Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for ...proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre.
The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry.
The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia.
The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.
Tumor necrosis factor-alpha (TNF) is a major mediator of apoptosis as well as immunity and inflammation. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated ...in the pathogenesis of a wide spectrum of human diseases, including cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TNF binds to two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals. Many findings suggest an important role of phosphorylation of the TNF-R1 by number of protein kinases. Role of TNF-R2 phosphorylation on its signaling properties is understood less than TNF-R1. Other cellular substrates as TRADD adaptor protein, TRAF protein family and RIP kinases are reviewed in relation to TNF receptor-mediated apoptosis or survival pathways and regulation of their actions by phosphorylation.
Q fever is a zoonotic disease caused by Coxiella burnetii . Easy aerosol dissemination, strong environmental persistence and high infectivity make the bacterium a serious threat for humans and ...animals. A rapid, sensitive and specific test for the infectious agent is still a challenge in the field. C. burnetii expresses a spectrum of amphophilic macromolecules on its surface. Among them, a lipopolysaccharide (LPS) is of particular biological, immunological and medical significance 1 . Upon serial laboratory passages in yolk sacs of embryonated hen eggs, C.
Abstract Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive51 chromium (51 Cr) release assay is a “gold standard” for assessment of natural ...killer (NK) cytolytic activity in vitro . Several disadvantages of this assay led us to design alternative tools based on flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, spontaneous release of fluorochromes and subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column purified NK cells were used as effectors coincubated with target K-562 cells at different E:T ratios for 3 h and 90 min, respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland–Altman statistical method was applied to measure true agreement for all CAM–51 Cr, CFSE–51 Cr, DiO–51 Cr and MTG–51 Cr pairs analyzed. Based on the data, none of the four proposed methods can be stated equivalent to the standard51 Cr release assay. Considering linear relationships between data obtained with four fluorochromes and51 Cr release assay as well as linear regression analysis with R2 =0.9393 value for CAM–51 Cr pair, we found the CAM assay to be the most closely related to the51 Cr assay.
Coxiella burnetii is the aetiological agent of Q fever. LPS is a major factor of virulence of the bacterium, and therefore studies of its structure/function relationship studies are of potential ...interest. In virulent phase I, C. burnetii biosynthesises smooth LPS I with an O-specific chain, whereas in avirulent phase II, it synthesises rough LPS II 1 . Both LPSs were isolated 1 from the C. burnetii isolates RSA 493, clone 7, and RSA 439, clone 4, respectively. We investigated an LPS from the C. burnetii clonal derivative RSA 514 named ‘Crazy’ (Cr), which was isolated from the placental tissue of a guinea pig infected with the RSA 493 isolate for 343 days 2 . The major emphasis was put on the lipid A as no data on its structure have been available thus far. It has been recognised recently that variation of the lipid A domain of LPS serves as one strategy utilised 3 by Gram-negative bacteria to promote survival by providing resistance to components of the innate immune system and helping to evade recognition by Toll-like receptor 4. Thus, it was of interest to see if the long-term survival of the microorganism in the host led to modifications in its lipid A in comparison with the known structures for lipid A from the C. burnetii isolate Priscilla 4 and also those established most recently in the LPS I and LPS II (P.V. Vadovic and R.T. Toman, unpublished results).
Neuropeptides are auxiliary messenger molecules that always co-exist in nerve cells with one or more small molecule (classic) neurotransmitters. Neuropeptides act both as transmitters and trophic ...factors, and play a role particularly when the nervous system is challenged, as by injury, pain or stress. Here neuropeptides and coexistence in mammals are reviewed, but with special focus on the 29/30 amino acid galanin and its three receptors GalR1, -R2 and -R3. In particular, galanin's role as a co-transmitter in both rodent and human noradrenergic locus coeruleus (LC) neurons is addressed. Extensive experimental animal data strongly suggest a role for the galanin system in depression-like behavior. The translational potential of these results was tested by studying the galanin system in
human brains, first in normal brains, and then in a comparison of five regions of brains obtained from depressed people who committed suicide, and from matched controls. The distribution of galanin and the four galanin system transcripts in the normal human brain was determined, and selective and parallel changes in levels of transcripts and DNA methylation for galanin and its three receptors were assessed in depressed patients who committed suicide:
of transcripts, e.g., for galanin and GalR3 in LC, paralleled by a
in DNA methylation, suggesting involvement of epigenetic mechanisms. It is hypothesized that, when exposed to severe stress, the noradrenergic LC neurons fire in bursts and release galanin from their soma/dendrites. Galanin then acts on somato-dendritic, inhibitory galanin autoreceptors, opening potassium channels and inhibiting firing. The purpose of these autoreceptors is to act as a 'brake' to prevent overexcitation, a brake that is also part of
to stress that protects against depression. Depression then arises when the inhibition is too strong and long lasting - a maladaption, allostatic load, leading to depletion of NA levels in the forebrain. It is suggested that disinhibition by a galanin antagonist may have antidepressant activity by restoring forebrain NA levels. A role of galanin in depression is also supported by a recent candidate gene study, showing that variants in genes for galanin and its three receptors confer increased risk of depression and anxiety in people who experienced childhood adversity or recent negative life events. In summary, galanin, a neuropeptide coexisting in LC neurons, may participate in the mechanism underlying resilience against a serious and common disorder, MDD. Existing and further results may lead to an increased understanding of how this illness develops, which in turn could provide a basis for its treatment.
Major depressive disorder (MDD) is a substantial burden to patients, families, and society, but many patients cannot be treated adequately. Rodent experiments suggest that the neuropeptide galanin ...(GAL) and its three G protein-coupled receptors, GAL1–3, are involved in mood regulation. To explore the translational potential of these results, we assessed the transcript levels (by quantitative PCR), DNA methylation status (by bisulfite pyrosequencing), and GAL peptide by RIA of the GAL system in postmortem brains from depressed persons who had committed suicide and controls. Transcripts for all four members were detected and showed marked regional variations, GAL and galanin receptor 1 (GALR1) being most abundant. Striking increases in GAL and GALR3 mRNA levels, especially in the noradrenergic locus coeruleus and the dorsal raphe nucleus, in parallel with decreased DNA methylation, were found in both male and female suicide subjects as compared with controls. In contrast, GAL and GALR3 transcript levels were decreased, GALR1 was increased, and DNA methylation was increased in the dorsolateral prefrontal cortex of male suicide subjects, however, there were no changes in the anterior cingulate cortex. Thus, GAL and its receptor GALR3 are differentially methylated and expressed in brains of MDD subjects in a region- and sex-specific manner. Such an epigenetic modification in GALR3, a hyperpolarizing receptor, might contribute to the dysregulation of noradrenergic and serotonergic neurons implicated in the pathogenesis of MDD. Thus, one may speculate that a GAL₃ antagonist could have antidepressant properties by disinhibiting the firing of these neurons, resulting in increased release of noradrenaline and serotonin in forebrain areas involved in mood regulation.
Most solid tumors display extracellular acidosis, which only partially overlaps with hypoxia and induces distinct adaptive changes leading to aggressive phenotype. Although acidosis is mainly ...attributable to excessive production of lactic acid, it also involves carbonic anhydrase (CA) IX-mediated conversion of CO(2) to an extracellular proton and a bicarbonate ion transported to cytoplasm. CA IX is pre-dominantly expressed in tumors with poor prognosis and its transcription and activity are induced by hypoxia. Here we investigated whether low extracellular pH in absence of hypoxia can influence CA IX expression in cell lines derived from glioblastoma, a tumor type particularly linked with acidosis. Our data show that extracellular acidosis increased the level of CA IX protein, mRNA and the activity of minimal CA9 promoter that contains binding sites for HIF-1 and SP-1 transcription factors. Mutation within each of these two biding sites reduced the promoter activity, but did not eliminate the increase by acidosis. Transfection of HIF-1alpha cDNA produced additive inducing effect with acidosis. Normoxic acidosis was accompanied by HIF-1alpha protein accumulation and transiently increased phosphorylation of ERK1/2. Expression of a dominant-negative mutant of ERK2 reduced the CA9 promoter activity in both standard and acidic conditions. Similar result was obtained by inhibitors of MAPK and PI3K pathways, whose combination completely suppressed CA IX expression and abolished induction by acidosis. Altogether, our results suggest that acidosis increases the CA IX expression via a hypoxia-independent mechanism that operates through modulation of the basic CA9 transcriptional machinery.
The ability of various strains of Coxiella burnetii (C.b.) and their phase I and II lipopolysaccharides (LPSs) to induce tumor necrosis factor alpha (TNF-alpha) in peritoneal Balb/c mouse macrophages ...in vitro was investigated. Considerable differences in the induction ability were observed in dependence on the strain applied. In a TNF-alpha bioassay, the most effective inducers were both corpuscles and LPSs of the strains Priscilla and Scurry, followed by Nine Mile, Luga, and Henzerling I. In contrast, in ELISA, the most effective inducers were LPSs of the strains Luga and Henzerling, followed by Nine Mile, Priscilla, and Scurry. The role of toll-like receptor 4 (TLR4) in the induction was confirmed by the use of C3H/HeJ mouse macrophages. Thus, the induction of TNF-alpha was much higher in Balb/c mouse macrophages than that in TLR4-deficient C3H/HeJ mouse macrophages. Differences in the results of the bioassay and those of ELISA suggest a role of another secreted factor(s) induced with C.b. in murine macrophages that could act synergically with TNF-alpha in L929 cells in the bioassay. The observed differences in TNF-alpha induction might play a role in the pathobiology of Q fever.
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