Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: primary pulmonary hypertension (PPH); vasoactive intestinal peptide (VIP); pulmonary artery smooth muscle cells (PASMCs); mean pulmonary arterial pressure (MPAP).

Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: primary pulmonary hypertension (PPH); vasoactive intestinal peptide (VIP); pulmonary artery smooth muscle cells (PASMCs); mean pulmonary arterial pressure (MPAP).

Defined angiographically, no-reflow (NR) manifests as an acute reduction in coronary flow in the absence of epicardial vessel obstruction. One candidate protein to cause coronary NR is tissue factor (TF), which is abundant in atherosclerotic plaque and a cofactor for activated plasma coagulation factor VII. Scrapings from atherosclerotic carotid arteries contained TF activity (corresponding to 33.03 ؎ 13.00 pg/cm 2 luminal plaque surface). Active TF was sedimented, indicating that TF was associated with membranes. Coronary blood was drawn from 6 patients undergoing coronary interventions with the distal protection device PercuSurge GuardWire (Traatek, Miami, FL). Fine particulate material that was recovered from coronary blood showed TF activity (corresponding to 91.1 ؎ 62.16 pg/mL authentic TF). To examine the role of TF in acute coronary NR, blood was drawn via a catheter from coronary vessels in 13 patients during NR and after restoration of flow. Mean TF antigen levels were elevated during NR (194.3 ؎ 142.8 pg/mL) as compared with levels after flow restoration (73.27 ؎ 31.90 pg/mL; P ‫؍‬ .02). To dissect the effects of particulate material and purified TF on flow, selective intracoronary injection of atherosclerotic material or purified relipidated TF was performed in a porcine model. TF induced NR in the model, thus strengthening the concept that TF is causal, not just a bystander to atherosclerotic plaque material. The data suggest that active TF is released from dissected coronary atherosclerotic plaque and is one of the factors causing the NR phenomenon. Thus, blood-borne TF in the coronary circulation is a major determinant of flow. (Blood. 2002;99:2794-2800)

Lipid rafts are detergent-resistant, cholesterol-and sphingolipid-rich membrane domains that are involved in important cellular processes such as signal transduction and intracellular trafficking. Stomatin, a major lipid-raft component of erythrocytes and epithelial cells, is also an abundant platelet protein. Microscopical methods and subcellular fractionation showed that stomatin is located mainly at the ␣-granular membrane. The lipid-raft marker proteins flotillin-1 and flotillin-2 were also present in platelets but excluded from ␣ granules. Stomatin and the flotillins were associated with Triton X-100-insoluble lipid rafts. Whereas stomatin was partly soluble in Triton X-100, it was insoluble in the detergents Lubrol and 3-[(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate (CHAPS). Flotation experiments after CHAPS lysis of platelets revealed a distinct set of lipidraft-associated proteins, which were identified by matrix-assisted laser desorption/ ionization mass spectrometry as stomatin, flotillin-1, flotillin-2, CD36, CD9, integrin ␣ IIb ␤ 3 , and the glucose transporter GLUT-3. Stomatin, the flotillins, and CD36 were exclusively present in this lipid-raft fraction. Activation of platelets by calcium ionophore A23187 or thrombin led to translocation of stomatin to the plasma membrane, cleavage by calpain, and specific sorting into released microvesicles. IntroductionStomatin (protein 7.2b, band 7.2), described as a major protein component of the erythrocyte membrane, [1][2][3][4] has been found to be absent from red cell membranes in patients with overhydrated hereditary stomatocytosis. 4,5 However, because normal stomatin messenger RNA is present in the reticulocytes 6 of these patients and stomatocytosis does not occur in stomatin knockout mice, 7 the absence of stomatin is an effect rather than the cause of the disease. Studies in UAC epithelial cells revealed that stomatin forms high-order oligomers and is associated with detergent-resistant membrane microdomains, which are also termed lipid rafts. 8,9 These characteristics of stomatin and its unusual monotopic structure 10 are reminiscent of typical features of the caveolin proteins, which are highly enriched at the cytoplasmic side of caveolae. In erythrocytes, which do not express caveolins, stomatin and the distantly related proteins flotillin-1 and flotillin-2 11 are the major integral membrane proteins of lipid rafts, suggesting important, yet distinct roles for these proteins at the interface between lipid rafts and the cytoskeleton or signaling components. 12 The concept of lipid rafts or membrane microdomains was originally proposed to explain the vectorial transport of glycosyl phosphatidylinositol (GPI)-anchored proteins to the apical surface in polarized cells. 13,14 In the past decade, numerous studies have established the general characteristics of lipid rafts. [15][16][17][18] These microdomains contain mainly cholesterol and sphingolipids as lipid constituents, which make them insoluble in nonionic detergents, and are specifically...

The trophoblast of human placenta is directly exposed to the maternal circulation. It forms the main barrier to maternal-fetal glucose transport. The present study investigated the effect of sustained hyperglycemia in vitro on the glucose transport system of these cells. Trophoblasts isolated from term placentas and immunopurified were cultured for 24, 48, and 96 h in DMEM containing either 5.5 (normoglycemia) or 25 mmol/l D-glucose (hyperglycemia), respectively. Initial uptake of glucose was measured using 3-O-[14C]methyl-D-glucose. Kinetic parameters were calculated as K(M) = 73 mmol/l and Vmax = 29 fmol s(-1) per trophoblast cell. Uptake rates of cells cultured under hyperglycemic conditions did not differ at exogenous D-glucose concentrations in the physiological range (1, 5.5, 10, and 15 mmol/l), but were significantly decreased by 25% (P<0.05) at diabetes-like concentrations (20 and 25 mmol/l) as compared to normoglycemic conditions. This effect was due to a decrease in Vmax (-50%), whereas K(M) remained virtually unaffected. GLUT1 mRNA levels were lower by 50% (P<0.05; Northern blotting) and GLUT1 protein was reduced by 16% (P<0.05; Western blotting) in trophoblast cells cultured under hyperglycemic vs. normoglycemic conditions. We conclude that prolonged hyperglycemia in vitro reduces trophoblast glucose uptake at substrate concentrations corresponding to blood levels of poorly controlled diabetic gravidas. This effect is due to diminished GLUT1 mRNA and protein expression in the trophoblast.

In patients with cystic fibrosis (CF), the progression of pulmonary disease differs considerably, even in identical cystic fibrosis transmembrane conductance regulator-genotypes which could reflect an additional influence of the host's immune response. This study therefore measured cytokine expression patterns in CF patients with different clinical presentation. Expression of interleukin (IL)-8, interferon gamma (IFN-gamma), IL-4, IL-10, and transforming growth factor (TGF)beta(I) was assessed in bronchial mucosal biopsies of eight CF patients with acute exacerbation (age 6.0-14.2 yrs), eight CF patients with chronic stable disease (age 7.3-17.4 yrs), and in five normal control subjects by semiquantitative and quantitative reverse transcriptase polymerase chain reaction combined with histopathological assessment and immunohistochemical staining. All CF patients expressed IL-8. In acute exacerbation, expression of TGF-beta1 and IFN-gamma was either absent or extremely low. In contrast, all patients with stable disease strongly expressed TGF-beta1. The highest expression of TGF-beta1 and IFN-gamma was found in CF patients with mild disease and a history of infrequent exacerbations. No correlation was found between the expression of IL-4 and IL-10 and patient history. In normal control subjects, only a weak expression of TGF-beta1 was observed. These results show a remarkable correlation between cytokine pattern and the clinical course of cystic fibrosis. High expression of transforming growth factor-beta1 and interferon gamma was associated with mild disease, whereas no or very weak expression of these cytokines was typical for patients with acute disease and frequent exacerbations suggesting a contribution of the immune response to the progression of pulmonary disease in cystic fibrosis.

PurposeTo investigate whether or not low intensity radio frequency electromagnetic field exposure (RF-EME) associated with mobile phone use can affect human cells, we used a sensitive proteome analysis method to study changes in protein synthesis in cultured human cells.MethodsFour different cell kinds were exposed to 2 W/kg specific absorption rate in medium containing 35S-methionine/cysteine, and autoradiography of 2D gel spots was used to measure the increased synthesis of individual proteins.ResultsWhile short-term RF-EME did not significantly alter the proteome, an 8-h exposure caused a significant increase in protein synthesis in Jurkat T-cells and human fibroblasts, and to a lesser extent in activated primary human mononuclear cells. Quiescent (metabolically inactive) mononuclear cells, did not detectably respond to RF-EME. Since RF exposure induced a temperature increase of less than 0.15°C, we suggest that the observed cellular response is a so called “athermal” effect of RF-EME.ConclusionOur finding of an association between metabolic activity and the observed cellular reaction to low intensity RF-EME may reconcile conflicting results of previous studies. We further postulate that the observed increased protein synthesis reflects an increased rate of protein turnover stemming from protein folding problems caused by the interference of radio-frequency electromagnetic fields with hydrogen bonds. Our observations do not directly imply a health risk. However, vis-a-vis a synopsis of reports on cells stress and DNA breaks, after short and longer exposure, on active and inactive cells, our findings may contribute to the re-evaluation of previous reports.Electronic supplementary materialThe online version of this article (doi:10.1007/s00420-010-0513-7) contains supplementary material, which is available to authorized users.

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