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Biomedical subjects

Maria Zellner

Publications and source records attributed to Maria Zellner.

8 recordsLinked to original sources
PubMed2005

Reduced stress tolerance of glutamine-deprived human monocytic cells is associated with selective down-regulation of Hsp70 by decreased mRNA stability.

In critically ill patients, clinicians observe a reverse correlation of survival and a decreased plasma concentration of the most abundant free amino acid, glutamine (Gln). However, in this context, the role of Gln remains largely elusive. Gln is used as an energy substrate by monocytes. Gln deprivation of these cells results in an increased susceptibility to cell stress and apoptosis, as well as in a reduced responsiveness to pro-inflammatory stimuli. We performed a systematic study to elucidate the molecular mechanism by which Gln depletion affects the heat stress response of the monocytic cell line U937. Proteomic analysis revealed that Gln depletion was associated with specific changes in the protein expression pattern. However, the overall level of tRNA-bound Gln remained unaffected. The stress protein heat shock protein (Hsp) 70 showed the highest reduction in protein synthesis. This was due to enhanced mRNA decay during Gln starvation while the transcriptional and the translational control of Hsp70 expression remained unchanged. A physiological Gln concentration and above was found to be necessary for maximum Hsp70 accumulation upon heat shock. Thus, the study shows a specific link between Gln metabolism and the regulation of heat shock proteins.

Cells, Cultured↗
PubMed2005

Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets.

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.

Adult↗
PubMed2005

Increased susceptibility of glutamine-depleted monocytes to fever-range hyperthermia: the role of 70-kDa heat shock protein.

OBJECTIVE: This study investigates the effect of fever-range hyperthermia on Gln-starving monocytes and the role of the 70-kDa heat shock protein Hsp70. SUMMARY BACKGROUND DATA: Fever is a protective acute-phase response to infection. However, in critically ill patients, the harmful effects of fever seem to be predominant. Critical illness is frequently associated with reduced plasma glutamine (Gln) levels, which contribute to the immune suppression in these patients due to impaired monocyte function. METHODS: Isolated monocytes were suspended in Gln-depleted medium and exposed to 41 degrees C. Cell survival was determined by an MTT-based assay, and phagocytosis of Escherichia coli was measured by flow cytometry. Expression of Hsp70 was determined by Western blot. RESULTS: Hyperthermia for 300 minutes strongly decreased the viability of Gln-depleted monocytes (85%), whereas it had only a moderate effect on Gln-supplied cells (45%, P < 0.05). Shorter treatments (45 minutes) of Gln-starving monocytes had almost no effects on viability but decreased the phagocytosis activity by 30.8%. In addition, the expression of Hsp70 was inhibited almost completely. CONCLUSION: These data show that Gln-starving monocytes have a reduced thermoresistance. This suggests that elevated body temperature damages monocytes in critically ill patients with reduced plasma Gln-levels possibly via an inhibition of the cytoprotective protein Hsp70.

Cell Survival↗
PubMed2004

Platelet function in mitochondriopathy with stroke and stroke-like episodes.

Stroke and stroke-like episodes are frequent complications in mitochondriopathy, particularly in MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes) which is a disorder of the mitochondrial oxidative metabolism in diverse cell types. To clarify a possible pathological aspect of stroke in these patients, we investigated platelet function before and after physical exercise. Ten patients with mitochondriopathy and stroke and ten healthy sex and age matched controls were investigated in an analyst blinded, prospective cross-sectional trial. Exercise decreased intraplatelet adenosine triphosphate (ATP) concentrations by -22% from baseline in patients with mitochondriopathy (p<0.01 between groups) while exercise increased ATP-levels by 28% healthy controls (p=0.01 vs baseline). Thrombin receptor activating peptide (TRAP) stimulated P-selectin expression increased up to 50% (p<0.05) in healthy subjects following exercise compared to 39% (p>0.05) in patients with mitochondriopathy. Exercise trendwise decreased platelet plug formation under shear stress by 24% in patients as measured by the platelet function analyzer PFA-100(R). Tromboelastography showed firm thrombus formation and delayed lysis in patients following exercise. In conclusion, this trial has shown that ATP depletion during and after exercise probably accounts for a defective oxidative metabolism in platelets of patients with mitochondriopathy and stroke. This might induce decreased platelet function in these patients but fails to explain the increased stroke rate. Therefore other mechanisms seem to be etiologically involved in the pathogenesis of stroke in patients with mitochondriopathy.

Adenosine Triphosphate↗
PubMed2003

Glutamine starvation of monocytes inhibits the ubiquitin-proteasome proteolytic pathway.

Peripheral blood monocytes utilize free glutamine (Gln) in addition to glucose as an important energy substrate. Although this demand increases upon activation, monocytes are commonly confronted with decreased plasma Gln during critical illness and thus suffer from Gln-starvation. Here we investigate the influence of Gln-starvation on protein stability and its effects on the monocyte proteome. Gln-starvation caused a reduction of protein degradation which was accompanied by an accumulation of ubiquitin-protein conjugates and a reduction of intracellular ATP. Similar effects were observed under ATP-reducing conditions and in the presence of a proteasome inhibitor. Using two-dimensional gel electrophoresis we identified the IL-1beta precursor protein (pIL-1beta) as the, by far, most induced protein in endotoxin-treated monocytes. The degradation of the short-lived pIL-1beta was strongly reduced during Gln-starvation, while the degradation of the long-lived, constitutively expressed beta-actin was less affected. This indicates that although Gln-starvation reduces protein breakdown on the overall proteasome level, it leads to differential changes in the stability of specific proteins. This selective effect is likely to contribute to the immunocompromised state of monocytes commonly observed during critical illness.

Actins↗
PubMed2002

Human monocyte stimulation by experimental whole body hyperthermia.

The thermal effect of fever, an evolutionarily conserved acute-phase response, has been associated with better survival and a shorter duration of disease in cases of infection. The molecular consequence of this beneficial fever response is poorly understood. To determine the influence of hyperthermia on human monocytes, which are important for the recognition and elimination of pathogens, twelve healthy volunteers were immersed in a 39.5 degrees C hot water bath to increase their body temperature. The expression of the endotoxin receptor CD14 and the complement receptor CD11b increased after the hot water bath (P < 0.05), whereas the expression of the selectin CD62L, which mediates the initial attachment of leukocytes at the endothelium during inflammation, was downregulated after hyperthermia (P < 0.05). Comparable changes in monocyte receptor expression were observed after in vitro hyperthermia. Furthermore, 3 hours after in vivo hyperthermia, the response of monocytes to endotoxin was enhanced in an ex vivo lipopolysaccharide stimulation assay, as expressed by a greater TNF-alpha release (P < 0.05). We conclude that the thermal effect of fever directly activates monocytes, which increases their ability to respond to bacterial challenge.

Adult↗
PubMed2002

Glutamine depletion impairs cellular stress response in human leucocytes.

During sepsis and major trauma the blood glutamine (Gln) level is reduced. The administration of Gln can improve the outcome of these patients. However, the mechanism of this beneficial effect of Gln is poorly understood. In the course of critical illness leucocytes are confronted with cytotoxic inflammatory mediators. To protect themselves against these factors, cells express heat shock proteins (HSP). Previous studies have shown that the expression of the major inducible HSP (HSP70) is improved by high Gln concentrations above 4 mM. In this study we investigated whether Gln depletion, such as observed during critical illness, has an effect on HSP70 expression. Human lymphocytes exposed for 2 h to 42 degrees C showed a 3-fold increase in HSP70 expression (P<0.01). A preceding Gln starvation period over 3 days had no influence on this increase. However, when Gln is reduced during the stress response, HSP70 expression is impaired. A reduction of Gln from 0.5 mM (physiological) to 0.125 mM (pathological) led to a 40% lower HSP70 level (P<0.002). In contrast, increasing Gln concentrations (up to 2 mM) had only minor stimulatory effects (about 15%). This Gln-dependency of heat mediated HSP70 expression was observed in resting as well as proliferating lymphocytes. Our data indicate that during periods of reduced plasma Gln levels the stress response of human lymphocytes is impaired. Thus, Gln may be essential to minimize the susceptibility of leucocytes to cytotoxic inflammatory mediators. This is a new aspect of the protective effect of Gln supplementation in critically ill patients.

Cell Division↗