Discovery of new molecular subtypes in oesophageal adenocarcinoma

A large number of patients suffering from oesophageal adenocarcinomas do not respond to conventional chemotherapy; therefore, it is necessary to identify new predictive biomarkers and patient signatures to improve patient outcomes and therapy selections. We analysed 87 formalin-fixed and paraffin-embedded (FFPE) oesophageal adenocarcinoma tissue samples with a reverse phase protein array (RPPA) to examine the expression of 17 cancer-related signalling molecules. Protein expression levels were analysed by unsupervised hierarchical clustering and correlated with clinicopathological parameters and overall patient survival. Proteomic analyses revealed a new, very promising molecular subtype of oesophageal adenocarcinoma patients characterised by low levels of the HSP27 family proteins and high expression of those of the HER family with positive lymph nodes, distant metastases and short overall survival. After confirmation in other independent studies, our results could be the foundation for the development of a Her2-targeted treatment option for this new patient subgroup of oesophageal adenocarcinoma.     

Present state and possibilities for improvement of cancer prevention and early detection in the Osijek-Baranya county

Cancer morbidity and mortality are on a steady increase in Croatia. Technologic possibilities for appropriate management are available for four cancer sites, i.e. cancer of the breast, cervix uteri, colorectum and prostate, and include cancer prevention and early detection in individuals yet free from manifest signs of the disease. The magnitude of the problem, the experience acquired to date, health care personnel available, and additional resources required to launch a systematic program of early detection of the disease are presented. The program should be initially launched in a county with greatest experience in early detection of cancer, where health care service is ready to immediately start its implementation. The role of family physician, gynecologic service at primary health care level, and polyclinic-consultation hospital service in program implementation is described. The following three possible options for early detection of cancer are analyzed and proposed: minimal program (early detection every 3 years), medium program (the same individuals examined every 2 years), and optimal program proposed by the American Cancer Society and other national and international organizations.     

Study of the oxidative stress in a rat model of chronic brain hypoperfusion

A multiple analysis of the cerebral oxidative stress was performed on a physiological model of dementia accomplished by three-vessel occlusion in aged rats. The forward rate constant of creatine kinase, k_for, was studied by saturation transfer ³¹P magnetic resonance spectroscopy in adult and aged rat brain during chronic hypoperfusion. In addition, free radicals in aging rat brain homogenates before and/or after occlusion were investigated by spin-trapping electron paramagnetic resonance spectroscopy (EPR). Finally, biochemical measurements of oxidative phosphorylation parameters in the above physiological model were performed. The significant reduction of k_for in rat brain compared to controls 2 and 10 weeks after occlusion indicates a disorder in brain energy metabolism. This result is consistent with the decrease of the coefficient of oxidative phosphorylation (ADP:O), and the oxidative phosphorylation rate measured in vitro on brain mitochondria. The EPR study showed a significant increase of the ascorbyl free radical concentration in this animal model. Application of -phenyl-N-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin traps revealed formation of highly reactive hydroxyl radical (OH) trapped in DMSO as the CH₃ adduct. It was concluded that the ascorbate as a major antioxidant in brain seems to be useful in monitoring chronic cerebral hypoperfusion.     

Opposite effects of serotonin and interferon-α on the membrane potential and function of human natural killer cells

Summary In an earlier article, we reported that serotonin (5-hydroxytryptamine, 5-HT) inhibits the natural killer cell (NK) cytotoxicity of human whole blood in a dose-dependent manner and that natural human interferon-α (IFN-α) partially eliminates this effect. Because natural IFN-α might contain factors other than IFN, we repeated these experiments with recombinant human interferon-α (rhIFN-α) and separated blood lymphocytes enriched with NK cells and then demonstrated that IFN really is responsible for this effect. Furthermore, this investigation was carried out to clarify the mechanisms of the action of 5-HT and of rhIFN-α on NK cells. The inhibition of the cytotoxicity was pronounced when 5-HT was added at the onset of the cytotoxic assay, whereas the pretreatment of lymphocytes for 18 h only led to a slight inhibition. Moreover, rhIFN-α applied 1 h before or 1 h after the addition of 5-HT decreased the inhibitory effect of 5-HT. Flow cytometric analysis involving the use of a voltage-sensitive dye, oxonol, revealed that 5-HT depolarized, whereas rhIFN-α hyperpolarized the plasma membrane of the lymphocytes. Thus, it seems likely that the inhibitory effect of 5-HT on the cytotoxicity of peripheral human lymphocytes is due to the depolarization on the plasma membrane of the effector cells and that rhIFN-α antagonizes this ability via its hyperpolarizing activity.     

Comparison of ELISA-based tyrosine kinase assays for screening EGFR inhibitors

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu,Tyr)l:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431 cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.     

Mitochondrial DNA of ancient Cumanians: culturally Asian steppe nomadic immigrants with substantially more western Eurasian mitochondrial DNA lineages

The Cumanians were originally Asian pastoral nomads who in the 13th century migrated to Hungary. We have examined mitochondrial DNA from members of the earliest Cumanian population in Hungary from two archeologically well-documented excavations and from 74 modern Hungarians from different rural locations in Hungary. Haplogroups were defined based on HVS I sequences and examinations of haplogroup-associated polymorphic sites of the protein coding region and of HVS II. To exclude contamination, some ancient DNA samples were cloned. A database was created from previously published mtDNA HVS I sequences (representing 2,615 individuals from different Asian and European populations) and 74 modem Hungarian sequences from the present study. This database was used to determine the relationships between the ancient Cumanians, modern Hungarians, and Eurasian populations and to estimate the genetic distances between these populations. We attempted to deduce the genetic trace of the migration of Cumanians. This study is the first ancient DNA characterization of an eastern pastoral nomad population that migrated into Europe. The results indicate that, while still possessing a Central Asian steppe culture, the Cumanians received a large admixture of maternal genes from more westerly populations before arriving in Hungary. A similar dilution of genetic, but not cultural, factors may have accompanied the settlement of other Asian nomads in Europe.     

The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infection

Persistence infection is the keystone of the ruminant and human diseases called brucellosis and Malta fever, respectively, and is linked to the intracellular tropism of Brucella spp. While described as non-motile, Brucella spp. have all the genes except the chemotactic system, necessary to assemble a functional flagellum. We undertook to determine whether these genes are expressed and are playing a role in some step of the disease process. We demonstrated that in the early log phase of a growth curve in 2YT nutrient broth, Brucella melitensis expresses genes corresponding to the basal (MS ring) and the distal (hook and filament) parts of the flagellar apparatus. Under these conditions, a polar and sheathed flagellar structure is visible by transmission electron microscopy (TEM). We evaluated the effect of mutations in flagellar genes of B. melitensis encoding various parts of the structure, MS ring, P ring, motor protein, secretion apparatus, hook and filament. None of these mutants gave a discernible phenotype as compared with the wild-type strain in cellular models of infection. In contrast, all these mutants were unable to establish a chronic infection in mice infected via the intraperitoneal route, raising the question of the biological role(s) of this flagellar appendage.     

Gene expression profiling in murine autoimmune arthritis during the initiation and progression of joint inflammation

We present here an extensive study of differential gene expression in the initiation, acute and chronic phases of murine autoimmune arthritis with the use of high-density oligonucleotide arrays interrogating the entire mouse genome. Arthritis was induced in severe combined immunodeficient mice by using adoptive transfer of lymphocytes from proteoglycan-immunized arthritic BALB/c mice. In this unique system only proteoglycan-specific lymphocytes are transferred from arthritic mice into syngeneic immunodeficient recipients that lack adaptive immunity but have intact innate immunity on an identical (BALB/c) genetic background.     

Incorporation of the V-ATPase inhibitors concanamycin and indole pentadiene in lipid membranes. Spin-label EPR studies

The incorporation of concanamycin A, a potent inhibitor of vacuolar ATPases, into membranes of dimyristoyl phosphatidylcholine has been studied by using EPR of spin-labelled lipid chains. At an inhibitor/lipid ratio of 1:1 mol/mol, concanamycin A broadens the chain-melting transition of the phospholipid bilayer membrane, and effects the lipid chain motion in the fluid phase. The outer hyperfine splitting of a spin label at the C-5 position and the line widths of a spin label at the C-14 position of the lipid chain are increased by concanamycin A. Considerably larger membrane perturbations are caused by equimolar admixture of a designed synthetic 5-(5,6-dichloro-2-indolyl)-2,4-pentadienoyl V-ATPase inhibitor. These results indicate that concanamycin A intercalates readily between the lipid chains in biological membranes, with minimal perturbation of the bilayer structure. Essentially identical results are obtained with concanamycin A added to preformed membranes as a concentrated solution in DMSO, or mixed with lipid in organic solvent prior to membrane formation. Therefore, the common mode of addition in V-ATPase inhibition assays ensures incorporation of concanamycin into the lipid bilayer milieu, which provides an efficient channel of access to the transmembrane domains of the V-ATPase.