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1.
Electrophoresis ; 35(4): 515-21, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24132587

ABSTRACT

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.


Subject(s)
Immunoglobulins/chemistry , Proteomics/methods , Recombinant Proteins/chemistry , beta 2-Glycoprotein I/chemistry , Electrophoresis, Gel, Two-Dimensional , Humans , Recombinant Proteins/standards , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
2.
Thromb Res ; 130(2): 226-36, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22289207

ABSTRACT

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.


Subject(s)
Blood Coagulation/drug effects , Hemorrhage/chemically induced , Hemostatics/adverse effects , Sheep/blood , Thrombin/adverse effects , Administration, Intravenous , Amino Acid Sequence , Animals , Blood Platelets/cytology , Blood Platelets/drug effects , Blood Proteins/analysis , Blood Proteins/metabolism , Fibrinogen/analysis , Fibrinogen/metabolism , Hemorrhage/pathology , Hemostatics/administration & dosage , Lung/drug effects , Lung/pathology , Mass Spectrometry , Molecular Sequence Data , Platelet Count , Platelet Function Tests , Proteomics , Sheep/metabolism , Thrombelastography , Thrombin/administration & dosage
3.
J Proteomics ; 74(12): 2866-80, 2011 Nov 18.
Article in English | MEDLINE | ID: mdl-21787891

ABSTRACT

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.


Subject(s)
Gene Expression Regulation/drug effects , Glucose/metabolism , Interferon Inducers/pharmacology , Lipid Metabolism/drug effects , Macrophages/metabolism , Poly I-C/pharmacology , Proteome/biosynthesis , TYK2 Kinase/metabolism , Animals , Carbohydrate Metabolism/drug effects , Carbohydrate Metabolism/genetics , Carbohydrate Metabolism/immunology , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Glucose/genetics , Glucose/immunology , Immunity, Innate/drug effects , Immunity, Innate/genetics , Immunity, Innate/immunology , Lipid Metabolism/genetics , Lipid Metabolism/immunology , Macrophages/cytology , Macrophages/immunology , Mice , Mice, Knockout , Proteome/genetics , Proteome/immunology , TYK2 Kinase/genetics , TYK2 Kinase/immunology
4.
Proteomics ; 11(10): 2120-3, 2011 May.
Article in English | MEDLINE | ID: mdl-21462342

ABSTRACT

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.


Subject(s)
Biological Products/chemistry , Biological Products/standards , Electrophoresis, Gel, Two-Dimensional/methods , Peptide Mapping/methods , Albumins/chemistry , Blood Proteins/chemistry , Drug Contamination , Humans
5.
J Chromatogr B Analyt Technol Biomed Life Sci ; 878(25): 2314-8, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20674523

ABSTRACT

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.


Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Fibrinogen/chemistry , Immunoblotting/methods , Proteomics/methods , Animals , Cats , Cattle , Chickens , Dogs , Fibrinogen/metabolism , Horses , Humans , Protein Isoforms , Protein Subunits , Species Specificity , Swine
6.
J Immunol ; 185(6): 3544-53, 2010 Sep 15.
Article in English | MEDLINE | ID: mdl-20713887

ABSTRACT

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.


Subject(s)
Interleukin-1beta/biosynthesis , Interleukin-1beta/genetics , Protein Biosynthesis/genetics , Protein Biosynthesis/immunology , TYK2 Kinase/physiology , Animals , Bone Marrow Cells/enzymology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Line, Tumor , Cells, Cultured , Interferon Type I/physiology , Macrophages/enzymology , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Precursors/biosynthesis , Protein Precursors/genetics , Signal Transduction/genetics , Signal Transduction/immunology , TYK2 Kinase/deficiency , TYK2 Kinase/genetics , Up-Regulation/genetics , Up-Regulation/immunology
7.
Mycotoxin Res ; 26(3): 171-80, 2010 Aug.
Article in English | MEDLINE | ID: mdl-23605381

ABSTRACT

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.

8.
Res Vet Sci ; 86(2): 362-7, 2009 Apr.
Article in English | MEDLINE | ID: mdl-18619635

ABSTRACT

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.


Subject(s)
Blood Proteins/analysis , Proteomics/methods , Swine Diseases/diagnosis , Swine/blood , Actinobacillus Infections/diagnosis , Actinobacillus Infections/veterinary , Animals , Circoviridae Infections/diagnosis , Circoviridae Infections/veterinary , Electrophoresis, Gel, Two-Dimensional/methods , Electrophoresis, Gel, Two-Dimensional/veterinary , Immunoblotting/veterinary , Swine Diseases/blood , Tandem Mass Spectrometry/veterinary
9.
Proteomics ; 8(17): 3469-85, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18683816

ABSTRACT

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.


Subject(s)
Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Proteome/drug effects , TYK2 Kinase/physiology , Animals , Cell Cycle Proteins/biosynthesis , Electrophoresis, Gel, Two-Dimensional , Female , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, Knockout , Peptide Elongation Factor 2/biosynthesis , Peroxiredoxins/biosynthesis , Plasminogen Activator Inhibitor 2/biosynthesis , RNA, Messenger/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , TYK2 Kinase/deficiency
10.
Lab Invest ; 88(1): 70-7, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17982471

ABSTRACT

Mitochondria are involved in the development of organ failure in critical care diseases. However, the mechanisms underlying mitochondrial dysfunction are not clear yet. Inducible hemoxygenase (HO-1), a member of the heat shock protein family, is upregulated in critical care diseases and considered to confer cytoprotection against oxidative stress. However, one of the products of HO-1 is Fe2+ which multiplies the damaging potential of reactive oxygen species catalyzing Fenton reaction. The aim of this study was to clarify the relevance of free iron metabolism to the oxidative damage of the liver in endotoxic shock and its impact on mitochondrial function. Endotoxic shock in rats was induced by injection of lipopolysaccharide (LPS) at a dose of 8 mg/kg (i.v.). We observed that the pro-inflammatory cytokine TNF-alpha and the liver necrosis marker aspartate aminotransferase were increased in blood, confirming inflammatory response to LPS and damage to liver tissue, respectively. The levels of free iron in the liver were significantly increased at 4 and 8 h after onset of endotoxic shock, which did not coincide with the decrease of transferrin iron levels in the blood, but rather with expression of the inducible form of heme oxygenase (HO-1). The proteins important for sequestering free iron (ferritin) and the export of iron out of the cells (ferroportin) were downregulated facilitating the accumulation of free iron in cells. The temporarily increased concentration of free iron in the liver correlated with the temporary impairment of both mitochondrial function and tissue ATP levels. Addition of exogenous iron ions to mitochondria isolated from control animals resulted in an impairment of mitochondrial respiration similar to that observed in endotoxic shock in vivo. Our data suggest that free iron released by HO-1 causes mitochondrial dysfunction in pathological situations accompanied by endotoxic shock.


Subject(s)
Endotoxins/pharmacology , Heme Oxygenase (Decyclizing)/metabolism , Iron/metabolism , Mitochondria, Liver/drug effects , Up-Regulation/drug effects , Adenosine Triphosphate/metabolism , Animals , Gene Expression Profiling , Iron/pharmacology , Lipopolysaccharides/toxicity , Male , Mitochondria, Liver/metabolism , Mitochondria, Liver/physiology , Oxidative Stress , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism
11.
Exp Mol Pathol ; 82(1): 33-41, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17150211

ABSTRACT

Spontaneously hypertensive stroke-prone rats (SHRSP) feature an established model for human cerebrovascular disease. SHRSP, kept on a high-salt permissive diet (JPD), develop hypertension, renal and brain damage. In this report we compared the behavior of female and male SHRSP regarding the main aspects of their pathological condition. Brain abnormalities, detected by magnetic resonance imaging, developed spontaneously in males after 42+/-3 days, in females after 114+/-14 days from the start of JPD. Survival was >3-fold longer for females than for males. The development of brain damage was preceded, in both genders, by an inflammatory condition characterized by the accumulation in serum and urine of acute-phase proteins. The increase in thiostatin level was significantly lower and delayed in female in comparison to male SHRSP. During JPD female and male SHRSP developed massive proteinuria, its worsening being significantly slower in females. The alterations of vasculature-bound barriers in kidney and brain were connected with endothelial dysfunction and relative deficiency in nitric oxide (NO). In thoracic aortic rings, basal release of NO was significantly higher in female than in male SHRSP, both if receiving and if not receiving JPD. The gender differences in SHRSP thus appear to be connected to a more efficient control in females of inflammation and of endothelial dysfunction.


Subject(s)
Biomarkers/analysis , Brain Diseases/etiology , Endothelium, Vascular/metabolism , Hypertension/complications , Inflammation/metabolism , Acute-Phase Proteins/analysis , Animals , Aorta/metabolism , Blood Proteins/analysis , Brain/blood supply , Brain Diseases/pathology , Electrophoretic Mobility Shift Assay , Female , Hypertension/mortality , Hypertension/physiopathology , Kidney/blood supply , Kininogens/analysis , Magnetic Resonance Imaging , Male , Nitric Oxide/metabolism , Organ Culture Techniques , Proteinuria/etiology , Rats , Rats, Inbred SHR , Sex Factors , Time Factors
12.
Biochem Biophys Res Commun ; 352(1): 91-6, 2007 Jan 05.
Article in English | MEDLINE | ID: mdl-17112473

ABSTRACT

In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis.


Subject(s)
Endoplasmic Reticulum/drug effects , Endotoxins/pharmacology , Mitochondria, Liver/drug effects , Animals , Biomarkers , Endoplasmic Reticulum/physiology , Lipopolysaccharides/pharmacology , Liver/drug effects , Liver/metabolism , Male , Mitochondria, Liver/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species , Superoxide Dismutase/metabolism
13.
Electrophoresis ; 27(8): 1626-9, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16532519

ABSTRACT

Low levels of fetal calf serum (FCS), used as protein supplement in cell culture medium, were traced in preparations of primary murine macrophages (bone-marrow-derived macrophages (BMM) and peritoneal macrophages (PM)). Main components of this common additive were mapped in 2-DE by means of differential image gel electrophoresis and immunoblotting. Additional washing steps in cell preparation helped to decrease the levels of the four highest abundance foetal serum proteins (serum albumin (SA), alpha1-fetoprotein (AFP), alpha1-antitrypsin (alpha1AT) and transferrin (Tf)) to less than 1% of total protein. Macrophage spot pattern was recorded in parallel and showed little variation. Results presented are supposed to be of general interest for cell preparations with similar background.


Subject(s)
Artifacts , Culture Media , Electrophoresis, Gel, Two-Dimensional/methods , Fetal Blood , Macrophages, Peritoneal/chemistry , Macrophages/chemistry , Proteome/chemistry , Animals , Cattle , Cell Culture Techniques , Mice
14.
FEBS Lett ; 580(5): 1257-62, 2006 Feb 20.
Article in English | MEDLINE | ID: mdl-16442530

ABSTRACT

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.


Subject(s)
Mitochondria, Liver/chemistry , Proteome/analysis , Shock, Septic/metabolism , Animals , Cell Respiration , Electrophoresis, Gel, Two-Dimensional , Lipopolysaccharides/pharmacology , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Oxygen/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Shock, Septic/genetics
15.
Virology ; 346(1): 139-50, 2006 Mar 01.
Article in English | MEDLINE | ID: mdl-16310820

ABSTRACT

Mouse mammary tumour virus (MMTV) encodes a viral superantigen (Sag) and a negative acting factor (Naf) which share parts of their coding sequence. Using 2-dimensional gel electrophoresis (2D-DIGE), we could show that at least 10 different cellular proteins were differentially expressed in Naf positive cells. Also, luciferase reporter expression was down-regulated in Naf expressing cells independent of the promoter used and further experiments suggested that this effect was due in part to a decrease in cellular growth rates. Although in Naf positive cells expression of the major sag containing transcript was strongly induced by the synthetic glucocorticoid dexamethasone, the hormone analogue neither influenced luciferase expression nor mRNA expression of selected cellular proteins identified by 2D-DIGE. Taken together, these data support the previous finding that Naf and Sag have separable activities and suggest that Naf may play a role in modulating host cell gene expression during MMTV infection.


Subject(s)
Gene Expression Regulation , Mammary Tumor Virus, Mouse/pathogenicity , Proteins/metabolism , Viral Proteins/metabolism , Animals , Cell Line , Electrophoresis, Gel, Two-Dimensional , Luciferases/metabolism , Mass Spectrometry/methods , Mice , Proteins/chemistry , Proteins/genetics , Superantigens/genetics , Superantigens/metabolism , Transfection , Viral Proteins/genetics
16.
Proteomics ; 4(10): 3227-34, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15378688

ABSTRACT

We constructed a reference two-dimensional protein map for horse (Equus caballus) serum. The serum proteins were separated by two-dimensional electrophoresis (2-DE); 29 different gene products were identified. Proteins represented by 25 spots/spot groups were identified by tandem nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-TOF technology. The identities of four proteins were deduced by similarity to the human plasma protein database. In selected cases, i.e. the immunoglobulins, immunoblotting with specific antibodies provided additional information about the respective proteins. Albumin was detected as the full-length protein and as fragments of various sizes. Spots representing products of different mass and charge were also detected for alpha1-antitrypsin, haptoglobin and transthyretin. Thus, despite the fact that the Equus caballus genome is incompletely characterized, we were able to identify almost all moderate to high abundance proteins stained in the serum 2-DE pattern.


Subject(s)
Databases, Protein , Horses/blood , Proteome , Proteomics/methods , Albumins/metabolism , Amino Acid Sequence , Animals , Electrophoresis, Gel, Two-Dimensional , Immunoglobulins/chemistry , Mass Spectrometry , Molecular Sequence Data , Peptides/chemistry , Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
17.
J Neurosci Res ; 78(1): 115-22, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15372505

ABSTRACT

Spontaneously hypertensive stroke-prone rats (SHRSP) develop brain abnormalities invariably preceded by the accumulation of acute-phase proteins in body fluids. This study describes the sequence of pathological events, and in particular the involvement of inflammation, at the onset of brain injury in this animal model. In SHRSP subjected to permissive dietary treatment, the appearance of brain damage and of altered permeability of the blood-brain barrier (BBB) was monitored over time by magnetic resonance imaging (MRI) after intravenous injection of gadolinium. The protein content in cerebrospinal fluid and brain extracts was analyzed by two-dimensional electrophoresis. Gadolinium diffusion showed impairment of the BBB after 42 +/- 3 days from the start of salt loading, simultaneously with the detection of brain abnormalities by MRI. Tissue lesions were initially localized at one or more small foci and then spread throughout the brain in the form of fibrinoid necrosis. This type of lesion is characterized by fibrin deposition, in particular around the vessels; loss of tissue texture; and infiltration of macrophages and lymphocytes. High levels of plasma-derived proteins of molecular mass up to >130 kDa were detected in the cerebrospinal fluid after MRI had revealed brain abnormalities. Plasma proteins extravasated from brain vessels were immunodetected in tissue homogenates from affected areas. The results obtained in this study provide new insights into the pathogenesis of the spontaneous brain damage in SHRSP and in particular on the involvement of the inflammatory cascade. These studies may be useful in evaluating new pharmacological strategies aimed at preventing/treating brain diseases.


Subject(s)
Brain/metabolism , Hypoxia, Brain/metabolism , Hypoxia, Brain/pathology , Magnetic Resonance Imaging/methods , Proteomics/methods , Stroke/metabolism , Stroke/pathology , Animals , Biomarkers/cerebrospinal fluid , Blood-Brain Barrier , Brain/pathology , Disease Susceptibility/cerebrospinal fluid , Disease Susceptibility/metabolism , Disease Susceptibility/pathology , Disease Susceptibility/physiopathology , Hypertension/metabolism , Hypertension/pathology , Hypoxia, Brain/cerebrospinal fluid , Hypoxia, Brain/physiopathology , Male , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Stroke/cerebrospinal fluid , Stroke/physiopathology
18.
Proteomics ; 4(1): 257-60, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14730687

ABSTRACT

Nonreducing two-dimensional gel electrophoresis (2-DE) is described for the study of immunoglobulin disorders with asynchronous production of single chains. Unlike classical reducing 2-DE, this method can distinguish between complex intact molecules and their free single chains (with different degrees of polymerization) and will thus be helpful for diagnosis of this type of disease. Examples are taken from canine patients, but the method may also be applied to both urine and serum specimens from other species. Nonreducing 2-DE thus represents a useful tool complementary to classical 2-DE, when further information about the appearance of free subunits or modifications of proteins are required, even in the presence of intact molecules.


Subject(s)
Bence Jones Protein/analysis , Paraproteinemias/diagnosis , Bence Jones Protein/urine , Electrophoresis, Gel, Two-Dimensional , Humans
19.
Viral Immunol ; 16(3): 335-46, 2003.
Article in English | MEDLINE | ID: mdl-14583148

ABSTRACT

Parameters of humoral and cellular immunity were investigated in pigs experimentally infected with a modified-live European porcine reproductive and respiratory syndrome virus (PRRSV, strain DV). PRRSV was detected by real-time RT-PCR and PRRSV-specific antibodies by a commercial ELISA test-kit, respectively. Interleukins IL-1alpha, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF-alpha) and interferon-gamma (IFN-gamma) as well as IL-2 receptor (IL-2R) were quantified at mRNA level using RT-PCR. Subpopulations of blood lymphocytes were assayed using flow cytometry. No significant changes neither in cytokine expression nor in shifts of CD4 and CD8 markers could be found, but similar curve diagrams concerning CD8 single positive T cells could be observed in all vaccinated animals with an initial decrease and an increase between post-infection days (PIDs) 7 and 14. In the vaccination group, TNF-alpha and IL-6 tended to be increased at PIDs 22 and 40, whereas no increase could be seen in IFN-gamma. When comparing the in vivo immune response to that being seen in in vitro experiments, similar shifts of CD4/CD8 lymphocyte subpopulations may be seen. Cytokine curve diagrams, however, do not reflect the in vitro findings to that extent.


Subject(s)
Porcine respiratory and reproductive syndrome virus/immunology , Animals , Antibodies, Viral/biosynthesis , Base Sequence , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytokines/biosynthesis , Cytokines/genetics , DNA Primers/genetics , DNA, Viral/genetics , Female , Immunity, Cellular , In Vitro Techniques , Male , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/prevention & control , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sus scrofa , Vaccination
20.
J Lab Clin Med ; 140(5): 303-11, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12434131

ABSTRACT

Inhibition of endogenous nitric oxide (NO) synthesis during early intestinal ischemia/reperfusion (I/R(i)) enhances remote organ damage related to I/R(i). However, the effects of NO synthase (NOS) inhibitors on NO formation in various organs have not yet been specified. We therefore investigated the effects of N-G-monomethyl-L-arginine (L-NMMA), a nonspecific NOS inhibitor, and L-arginine, the NOS substrate, on NO formed in ischemic intestine versus normoxic remote organs (lung and liver). We used electron paramagnetic resonance spectroscopy and a specific NO trap to assay NO in blood, intestine, lung, and liver of rats subjected to local I/R(i), with and without L-NMMA and L-arginine supplementation. We found that I/R(i) increased NO levels in the intestine and blood, but not in the remote organs lung and liver. Administration of L-NMMA before I/R(i) decreased I/R(i)-independent basal NO levels in normoxic lung and liver without influencing I/R(i)-induced increase in NO levels in intestinal tissue or in blood. L-arginine supplementation increased circulating levels of NO, with sensitivity to L-NMMA, without affecting NO levels in normoxic or ischemic tissue. Our data suggest that NOS activity controls the NO generated in normally perfused remote organs during early I/R(i). Hence NOS inhibitors, when administered during I/R(i), decrease physiological NO levels in normoxic remote organs without affecting increased NO levels originating from ischemic intestine. This may explain the harmful effect of nonspecific NOS inhibitors during early I/R(i). In addition, the generation of NO in remote organs is not limited by tissue L-arginine concentrations and, therefore, not influenced by exogenous L-arginine. The protective effect of L-arginine supplementation during I/R(i) is probably related to increasing intravascular NO formation.


Subject(s)
Enzyme Inhibitors/pharmacology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide/biosynthesis , Reperfusion Injury/enzymology , omega-N-Methylarginine/pharmacology , Animals , Arginine/pharmacology , Disease Models, Animal , Electron Spin Resonance Spectroscopy , Intestines/drug effects , Intestines/enzymology , Liver/drug effects , Liver/enzymology , Lung/drug effects , Lung/enzymology , Male , Nitric Oxide/blood , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction
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