Background Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. Methods We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. Results We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. Conclusion PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm.

Hepatic nerves have a complex role in synchronizing liver metabolism. Here, we used three-dimensional (3D) immunoimaging to explore the integrity of the hepatic nervous system in experimental and human nonalcoholic fatty liver disease (NAFLD). We demonstrate parallel signs of mild degeneration and axonal sprouting of sympathetic innervations in early stages of experimental NAFLD and a collapse of sympathetic arborization in steatohepatitis. Human fatty livers display a similar pattern of sympathetic nerve degeneration, correlating with the severity of NAFLD pathology. We show that chronic sympathetic hyperexcitation is a key factor in the axonal degeneration, here genetically phenocopied in mice deficient of the Rac-1 activator Vav3. In experimental steatohepatitis, 3D imaging reveals a severe portal vein contraction, spatially correlated with the extension of the remaining nerves around the portal vein, enlightening a potential intrahepatic neuronal mechanism of portal hypertension. These fundamental alterations in liver innervation and vasculature uncover previously unidentified neuronal components in NAFLD pathomechanisms.

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 postmortem 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: upregulation of transcripts, e.g., for galanin and GalR3 in LC, paralleled by a decrease 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 resilience 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.

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 postmortem 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: upregulation of transcripts, e.g., for galanin and GalR3 in LC, paralleled by a decrease 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 resilience 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.

Sickness responses to lipopolysaccharide (LPS) were examined in mice with deletion of the interleukin (IL)-1 type 1 receptor (IL-1R1). IL-1R1 knockout (KO) mice displayed intact anorexia and HPA-axis activation to intraperitoneally injected LPS (anorexia: 10 or 120µg/kg; HPA-axis: 120µg/kg), but showed attenuated but not extinguished fever (120µg/kg). Brain PGE2 synthesis was attenuated, but Cox-2 induction remained intact. Neither the tumor necrosis factor-α (TNFα) inhibitor etanercept nor the IL-6 receptor antibody tocilizumab abolished the LPS induced fever in IL-1R1 KO mice. Deletion of IL-1R1 specifically in brain endothelial cells attenuated the LPS induced fever, but only during the late, 3rd phase of fever, whereas deletion of IL-1R1 on neural cells or on peripheral nerves had little or no effect on the febrile response. We conclude that while IL-1 signaling is not critical for LPS induced anorexia or stress hormone release, IL-1R1, expressed on brain endothelial cells, contributes to the febrile response to LPS. However, also in the absence of IL-1R1, LPS evokes a febrile response, although this is attenuated. This remaining fever seems not to be mediated by IL-6 receptors or TNFα, but by some yet unidentified pyrogenic factor.

The galanin family currently consists of four peptides, namely galanin, galanin-message associated peptide, galanin-like peptide and alarin. Unlike galanin that signals through three different G protein-coupled receptors; GAL1, GAL2, and GAL3, binding at its N-terminal end, the cognate receptors for other members of the galanin family are currently unknown. Research using short N-terminal galanin fragments generated either by enzymatic cleavage or solid-phase synthesis has revealed differences in their receptor binding properties exerting numerous biological effects distinct from galanin(1-29) itself. Our studies on tissue extracts derived from rat small intestine and bovine gut using chromatographic techniques and sensitive galanin(1-16)-specific radioimmunoassay revealed the presence of immunoreactive compounds reacting with antiserum against galanin(1-16) distributed in distinct elution volumes. These results suggested a possible presence of short N-terminal galanin fragments also in vivo. Moreover, employing immunoaffinity chromatography and reverse-phase high performance liquid chromatography (HPLC) followed by mass spectrometry allowed specific enrichment of these immunoreactive compounds from rat tissues and identification of their molecular structure. Indeed, our study revealed presence of several distinct short N-terminal galanin sequences in rat tissue. To prove their receptor binding, four of the identified sequences were synthetized, namely, galanin(1-13), galanin(1-16), galanin(1-20), galanin(6-20), and tested on coronal rat brain sections competing with 125I-labeled galanin(1-29). Our autoradiographs confirmed that galanin(1-13), galanin(1-16), and galanin(1-20) comprehensively displaced 125I-galanin(1-29) but galanin(6-20) did not. Here we show, for the first time, that short N-terminal galanin fragments occur naturally in rat tissues and that similar or identical galanin sequences can be present also in tissues of other species. BIOLOGICAL SIGNIFICANCE:This study is first to provide an evidence of the presence of short N-terminal galanin fragments in vivo in a biological system and provides further foundations for the previous studies using synthetized short N-terminal galanin fragments.

Aniridia is a congenital pan-ocular disorder caused by haplo-insufficiency of Pax6, a crucial gene for proper development of the eye. Aniridia affects a range of eye structures, including the cornea, iris, anterior chamber angle, lens, and fovea. The ocular surface, in particular, can be severely affected by a progressive pathology termed aniridia-associated keratopathy (AAK), markedly contributing to impaired vision. The purpose of this review is to provide an update of the current knowledge of the genetic, clinical, micro-morphological, and molecular aspects of AAK. We draw upon material presented in the literature and from our own observations in large aniridia cohorts. We summarize signs and symptoms of AAK, describe current options for management, and discuss the latest research findings that may lead to better diagnosis and new treatment or prevention strategies for this debilitating ocular surface condition.

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.

Aniridia is a rare congenital genetic disorder caused by haploinsuffiency of the PAX6 gene, the master gene for development of the eye. The expression of tear proteins in aniridia is unknown. To screen for proteins involved in the aniridia pathophysiology, the tear fluid of patients with diagnosed congenital aniridia was examined using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two-dimensional map of tear proteins in aniridia has been established and 7 proteins were differentially expressed with P < 0.01 between aniridia patients and control subjects. Five of them were more abundant in healthy subjects, particularly alpha-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1 and two other proteins, zinc-alpha 2-glycoprotein and lactoferrin were more expressed in the tears of aniridia patients. Moreover, immunoblot analysis revealed elevated levels of vascular endothelial growth factor (VEGF) in aniridia tears which is in concordance with clinical finding of pathological blood and lymph vessels in the central and peripheral cornea of aniridia patients. The proteins with different expression in patients' tears may be new candidate molecules involved in the pathophysiology of aniridia and thus may be helpful for development of novel treatment strategies for the symptomatic therapy of this vision threatening condition.Biological significanceThis study is first to demonstrate protein composition and protein expression in aniridic tears and identifies proteins with different abundance in tear fluid from patients with congenital aniridia vs. healthy tears. (C) 2013 Elsevier B.V. All rights reserved.

Background: 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.Methods: 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.Results: 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 G(alpha 0) 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.Conclusion: The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

Recent proteomic studies of C. burnetii, the etiological agent of Q fever, have brought a deeper insight into the pathogen's physiology and offered new possibilities in investigations of inter- or intra-species relatedness. The data generated from these studies in conjunction with the current genomic sequence databases may reveal additional identities for conserved and unique C. burnetii biomarkers and aid in creating algorithms and/or databases that could develop into diagnostic and detection tools for the pathogen. Moreover, wide scale screening and further characterization of potential C. burnetii protein antigens along with a comprehensive evaluation of the humoral immune response will be of fundamental importance towards research and development of a safe and efficacious vaccine as well as improved serodiagnostic tests for rapid and sensitive detection of the Q fever pathogen. Given these advances, proteomics may make marked contributions to the improvement of human health protection against C. burnetii in the coming years.

This chapter contains sections titled: Introduction Composition and Structure of Lipid A of P. salmonis Composition and Structure of Lipid A of C. burnetii Conclusion Acknowledgments References

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacterium and a highly infectious pathogen. The disease is a widespread zoonosis and is endemic throughout the world. An easy aerosol dissemination, environmental persistence, and high infectivity make the bacterium a serious threat for humans and animals. Lipopolysaccharide is considered one of the major factors of virulence expression and infection of the bacterium. Detailed glycomic studies enabled to better understand structural and functional peculiarities of this biopolymer and its role in pathogenesis and immunity of Q fever. Recent proteomic studies of C. burnetii have brought new approaches in accurate detection of the infectious agent and offered new insights into the inter- or intra-species relatedness. Thus, structure/function relationship studies are currently of utmost importance in the field. This paper will focus on glycomic and proteomic approaches providing information on unique glycan and protein species of the microorganism as the candidate molecules for the use in detection/diagnosis, therapy, and prophylaxis.

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 [1Toman R Lipopolysaccharides from virulent and low-virulent phases of Coxiella burnetii.in: Raoult D Brouqui P Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium. Elsevier, Paris1999: 84Google Scholar]. Upon serial laboratory passages in yolk sacs of embryonated hen eggs, C. burnetii undergoes a virulent (phase I) to avirulent (phase II) variation. This is accompanied by modifications in both composition and structure of the LPS [1Toman R Lipopolysaccharides from virulent and low-virulent phases of Coxiella burnetii.in: Raoult D Brouqui P Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium. Elsevier, Paris1999: 84Google Scholar]. In virulent phase I, the bacterium biosynthesises an LPS that contains in the O-specific chain besides some frequently occurring sugars two unusual ones, virenose (Vir, 6-deoxy-3-C-methyl-D-gulose) and dihydrohydroxystreptose [Strep, 3-C-(hydroxymethyl)-L-lyxose] [1Toman R Lipopolysaccharides from virulent and low-virulent phases of Coxiella burnetii.in: Raoult D Brouqui P Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium. Elsevier, Paris1999: 84Google Scholar]. Both sugars have not been found in LPSs of other enterobacterial species and are considered unique biomarkers of C. burnetii. In previous work it was suggested that Vir and Strep are located in terminal positions [1Toman R Lipopolysaccharides from virulent and low-virulent phases of Coxiella burnetii.in: Raoult D Brouqui P Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium. Elsevier, Paris1999: 84Google Scholar] but recent findings show that Vir is also (1 → 4)-linked [2Vadovic P Slaba K Fodorova M et al.Structural and functional characterization of the glycan antigens involved in immunobiology of Q fever.Ann N Y Acad Sci. 2005; 1063: 149-153Crossref PubMed Scopus (14) Google Scholar]. The presence of unique biomarkers Vir and Strep in the C. burnetii LPS and their favourable location in its O-specific chain provide an excellent opportunity to generate monoclonal antibodies (mAbs) against them that would enable rapid, sensitive and specific detection of the virulent form of the bacterium. In this paper, we report on such a mAb that was highly specific for Vir in the C. burnetii LPS. The mAb (IgG2b subclass) was generated in response to the Vir-rich O-polysaccharide fraction of the C. burnetii isolate Nine Mile (NM) LPS conjugated to tetanus toxoid. The following C. burnetii isolates were tested with the mAb: NM, Priscilla (Pr), S, 48, 1/IIA, IXO, DER, L35, 27, RAK8, L, Henzerling (He), Florian and LUGA (all in phase I), NM, Pr, He (all in phase II), and a laboratory variant of the NM isolate 'Crazy' (Cr, intermediate to phases I and II). Their origin is given [3Svraka S Toman R Skultety L et al.Establishment of a genotyping scheme for Coxiella burnetii.FEMS Microbiol Lett. 2006; 254: 268-274Crossref PubMed Scopus (75) Google Scholar, 4Hackstadt T Peacock MG Hitchcock PJ et al.Lipopolysaccharide variation in Coxiella burnetii: Intrastrain heterogeneity in structure and antigenicity.Infect Immun. 1985; 48: 359-365Crossref PubMed Google Scholar] elsewhere. In addition, the phase I LPSs from the isolates NM, Pr, S, 48, 1/IIA, IXO, He and LUGA; the phase II LPSs from NM, Pr and He; and the LPS from Cr were tested, too. Further, the cells of some selected members of the families Rickettsiaceae (Rickettsia conorii, R. rickettsii, R. slovaca, R. typhi and R. sibirica), Piscirickettsiaceae (Piscirickettsia salmonis), Chlamydiaceae (Chlamydophila psittaci and C. abortus) and Enterobacteriaceae (Salmonella typhosa and Escherichia coli) were examined for possible cross-reactivity with the mAb. Likewise, LPSs from R. typhi, R. canada, R. prowazekii, R. tsutsugamushi and S. typhosa were submitted to analyses with the mAb in ELISA and immunoblot assays. In immunoblots, cells and LPSs were separated in 15% polyacrylamide gels, transferred to PVDF membrane and examined with the mAb diluted 1:5000 in blocking buffer at 20°C for 1 h. After washing, the membrane was incubated with anti-mouse IgG Ab conjugated with horseradish peroxidase. The immunoreaction was visualised with the light-emitting substrate detection system. ELISA was carried out with the same samples according to Slaba et al. [5Slaba K Skultety L Toman R Efficiency of various serological techniques for diagnosing Coxiella burnetii infection.Acta Virol. 2005; 49: 123-127PubMed Google Scholar]. The mAb serially diluted from 1:1000 (220 ng/well) was used as the primary Ab. Immunoblot and ELISA studies have shown that only Vir-containing C. burnetii cells and LPSs were recognised with the mAb. In immunoblot, variability in the immunoreactivity of phase I LPSs and isolates was seen in both distribution of the individual bands and their intensities in the most reactive region of about 14–36 kDa (Fig. 1). In contrast, no binding was observed with the phase II and Cr cells or their LPSs. Vir was absent in all these samples. Likewise, no signals were observed with the cells and LPSs from the selected species of the families Rickettsiaceae, Piscirickettsiaceae, Chlamydiaceae and Enterobacteriaceae. The same results were obtained in ELISA. Positive reactions of the cells and LPSs of C. burnetii isolates in phase I with the mAb were reflected in their corresponding titres. As in immunoblot studies, no binding was observed with the cells and LPSs of phase II isolates or the Cr variant, nor with other bacterial species mentioned above. In conclusion, the results have shown that immunoblotting and ELISA with the newly generated mAb could be useful tools for both sensitive and specific detection of the virulent form of C. burnetii and for differentiation of the individual isolates. This work was supported in part by the Slovak Research and Development Agency, Slovak Republic, under the contract No. APVT-51-032804 and by grant No. 2/6044/27 of the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences.

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 [1Toman R Lipopolysaccharides from virulent and low-virulent phases of Coxiella burnetii.in: Raoult D Brouqui P Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium. Elsevier, Paris1999: 84Google Scholar]. Both LPSs were isolated [1Toman R Lipopolysaccharides from virulent and low-virulent phases of Coxiella burnetii.in: Raoult D Brouqui P Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium. Elsevier, Paris1999: 84Google Scholar] 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 [2Hackstadt T Peacock MG Hitchcock PJ et al.Lipopolysaccharide variation in Coxiella burnetii: Intrastrain heterogeneity in structure and antigenicity.Infect Immun. 1985; 48: 359-365Crossref PubMed Google Scholar]. 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 [3Alexander C Rietschel ET Bacterial lipopolysaccharides and innate immunity.J Endotoxin Res. 2001; 7: 167-202PubMed Google Scholar] 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 [4Toman R Garidel P Andra J et al.Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities.BMC Biochem. 2004; (Accessed 12 Nov 2008.)http://www.biomedcentral.com/1471-2191/5/1PubMed Google Scholar] and also those established most recently in the LPS I and LPS II (P.V. Vadovic and R.T. Toman, unpublished results). The isolate Cr underwent large deletions in genes predicted to function in LPS or lipooligo-saccharide biosynthesis [5Hoover TA Culp DW Vodkin MH et al.Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (Crazy), of the Coxiella burnetii Nine Mile strain.Infect Immun. 2002; 70: 6726-6733Crossref PubMed Scopus (88) Google Scholar]. Thus, it was of interest to compare chemical compositions of the lipid A proximal regions in the LPS Cr and the LPS II from the isolate RSA 439, which was also shown to undergo similar deletions [5Hoover TA Culp DW Vodkin MH et al.Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (Crazy), of the Coxiella burnetii Nine Mile strain.Infect Immun. 2002; 70: 6726-6733Crossref PubMed Scopus (88) Google Scholar]. The LPS Cr was isolated by the hot phenol/water method and run on SDS-PAGE. Lipid A was obtained on mild acid hydrolysis of the LPS Cr. Sugars were analysed as alditol acetates and fatty acids were examined directly and after trimethylsilylation by GC-MS. For its detailed structure, the lipid A was analysed by MALDI-TOF- and MALDI-TOF/TOFS techniques. In contrast to the LPS I and LPS II [1Toman R Lipopolysaccharides from virulent and low-virulent phases of Coxiella burnetii.in: Raoult D Brouqui P Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium. Elsevier, Paris1999: 84Google Scholar], the LPS Cr gave one band at about 14 kDa on SDS-PAGE. Sugar analysis revealed the presence of D-mannose, D-glucose, D-glycero-D-manno-heptose, dihydrohydroxystreptose [3-C-(hydroxymethyl)-L-lyxofuranose], and D-glucosamine (GlcN) in a molar ratio 3.1:0.1:1.0:1.5:1.2, respectively. No virenose (6-deoxy-3-C-methyl-D-gulopyranose) was found in contrast with the LPS I [1Toman R Lipopolysaccharides from virulent and low-virulent phases of Coxiella burnetii.in: Raoult D Brouqui P Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium. Elsevier, Paris1999: 84Google Scholar]. Analyses of fatty acids indicated a high degree of heterogeneity in their nature and length. From the hydroxy fatty acids, the most abundant were 3-hydroxyhexa- and pentadecanoic acids, and from the non-hydroxy fatty acids, hexa- and pentadecanoic acids were the most prevalent. The MALDI-MS analyses of lipid A revealed the presence of clusters of five diphosphorylated and five monophosphorylated singly charged [M-H]– ions. The ions in both clusters differed one from another by 14 Da. The tandem mass spectrometry (MS/MS) analyses of the selected ions gave information on fatty acid distribution along the D-GlcN disaccharide. From the data obtained we conclude that the major lipid A species comprises a tetraacyl form that consists of the ß-(1 → 6)-linked D-GlcN disaccharide backbone carrying two phosphate groups (Fig. 1). One is linked to the glycosidic hydroxyl group of GlcN I and the other is ester-linked to the O-4' position of GlcN II. The amide-linked fatty acids are represented exclusively by 3-hydroxyhexadecanoic acids located at the N-2 and N-2' positions and the ester-linked fatty acids are represented by one 3-hydroxypentadecanoic and one pentadecanoic acids at the O-3' and O-3 positions, respectively. We also presume that the observed high heterogeneity of lipid A is due to the fatty acid length variations at the O-3 and O-3' positions. This was clearly seen in both MS and MS/MS spectra. It is evident that structural features of the investigated lipid A (Fig. 1) resemble those found [4Toman R Garidel P Andra J et al.Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities.BMC Biochem. 2004; (Accessed 12 Nov 2008.)http://www.biomedcentral.com/1471-2191/5/1PubMed Google Scholar] for lipid A from the C. burnetii isolate Priscilla, and also those established most recently for lipids A present in the LPS I and LPS II [PV Vadovic and RT Toman, unpublished results]. All these lipids A exhibited low endotoxic activity. It appears that the lipid A represents the most conserved region also in the LPSs of C. burnetii isolates. In conclusion, the investigation of lipid A revealed no substantial structural modifications when compared with lipids A of other C. burnetii isolates despite the long-term survival of the microorganism in the host. The lipid A proximal region of the LPS Cr is more complex in chemical composition than that of the LPS II despite the fact that chromosomal deletions in the RSA 514 isolate were larger, extending on both ends beyond the RSA 439 isolate deletion junctions [5Hoover TA Culp DW Vodkin MH et al.Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (Crazy), of the Coxiella burnetii Nine Mile strain.Infect Immun. 2002; 70: 6726-6733Crossref PubMed Scopus (88) Google Scholar]. The reason for this discrepancy is unknown at present. This research was supported in part by the grant VEGA, No. 2/6044/27, Slovak Republic.

Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive (51)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 3h 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 standard (51)Cr release assay. Considering linear relationships between data obtained with four fluorochromes and (51)Cr release assay as well as linear regression analysis with R(2)=0.9393 value for CAM-(51)Cr pair, we found the CAM assay to be the most closely related to the (51)Cr assay.

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.