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.     

Comparative early and midterm results of open juxtarenal and infrarenal aneurysm repair

Background and aims Since the introduction of endovascular aortic aneurysm repair (EVAR) for aortic aneurysms, the number of juxtarenal aortic aneurysms (JRA) has been growing steadily due to selection bias (neck morphology for EVAR). This case-match study compares the perioperative outcome and midterm results of suprarenally clamped JRA with infrarenal aortic aneurysms (AAA). Methods From 1997 to 2004, patients who received open surgery with suprarenal clamping for JRA were included in the study and compared to matched patients with infrarenal clamping (AAA). Measurements analyzed were the in-hospital mortality and morbidity. Midterm results were obtained through clinical investigation and magnetic resonance angiography imaging. Results Thirty-five patients (mean age, 68.4 years; 30 male and 5 female) received suprarenal cross-clamping for JRA. The overall in-hospital mortality for JRA and for the controls (AAA) with elective aortic repair was 4.5% (6.1% JRA; 3% AAA, p = 0.058). The morbidity of JRA was elevated according to the rate of pulmonary complications (p = 0.021) and the need for re-operation (p = 0.019). The mean follow-up time was 2.3 years (range, 8–96 months). At follow-up, 28 patients (80%) from the JRA group and 29 patients from the AAA group (82.9%) were alive. Conclusion Open aortic surgery for JRA with the need for suprarenal cross-clamping shows a slightly elevated in-hospital mortality rate without statistical significance and equal midterm mortality results in comparison with infrarenally clamped aortic aneurysms.     

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.     

Early and midterm results after open and endovascular repair of ruptured abdominal aortic aneurysms in a comparative analysis.

PURPOSE: To compare early and midterm results of open versus endovascular aortic repair of ruptured abdominal aneurysms (rAAA). METHODS: A retrospective analysis was performed of 58 consecutive patients with rAAA who were treated with open or endovascular aneurysm repair (EVAR) at a single center between January 2000 and December 2005. Patients without definitive signs of rupture (symptomatic patients) were excluded from the study. Twenty-nine patients (21 men; median age 71 years) were treated using endovascular techniques (EVAR group) and 29 (28 men; median age 71 years) with open repair (OR group).The hemodynamic status at the time of admission was evaluated with respect to blood pressure, pulse rate, and hemoglobin level to reduce selection bias. Patients underwent follow-up by clinical examination and computed tomography. RESULTS: The 30-day mortality rate was 31% (9/29) in each group (p = 1.0); the morbidity rates also did not differ between groups [16 (55.2%) EVAR vs. 18 (62.1%) OR; p = 0.9]. There was 1 (3.4%) primary conversion in the EVAR group and 7 (24.1%) endoleaks [3 (10.3%) primary; 4 (13.8%) secondary]. There was no difference between the groups with regard to intensive care unit stay (4 days for EVAR vs. 3 days for OR, p = 0.98) or total hospital stay (9 days for EVAR vs. 12 days for OR, p = 0.69). After a mean follow-up of 40.25 months (range 1-70), the midterm mortality rates did not differ [5 (17.2%) EVAR vs. 3 (10.3%) OR, p = 0.41]. CONCLUSION: EVAR of rAAAs is feasible, with equal early and midterm mortality rates compared to open repair. When a defined patient selection is used for rupture, including hemodynamic status, there is no evidence of a better outcome with EVAR in emergency cases.     

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.     

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.     

Sehnenverletzungen an der Schulter

Die Unfallchirurgie -     

Therapy of deep leg vein thrombosis. When is surgical therapy indicated?

In spite of quite a few clinical trials the benefit of venous thrombectomy is seen controversially. The primary objectives of treating venous thrombosis are survival rate, prevention of pulmonary embolism and of postthrombotic syndrome. We report our experience with 47 patients who underwent venous thrombectomy. The mortality rate was 0%. We did not observe clinically relevant pulmonary embolism. After two years 90% of thrombectomised veins were patent. The mortality rates given in the literature of conservative treatment with heparin and following oral anticoagulation are 0.4 to 1.6%. Fibrinolysis shows mortality rates of 1 to 2.4, and thrombectomy of 3.8%, respectively. Venous thrombectomy is an effective treatment to prevent pulmonary embolism. In our own experience we saw no clinically significant pulmonary event. The danger of embolism rises with the proximity of the venous thrombus. Therefore those patients may have the greatest potential benefit from thrombectomy who present with a mobile inguinal thrombus or a thrombus in the iliac vein. So far there are no statistically sufficient data to support the indication of thrombectomy to prevent a postthrombotic syndrome.     

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.