products of different origin. The experimental setup included nonreducing 2DE or DIGE combined with MALDI-TOF and the thrombin generation assay, a routine safety test for pharmaceutical Ig preparations, and was complemented by a specific immunoassay. 2DE patterns displayed contaminations with trace amounts of human apolipoprotein H (Apo-H), transferrin, albumin, and its fragments. In contrast to the latter, Apo-H is a protein that is active in the coagulation cascade, and thus a potential involvement in thromboembolic events in vivo cannot be excluded. It was found by 2DE and MALDI-TOF to be a contaminant of several Ig preparations. Spiking experiments of Ig preparations with pure Apo-H demonstrated an Apo-H concentration dependent increase in thrombin generation assay values. Traces of Apo-H are possibly also contributing to unwanted side effects, as already known for factor XIa. The significance of Apo-H contaminations for these side effects might be verified by detailed analyses of pharmacovigilance data., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo., Methods: Thrombin (0.0004-0.42 IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n=5 animals; 15 experiments). In the dose verification study animals received 0.42 IU/kg/min of thrombin vs. saline solution in a cross-over design (n=3 animals; 7 experiments)., Results: Thrombin at an infusion rate of 0.42 IU/kg/min decreased fibrinogen levels by 75% (p<0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p=0.006), prolonged thrombin time by 70% (p=0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p<0.05) and decreased the activity of the coagulation factors VIII and XIII (p<0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p=0.019) and induced a 65% decrease in maximal clot firmness (p<0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism., Conclusion: Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tyrosine kinase 2 (Tyk2) is an integral part of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway which relays intracellular signals of various cytokines. Tyk2 crucially contributes to host defense mechanisms against microbial pathogens and to tumor surveillance but also facilitates immune pathologies. Here we investigated the impact of Tyk2 on the macrophage proteome using the synthetic double-stranded RNA analog polyinosinic acid-polycytidylic acid (poly(I:C)) as a mimicry of viral infections. By means of 2D-DIGE in connection with PMF obtained by MALDI-MS and sequence tag determination by MS/MS we unambiguously identified eighteen protein spots corresponding to sixteen distinct proteins that are regulated by poly(I:C) and differentially expressed between wildtype (WT) and Tyk2-deficient macrophages. The majority of these proteins are functionally assigned to cellular immune responses and to metabolism. We show for selected metabolic enzymes, i.e. triosephosphate isomerase (TIM), ATP-citrate synthase (ACLY) and long-chain-fatty-acid-CoA ligase 4 (ACSL4), that Tyk2 affects protein expression transcriptionally and post-transcriptionally. We furthermore confirm the involvement of Tyk2 in the regulation of lipid and carbohydrate metabolism at the level of metabolites. Taken together, our results provide new evidence for important functions of Tyk2 at the molecular interface between innate immunity and cellular metabolism., (Copyright © 2011 Elsevier B.V. All rights reserved.)

2-D DIGE was used to investigate 'fingerprint proteins' in biological medicines. A presumably non-originator human albumin was analysed, and the 2-D DIGE patterns of the non-genuine and the authentic product were compared. The products could be clearly distinguished based on the pattern of minor components, which represent plasma proteins and degradation products remaining in the final products after fractionation and purification. The approach demonstrated that 2-D DIGE is an excellent tool for the analysis of biologicals of different sources and for ensuring the identity and quality of blood products., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

IL-1beta is an important proinflammatory cytokine with a major role in several inflammatory diseases. Expression of IL-1beta is tightly regulated at the level of transcription, mRNA stability, and proteolytic processing. In this study, we report that IL-1beta expression in response to LPS is also regulated at the translational level. LPS-induced IL-1beta protein levels in macrophages derived from murine bone marrow are markedly increased in the absence of tyrosine kinase 2 (Tyk2). Increased IL-1beta is found intra- and extracellularly, irrespective of the efficiency of IL-1beta processing. We show that the absence of Tyk2 results both in higher translational rates and in enhanced association of IL-1beta mRNA with polysomes. Induction and stability of IL-1beta mRNA are not affected by the lack of Tyk2. We show further that the Tyk2-dependent translational inhibition is mediated by autocrine/paracrine type I IFN signaling and requires signal transducer and activator of transcription 1. Enhanced IL-1beta production in Tyk2- and IFN receptor 1-deficient macrophages is also observed following Listeria monocytogenes infection. Taken together, the data describe a novel mechanism for the control of IL-1beta synthesis.

We characterized the two-dimensional electrophoretic patterns of fibrinogen chains alpha, beta, and gamma from the plasma of six animal species -Bos taurus, Canis familiaris, Equus caballus, Felis catus, Gallus domesticus and Sus scrofa. Comparing the spots resolved from serum and plasma samples, or exploiting the cross-reactivity of animal fibrinogen with an antiserum raised against the human protein could detect only some of the fibrinogen chains. Conversely, the analysis of the precipitate obtained by heating plasma for some minutes at 56 degrees C was adequate for the recognition of all fibrinogen chains in all samples. Physicochemical properties of the homologous proteins were found to extensively vary across species, with complete separation among the mapping areas for alpha, beta and gamma chains and maximal heterogeneity among beta chains., (2010 Elsevier B.V. All rights reserved.)

Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.

A reference protein map for pig serum was set up using two-dimensional electrophoresis (2-DE). Thirty-nine protein chains or spots deriving from 26 different proteins were identified by immunological and mass spectrometric methods. Thus, the positions of most medium to higher abundance serum proteins could be determined on the 2-DE gels. The plasma protein fibrinogen was also included in our study. The overall pig protein pattern differs in some respect to serum/plasma maps of other mammalian species, e.g. in levels and properties of single proteins such as haptoglobin or IgM or in species-specific proteins like pig major acute phase protein. Serum protein maps are a useful tool to get an overview on expressed proteins, and to monitor changes in concentration as well as isotype distribution of the identified proteins. As a consequence, more detailed knowledge on protein pattern changes may give deeper insights into the metabolic development of some pathologic conditions and may lead to putative biomarkers for further investigation. Selected examples for protein pattern changes in pigs infected by a viral (porcine circovirus type 2) and a bacterial (Actinobacillus pleuropneumoniae) pathogen illustrate the usefulness of the method.

Tyrosine kinase 2 (Tyk2) belongs to the Janus kinase (Jak) family and is involved in signalling via a number of cytokines. Tyk2-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxin shock. Macrophages are key players in the pathogenesis of endotoxin shock and, accordingly, defects in the LPS responses of Tyk2(-/-) macrophages have been reported. In the present study, the molecular role of Tyk2 is investigated in more detail using a proteomics approach. 2-D DIGE was applied to compare protein patterns from wild-type and Tyk2(-/-) macrophages and revealed significant differences in protein expression patterns between the genotypes before and after LPS treatment. Twenty-one proteins deriving from 25 differentially expressed spots were identified by MALDI/ESI MS. Among them, we show for N-myc interactor that its mRNA transcription/stability is positively influenced by Tyk2. In contrast, LPS-induced expression of plasminogen activator 2 protein but not mRNA is strongly enhanced in the absence of Tyk2. Our data furthermore suggest an influence of Tyk2 on the subcellular distribution of elongation factor 2 and on LPS-mediated changes in the peroxiredoxin 1 spot pattern. Thus, our results imply regulatory roles of Tyk2 at multiple levels and establish novel connections between Tyk2 and several cellular proteins.