Manfred Gemeiner

Proteins of rat serum, urine, and cerebrospinal fluid : VI. Further protein identifications and interstrain comparison

We have investigated the biological fluids – serum, cerebrospinal fluid, and urine – of three strains of rats; the present data extend our database (also available on‐line) and may be of interest for pharmacological and toxicological investigation. Specifically, we have defined reference maps of the major protein components in cerebrospinal fluid and urine. Compartment‐specific isoforms were recognized for transferrin and transthyretin. Mass spectrometric data established the cleavage site of the signal peptide and identified the N‐terminal blocking group of prostaglandin D synthase from rat cerebrospinal fluid. A previously undescribed member of the family of low molecular mass rat urinary proteins was characterized as containing a sequence similar, but not identical, to the N‐terminal region of rat urinary protein‐2 (RUP‐2), and divergent from RUP‐1.

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.

Acute-Phase Proteins Before Cerebral Ischemia in Stroke-Prone Rats Identification by Proteomics

Background and Purpose— A high degree of proteinuria has been reported in stroke-prone spontaneously hypertensive rats (SHRSP). We studied the effect of salt loading on the detailed protein pattern of serum and urine in 3 rat strains: Wistar-Kyoto, spontaneously hypertensive rats, and SHRSP, an inbred animal model for a complex form of cerebrovascular disorder resembling the human disease. Methods— Rats were given a permissive diet and received 1% NaCl in drinking water. The protein pattern in body fluids was assessed over time by 2-dimensional electrophoretic analysis. Brain alterations were monitored by MRI and histology. Results— Several proteins were excreted in urine after weeks of treatment and in advance of stroke: transferrin, hemopexin, albumin, &agr;2-HS-glycoprotein, kallikrein-binding protein, &agr;1-antitrypsin, Gc-globulin, and transthyretin. Markers of an inflammatory response, including very high levels of thiostatin, were detected in the serum of SHRSP at least 4 weeks before a stroke occurred. Conclusions— In SHRSP subjected to salt loading, an atypical inflammatory condition and widespread alterations of vascular permeability developed before the appearance of anomalous features in the brain detected by MRI. Urinary concentrations of each of the excreted serum proteins correlated positively with time before stroke occurred.

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction

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-α 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.

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.

A proteomic reference map for pig serum proteins as a prerequisite for diagnostic applications.

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.

Delay in processing porcine whole blood affects cytokine expression.

Quantification of cytokine messenger ribonucleic acid (mRNA) in blood samples has become an important tool in the investigation of immune cell activation in a variety of clinical settings. It has been shown that the method of sample collection and processing influences the levels of several cytokine mRNAs. Therefore, it is generally accepted that blood samples for analysis of cytokine expression be processed as soon as possible and under standardised conditions. Since immediate sample processing is not always possible, we investigated the effect of different storage conditions (room temperature (Rt) and 4 degrees C) and storage times (1, 2, 4, 6 and 24 h) on the mRNA level of different cytokines (IL-1alpha, IL-2, IL-6, IL-8, IL-10, IFN-gamma), as well as the IL-2 receptor (IL-2R) in porcine whole blood samples (n=8). Quantification of cytokine expression was performed using simultaneous reverse transcription PCR (RT-PCR) combined with the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference. Our data demonstrate that delays in sample processing longer than 1 h result in significant changes of the mRNA levels of individual cytokines. Expression of the monokines IL-1alpha, IL-6 and IL-10 were increased by storage at both room temperature and 4 degrees C. Expression of IL-8 was increased only in the samples stored at room temperature, and expression of IFN-gamma was raised exclusively in the samples stored at 4 degrees C. We conclude that porcine blood samples should be processed within 2 h to prevent undesired stimulatory effects on the cytokine expression pattern. However, if only selected cytokines are investigated, the undesired effects of prolonged storage can be selectively suppressed by choosing the appropriate temperature of sample storage.

Proteins of rat serum: III. Gender‐related differences in protein concentration under baseline conditions and upon experimental inflammation as evaluated by two‐dimensional electrophoresis

We have previously described the major components of rat serum (Electrophoresis 1998, 19, 1484—1492 and 1493—1500). In this report we examine sex‐related differences in protein concentrations, both in control animals and upon experimentally induced inflammation. Under baseline conditions approximately one third of the spots resolved in serum by two‐dimensional electrophoresis (2‐DE) are expressed at levels ≥ 25% higher in female rats than in male rats and a further 10% at levels ≥ 25% lower. Inflammation increases the expression of the positive acute‐phase reactants: hemopexin, ceruloplasmin, α1‐antitrypsin (all approximately 2‐fold), C‐reactive protein (3‐ to 5‐fold), serine protease inhibitor‐3 (4‐ to 5‐fold), thiostatin (> 5‐fold in females, > 20‐fold in males), clusterin, orosomucoid, haptoglobin chains and α2‐macroglobulin. The baseline level of the last four markers is below the detection limit, hence no percent increase can be computed. Conversely, negative acute‐phase reactants are reduced on inflammation: α1‐inhibitor III, α2‐HS‐glycoprotein, kallikrein‐binding protein and transthyretin (all reduced to between 1/2 to 1/3 of the baseline levels), retinol‐binding protein (to about 1/2 to 1/4) and albumin (to 2/3). Except for thiostatin, the changes in acute‐phase protein levels are similar in male and female rats.

Proteins of rat serum V: adjuvant arthritis and its modulation by nonsteroidal anti-inflammatory drugs.

The effect of adjuvant arthritis (AA) on the pattern of rat serum proteins includes the upregulation of haptoglobin, orosomucoid, α2‐macroglobulin, serine protease inhibitor‐3, thiostatin, α1‐antitrypsin, C‐reactive protein, and the downregulation of kallikrein‐binding protein, α1‐inhibitor III, apolipoprotein A‐I, α2‐HS‐glycoprotein, albumin, apolipoprotein A‐IV, transthyretin and transferrin. Minor changes (± 20%) are observed for Gc‐globulin, ceruloplasmin, and α1‐macroglobulin. AA thus grossly resembles the acute inflammatory response elicited by the injection of turpentine, although the changes in the levels of negative acute‐phase proteins (APP) are smaller in acute inflammation. Indomethacine and ibuprofen inhibit the effects of arthritis on the synthesis of rat serum proteins in different ways: The former is, on average, three times as effective as the latter. Each drug interferes differently with different proteins. In animals without AA, both nonsteroidal anti‐inflammatory drugs (NSAID) mimick the inflammatory pattern to a certain extent, with more effect on the negative than on the positive APPs. Overall, the shifts in serum protein levels parallel changes in inflammatory parameters such as joint swelling and serum interleukin‐6 (IL‐6) activity. Protein quantitation after two‐dimensional electrophoresis (2‐DE) reveals some effects of the drugs per se which escape detection by other routine tests.

Organ specific formation of nitrosyl complexes under intestinal ischemia/reperfusion in rats involves NOS-independent mechanism(s).

Intestinal ischemia/reperfusion may lead to local and distant organ damage involving nitric oxide (NO). NO rapidly reacts with heme/non-heme-iron-yielding nitrosyl complexes, which can be determined directly by electron paramagnetic resonance spectroscopy. The aim of the present study was to characterize nitrosylation reactions induced by transient intestinal ischemia in blood and tissues. We used electron paramagnetic resonance spectroscopy and reverse transcription polymerase chain reaction analyses to estimate nitrosyl complex levels and inducible NO synthase mRNA expression in rats subjected to superior mesenteric artery occlusion for 60 min followed by the reperfusion. Nitrosyl hemoglobin concentrations in circulating blood were significantly increased during ischemia and reperfusion. Nitrosyl hemoglobin complexes were detected in ischemic intestine, but not in normoxic lung and liver or reperfused intestine. Administration of N-G-monomethyl-L-arginine, a non-specific NO synthase inhibitor, did not affect the formation of circulating nitrosyl complexes. Moreover, inducible NO synthase mRNA was not found in intestinal tissues at 30 min of reperfusion. Our data suggest an organ-specific NO formation indicated by the increased nitrosylation reaction in ischemic intestinal tissue, but not in the distant normoxic organs, in spite of high circulating nitrosyl hemoglobin levels. NO involved in nitrosylation under intestinal ischemia/reperfusion is probably formed by NO synthase-independent mechanism(s).