Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Cardiac efficiency and oxygen consumption measured with 11C-acetate PET after long-term cardiac resynchronization therapy.

Cardiac resynchronization therapy (CRT) is a treatment option in patients with severe heart failure and left bundle-branch block (LBBB). This study evaluated the effects of 4 and 13 mo of CRT on myocardial oxygen consumption (MVO2) and cardiac efficiency as compared with mild heart failure patients without LBBB. METHODS: Sixteen patients with severe heart failure and LBBB due to idiopathic cardiomyopathy were studied at baseline and after 4 and after 13 mo of therapy. Thirteen patients with mild heart failure without LBBB served as a comparison group. The clearance rate (k2) of 11C-acetate was measured with PET to assess MVO2. Stroke volume was derived from the dynamic PET data according to the Stewart-Hamilton principle and, furthermore, cardiac efficiency using the work metabolic index. RESULTS: After 4 mo of CRT, stroke volume index (SVI) increased by 50% (P = 0.012) and cardiac efficiency increased by 41% (P < 0.001). Global k2 remained unchanged but regional k2 demonstrated a more homogeneous distribution pattern. The parameters showed no significant changes during therapy. Under CRT, cardiac efficiency, SVI, and the distribution pattern of regional k2 did not differ from mild heart failure patients without LBBB. CONCLUSION: CRT improves cardiac efficiency for at least 13 mo, as demonstrated by a higher SVI, whereas MVO2 remains unchanged. Cardiac efficiency, SVI, and the MVO2 distribution pattern reach the level of patients with mild heart failure without LBBB. The unfavorable hemodynamic performance in heart failure with LBBB is effectively restored by long-term CRT to the level of an earlier disease state.     

Lifetime and six-month prevalence of mental disorders in the Munich follow-up study

Summary The Lifetime and 6 month DSM-III prevalence rates of mental disorders from an adult general population sample of former West Germany are reported. The most frequent mental disorders (lifetime) from the Munich Follow-up Study were anxiety disorders (13.87%), followed by substance (13.51%) and affective (12.90%) disorders. Within anxiety disorders, simple and social phobia (8.01%) were the most common, followed by agoraphobia (5.47%) and panic disorder (2.39%). Females had about twice the rates of males for affective (18.68% versus 6.42%), anxiety (18.13% versus 9.07%), and somatization disorders (1.60% versus 0.00%); males had about three times the rates of substance disorders (21.23% versus 6.11%) of females. Being widowed and separated/divorced was associated with high rates of major depression. Most disordered subjects had at least two diagnoses (69%). The most frequent comorbidity pattern was anxietyand affective disorders. Simple and social phobia began mostly in childhood or early adolescence, whereas agoraphobia and panic disorder had a later average age of onset. The majority of the cases with both anxiety and depression had depression clearly after the occurrence of anxiety. The DIS-DSM-III findings of our study have been compared with both ICD-9 diagnoses assigned by clinicians independently as well as other epidemiological studies conducted with a comparable methodology.     

Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS).

During myogenesis in Drosophila embryos, a prominent adhesive structure is formed between precursor cells and fusion-competent myoblasts (fcms). Here, we show that Duf/Kirre and its interaction partners Rols7 (found in founder myoblasts and growing myotubes) and Sns (found in fcms) are organized in a ring-structure at the contact points of fcms with precursor cells, while cytoskeletal components like F-actin and Titin are centered in this ring in both cell types. The cytoplasmic protein Blow colocalizes with the actin plugs in fcms after cell adhesion. Furthermore, the requirement of additional as yet unidentified components was demonstrated by using mammalian C2C12 myoblasts. In this study, we propose that the fusion-restricted myogenic-adhesive structure (FuRMAS) is pivotal in linking cell adhesion as well as local F-actin assembly and dynamics to downstream events that ultimately lead to plasma membrane fusion. Moreover, we suggest that the FuRMAS may restrict the area of membrane breakdown.     

The Epstein-Barr virus latent membrane protein 1 induces interleukin-10 in Burkitt's lymphoma cells but not in Hodgkin's cells involving the p38/SAPK2 pathway

Infection of B cells with Epstein-Barr Virus (EBV) induces interleukin-10 (IL-10) production, which may contribute to transformation. IL-10 can modulate the immune response at certain levels, playing a crucial role in balancing humoral and cellular responses. Moreover, it can function as a growth and differentiation factor for B cells. However, the mechanism of IL-10 induction is still unclear. Here we demonstrate that IL-10 was specifically induced by the EBV-latent membrane protein 1 (LMP1) in Burkitt's lymphoma (BL) cell lines BL2 and BL41. In two T cell lines (Jurkat, MOLT3), two NHL cell lines (U266, MHH-PREB1), or three Hodgkin's disease (HD) cell lines (L428, L540, and KMH2), LMP1 did not induce IL-10 expression. In contrast, LMP1 activated CD40 or CD54 (ICAM1) expression in the analyzed cell lines. LMP1 derivatives lacking the C-terminal activation regions (CTAR), by deletion of the amino acids between 187 and 351 (Delta CTAR1) or 232 and 386 (Delta CTAR2), alone, or together induced IL-10 at very low amounts compared to wild-type LMP1. Inhibition of LMP1-mediated NF kappa B activation by constitutive repressive I kappa B-alpha only marginally impaired IL-10 expression in BL2 cells, while SB2035080 at 5 microM (a specific p38/SAPK2 inhibitor) led to reduced IL-10 expression. Our findings confirm the role of LMP1 in transactivation of cellular genes possibly important for tumor immunoescape but show that more than one signaling pathway is involved in this activation and suggests the necessity of a defined conformation of CTARs to activate IL-10 involving p38/SAPK2.     

Beta2-adrenergic receptors mediate the differential effects of catecholamines on cytokine production of PBMC.

We determined characteristics of beta2-adrenergic receptors (beta2R) on peripheral blood mononuclear cells (PBMC) and cytokine production after mitogenic stimulation and coincubation with catecholamines. PBMCs were stimulated with interleukin-2 (IL-2), tetanus toxoid (TT), anti-CD3 antibody, or phytohemagglutinin (PHA). The cytokines interferon-gamma (IFN-gamma), IL-4, and IL-6 were determined by ELISA following coincubation with high-dose (10(-5) M) and low-dose (10(-9) M) epinephrine (EPI) and norepinephrine (NE). Intracellular IFN-gamma and IL-4 were studied by FACS analysis. The beta2R density was investigated using a radioligand binding assay. The stimuli induced various cytokine profiles in PBMCs. Synthesis of IFN-gamma was induced by all mitogens and could be suppressed by catecholamines (26%-85% reduction). In PHA-stimulated PBMCs, IL-4 synthesis was decreased by high-dose catecholamines (24%-28% reduction). Adding a beta-blocking agent attenuated most catecholamine effects. A highly significant negative correlation between the density of beta2R with IFN-gamma and IL-6 levels of PHA-activated PBMCs (r = -0.88 to -0.96, p < 0.01-< 0.001) was observed. The results indicate that the density of beta2R on PBMC plays a role in mediating the differential catecholamine effects on cytokine production of PBMC. Furthermore, changes in cytokine expression induced by catecholamines favor Th2 responses.     

Tula Virus as Causative Agent of Hantavirus Disease in Immunocompetent Person,Germany

We report molecular evidence of Tula virus infection in an immunocompetent patient from Germany who had typical signs of hantavirus disease. Accumulating evidence indicates that Tula virus infection, although often considered nonpathogenic, represents a threat to human health.     

Consciousness and disturbances of consciousness

Summary The problem of consciousness is discussed briefly, including the contrary views of consciousness as a transcendental phenomenon and as an animistic fiction. Measurement of consciousness is possible only indirectly by means of quantitative assessment of accompanying behavioral deficits.Knowledge of the structural basis of consciousness is incomplete. The ascending reticular activating system (ARAS) is necessary for the maintenance of the state of consciousness. The monoamine and a great number of descending projections modulate the ARAS. The contents of consciousness depend also on telencephalic structures, primarily on the telencephalic cortex. Certain localized telencephalic lesions bring about disturbances of consciousness. The role of the corpus callosum in the problem of consciousness is discussed (one brain—two minds hypothesis).Then a classification of the various disturbances of consciousness is proposed. The term disturbances of vigilance is used for all disturbances of consciousness which are caused by a lesion in or a functional disorder of the ARAS or any of its modulating subsystems. The term disturbances of the contents of consciousness refers to disturbances of consciousness due to global or localized lesions or functional disorders of telencephalic structures. A list of characteristic features is given for each class of disturbance of consciousness.

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Zusammenfassung Das Problem Bewußtsein wird kurz skizziert. Dabei werden die gegensätzlichen Auffassungen über Bewußtsein als transzendentales Phänomen bzw. als animistische Fiktion erörtert. Die Messung von Bewußtsein ist indirekt nur über die Bestimmung des begleitenden Verhaltensdefizits möglich. Die Kenntnis über die strukturellen Grundlagen des Bewußtseins ist lückenhaft. Für die Aufrechterhaltung des Bewußtseinsgrades kommt dem aufsteigenden retikulären aktivierenden System (ARAS) eine notwendige — wenngleich nicht hinreichende — Bedeutung zu. Das ARAS wird durch monoaminerge und eine große Zahl vom Großhirn absteigender Projektionssysteme moduliert.Telenzephale Strukturen und vor allem der telenzephale Kortex scheinen für die Entfaltung von Bewußtseinsinhalten unentbehrlich. Auch umschriebene telenzephale Läsionen können partielle Störungen der Bewußtseinsinhalte verursachen. Die Bedeutung des Balkens für das Bewußtsein wird diskutiert (one brain—two minds-Hypothese).Im letzten Abschnitt wird eine Taxonomie der verschiedenartigen Bewußtseinsstörungen versucht. Unter dem Begriff Vigilanzstörungen werden alle Bewußtseinsstörungen zusammengefaßt, die durch Läsion oder Funktionsstörungen des aufsteigenden retikulären aktivierenden Systems (ARAS) — einschließlich seiner modulierenden Subsysteme — bedingt sind. Unter Störung der Bewußtseinsinhalte werden Bewußtseinsstörungen verstanden, die durch globale oder lokale Läsion bzw. Funktionsstörung telenzephaler Strukturen verursacht werden. Für beide Klassen von Bewußtseinsstörungen wird ein Merkmalskatalog beschrieben.