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.     

Phorbol 12-myristate 13-acetate induces beta-adrenergic receptor uncoupling and non-specific desensitization of adenylate cyclase in human mononuclear leukocytes

Tumour-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate beta-adrenergic receptor responses in various cell types, presumably by the activation of protein kinase C. In the present investigation we assessed the effect of PMA on the beta-adrenergic receptor-adenylate cyclase system of human mononuclear leukocytes (MNL). It was found that incubation of MNL with PMA resulted in a time- and concentration-dependent desensitization of isoproterenol-induced adenylate cyclase activity. However, the effect of PMA was not restricted to the beta-adrenergic receptor system, since basal adenylate cyclase activity and histamine-, prostaglandin E1-, 5'-guanylylimidodiphosphate (GppNHp)-, and NaF-stimulated values were also reduced. By contrast, no effect was found on the forskolin-induced adenylate cyclase activity. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate had no effect on adenylate cyclase at all, suggesting that the observed PMA effect was specifically mediated by activation of protein kinase C. The reduced beta-adrenergic response induced by PMA was not associated with a reduced beta-adrenergic receptor number, indicating uncoupling of this receptor from adenylate cyclase. Isoproterenol competition curves for 3H-dihydroalprenolol binding to membranes from untreated and PMA-treated cells demonstrated that the uncoupling was due to a reduced ability of the agonist to promote formation of the guanine nucleotide-sensitive high affinity state of the receptor. The results indicate that PMA may cause desensitization of catecholamine-responsive adenylate cyclase in MNL, and that the major locus of alteration is the guanine nucleotide regulatory protein.     

Complement component C5a predicts restenosis after superficial femoral artery balloon angioplasty.

PURPOSE: To investigate whether balloon angioplasty of the superficial femoral artery (SFA) increases serum levels of C5a and whether C5a predicts risk of restenosis. METHODS: C5a antigen was measured at baseline and 8 hours after intervention in 131 consecutive patients (76 women; median age 72 years) with intermittent claudication who underwent successful primary SFA balloon angioplasty. Patients were followed for a median 10 months [interquartile range (IQR) 6 to 14] for the occurrence of >50% restenosis by duplex ultrasound. RESULTS: Median C5a levels increased significantly from 39.7 ng/mL (IQR 27.8 to 55.0) at baseline to 53.8 ng/mL (IQR 35.6 to 85.1, p<0.001) 8 hours post intervention. During the follow-up period, 70 (53%) patients developed restenosis. Increasing levels of C5a (quartiles) at baseline were significantly associated with an increased risk for restenosis (p=0.0092). Adjusted hazard ratios (95% confidence intervals) for restenosis with increasing quartiles of baseline serum C5a levels were 1.24 (0.60 to 2.58), 1.93 (0.95 to 3.93), and 2.08 (1.02 to 4.21), respectively, compared to the lowest quartile. This effect was independent of nonspecific inflammation as reflected by plasma levels of C-reactive protein. CONCLUSION: Inflammatory mechanisms play a major role in the development of restenosis after angioplasty. The complement component C5a exerts strong chemotactic and proinflammatory effects. Enhanced complement activation prior to PTA, as measured by higher levels of C5a, was significantly associated with restenosis after SFA balloon angioplasty. Pathways of complement inhibition thus may be worth investigating with respect to improving patency rates.     

Bedeutung der B-Vitamine in der Augenheilkunde

Ohne Zusammenfassung     

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.     

Short-term patient-reported outcomes after different exercise-based cardiac rehabilitation programmes.

BACKGROUND: An objective of exercise-based cardiac rehabilitation is improvement in patient-reported outcomes such as health-related quality of life as well as anxiety and depressive symptoms. There are no direct comparisons of the effectiveness of inpatient and outpatient exercise-based cardiac rehabilitation programmes on patient-reported outcomes. METHODS: In this non-randomized study we collected patient-reported outcomes data with the MacNew Heart Disease health-related quality of life questionnaire and the Hospital Anxiety and Depression Scale at baseline, 1 month and again 3 months after admission to exercise-based cardiac rehabilitation in a cohort of 216 consecutive patients enrolled either in a 4-week inpatient exercise-based cardiac rehabilitation (n=62) or a 3-month outpatient exercise-based cardiac rehabilitation (n=87) and in a usual care group (n=67) to document the natural course in patient-reported outcome variables without exercise-based cardiac rehabilitation. RESULTS: Although MacNew health-related quality of life scores improved more with inpatient than outpatient exercise-based cardiac rehabilitation by month 1, the improvement was still significant in both groups at month 3 and also in the usual care group when compared to baseline. The health-related quality of life scores in the inpatient group, however, decreased between month 1 and 3 whereas they continued to improve in the outpatient group. The significant reduction in both anxiety and depressive symptoms in both exercise-based cardiac rehabilitation groups by month 1 was maintained at month 3 only with outpatient exercise-based cardiac rehabilitation. No significant changes over the 3 months were observed in the usual care group. CONCLUSION: Significant improvements of 1-month patient-reported outcomes are achieved in patients attending inpatient as well as outpatient exercise-based cardiac rehabilitation when compared with no exercise-based cardiac rehabilitation. In contrast to inpatient exercise-based cardiac rehabilitation, however, outpatient exercise-based cardiac rehabilitation leads to a further improvement of patient-reported outcomes. These results suggest that, if patients have to be admitted for inpatient exercise-based cardiac rehabilitation, this programme should be followed by an outpatient exercise-based cardiac rehabilitation to further improve and stabilize these patient-reported outcome variables.