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Regulation of food intake and energy homeostasis is controlled by a delicate balancing of numerous central and peripheral factors, including circulating peptide hormones. This study investigated the proteome of saliva using SELDI-TOF-MS in relation to satiety and body mass index (BMI) in humans. Within a longitudinal test session, 18 subjects were exposed to a lunch-induced hunger-satiety shift, while every 15 min collecting their whole saliva and rating their hunger and satiety. Saliva was analysed by SELDI-TOF-MS using IMAC arrays with a chromatographic copper surface (IMAC-Cu). From all subjects and time points measured, peptide and protein profiles showed 190 common peaks. Their interrelationships show that 37% of the variation was accounted for in one dimension. About 30 means had a strong association (0.70

Background: The development of neutralizing antibodies (inhibitors) towards factor VIII is a major complication in non-severe hemophilia A, profoundly aggravating the bleeding pattern. Identification of high risk patients is hampered by lack of data on the association between factor VIII gene (F8) mutations and the development of inhibitors that take exposure days to therapeutic factor VIII concentrates into account. Aims: To determine the risk of inhibitor development in patients with non-severe hemophilia A and to analyze the association with F8 mutation, taking exposure days to therapeutic factor VIII concentrates into account. Methods: The study population was derived from a source population of 2711 non-severe hemophilia A patients (factor VIII 2-40%), treated in 34 hemophilia treatment centers in Europe and Australia (the INSIGHT consortium). The association between F8 mutation and inhibitor development was assessed in 1112 patients, only recruited from centers that had genotyped at least 70% of their patients. Inhibitor risk was calculated as Kaplan-Meier incidence with cumulative number of exposure days as time variable. Thus, risk was calculated as the proportion of patients that developed an inhibitor after a certain number of exposure days (e.g. 20 or 50) to therapeutic factor VIII concentrates. Results: During 44,800 exposure days (median 24 exposure days per patient; Inter Quartile Range (IQR), 7-90), 59 of the 1112 patients developed an inhibitor; cumulative incidence of 5.3% (95% confidence interval (CI), 4.0-6.6) after a median of 28 exposure days (IQR, 12- 71). The inhibitor risk at 50 exposure days was 6.7% (95% CI, 4.5- 8.9) and at 100 exposure days this risk was further increased to 13.3% (95% CI, 9.6-17.0). Among a total of 221 different F8 point mutations 19 were associated with inhibitor development. The inhibitor risk was highest for R593C, D2074G, R2159C and W2229C, reaching 19%, 21%, 39% and 42%, respectively, at 50 exposure days. Conclusion: Among a total of 221 different point mutations, 19 mutations were associated with inhibitor development. Longitudinal analysis revealed that the inhibitor incidence in non-severe hemophilia A patients with certain F8 mutations approaches the incidence observed in patients with severe hemophilia. These results emphasize the importance of F8 genotyping in non-severe hemophilia A. New preventive and therapeutic approaches in this patient group are urgently needed.

Cell surface exposure of phosphatidylserine (PS) is shown to be part of normal physiology of skeletal muscle development and to mediate myotube formation. A transient exposure of PS was observed on mouse embryonic myotubes at E13, at a stage of development when primary myotubes are formed. The study of this process in cell cultures of differentiating C2C12 and H9C2 myoblasts also reveals a transient expression of PS at the cell surface. This exposure of PS locates mainly at cell-cell contact areas and takes place at a stage when the structural organization of the sarcomeric protein titin is initiated, prior to actual fusion of individual myoblast into multinucleated myotubes. Myotube formation in vitro can be inhibited by the PS binding protein annexin V, in contrast to its mutant M1234, which lacks the ability to bind to PS. Although apoptotic myoblasts also expose PS, differentiating muscle cells show neither loss of mitochondrial membrane potential nor detectable levels of active caspase-3 protein. Moreover, myotube formation and exposure of PS cannot be blocked by the caspase inhibitor zVAD(OMe)-fmk. Our findings indicate that different mechanisms regulate PS exposure during apoptosis and muscle cell differentiation, and that surface exposed PS plays a crucial role in the process of myotube formation

Phosphatidylserine (PS) externalization is regarded as one of the earliest hallmarks of cells undergoing programmed cell death. We studied the use of labeled human recombinant annexin-V, a protein selectively binding to PS, to detect cardiomyocyte death in an in vivo mouse model of cardiac ischemia and reperfusion (I/R). I/R was induced in mouse hearts by ligation and subsequent release of a suture around the left anterior descending coronary artery. Annexin-V (25 mg/kg) fused to a marker molecule was injected intra-arterially 30 minutes before euthanasia. After 15 minutes of ischemia followed by 30 minutes of reperfusion, 1.4+/-1. 2% (mean+/-SD) of the cardiomyocytes in the area at risk were annexin-V positive (n=6). This increased to 11.4+/-1.9% after 15 minutes of ischemia followed by 90 minutes of reperfusion (n=7) and to 20.2+/-3.3% after 30 minutes of ischemia followed by 90 minutes of reperfusion (n=7). In control mice, including those injected with annexin-V at the binding site of PS, no annexin-V-positive cells were observed. DNA gel electrophoresis showed typical laddering starting after 15 minutes of ischemia followed by 30 minutes of reperfusion, suggesting activation of the cell death program. Intervention in the cell death program by pretreatment with a novel Na(+)-H(+) exchange inhibitor substantially decreased annexin-V-positive cardiomyocytes from 20.2% to 2.2% in mice after 30 minutes of ischemia followed by 90 minutes of reperfusion. These data suggest that labeled annexin-V is useful for in situ detection of cell death in an in vivo model of I/R in the heart and for the evaluation of cell death-blocking strategies