Biomedical subjects

Manfred Gemeiner

Publications and source records attributed to Manfred Gemeiner.

12 recordsLinked to original sources

PubMed2006

Gender differences in endothelial function and inflammatory markers along the occurrence of pathological events in stroke-prone rats.

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.

Acute-Phase Proteins↗

PubMed2006

Opposite effects of endotoxin on mitochondrial and endoplasmic reticulum functions.

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.

Animals↗

PubMed2006

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock.

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.

Animals↗

PubMed2006

Contribution of cell culture additives to the two-dimensional protein patterns of mouse macrophages.

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.

Animals↗

PubMed2005

MMTV accessory factor Naf affects cellular gene expression.

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.

Animals↗

PubMed2004

Analysis of pathological events at the onset of brain damage in stroke-prone rats: a proteomics and magnetic resonance imaging approach.

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.

Animals↗

PubMed2004

Nonreducing two-dimensional gel electrophoresis for the detection of Bence Jones proteins in serum and urine.

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.

Bence Jones Protein↗

PubMed2004

The serum proteome of Equus caballus.

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.

Albumins↗

PubMed2003

Parameters of humoral and cellular immunity following vaccination of pigs with a European modified-live strain of porcine reproductive and respiratory syndrome virus (PRRSV).

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.

Animals↗

PubMed2002

Monitoring the effects of drug treatment in rat models of disease by serum protein analysis.

In this review we list from literature investigations on rat serum proteins using electrophoretic techniques in connection with drug testing. From our own research work, we provide annotated two-dimensional maps of rat serum proteins under control and experimental conditions. Emphasis is on species-specific components and on the effects of acute and chronic inflammation. We discuss our project of structural proteomics on rat serum as a minimally invasive approach to pharmacological investigation, and we outline a typical experimental plan for drug testing according to the above guidelines. We then report in detail on the results of our trials of anti-inflammatory drugs on adjuvant arthritis, an animal model of disease resembling in many aspects human rheumatoid arthritis. We demonstrate a correlation between biochemical parameters and therapeutic findings and outline the advantages of the chosen methodological approach, which proved also sensitive in revealing "side effects" of the test drugs. In an appendix we describe our experimental protocol when performing two-dimensional electrophoresis of rat serum.

Animals↗

PubMed2002

Strategies for proteomics with incompletely characterized genomes: the proteome of Bos taurus serum.

A reference map for Bos taurus serum was obtained using proteomic tools: 21 proteins, plus several serum albumin fragments, have been identified in 47 spots. One of the major acute-phase reactants, haptoglobin, was also detected in a pathological serum. A number of technical problems had to be solved. (i) Spot resolution in two-dimensional electrophoresis (2-DE) is not easily optimized, as several proteins have similar molecular mass; different polyacrylamide concentration gradients were used for the analysis of various size ranges. (ii) Identification of proteins through mass spectrometry (MS) procedures is also difficult as the genome of Bos taurus is incompletely characterized. The program FASTS proved particularly useful, since it allows simultaneous searching of several unordered sequence fragments, which may be individually too short to provide a statistically valid match using BLAST.

Amine Oxidase (Copper-Containing)↗

PubMed2002

Nitric oxide synthase inhibitors affect nitric oxide synthesis in normoxic but not in ischemic organs during intestinal ischemia and early reperfusion.

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.

Animals↗