Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A.

nefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria.

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of $\textit{Synechocystis }$ sp. PCC 7002 and $\textit{Synechocystis }$ sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that $\textit{Synechocystis }$ sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.T.L. was supported by BBSRC Research Grant BB/J016985/1 to C.W.M. D.J.L-S. was supported by the Environmental Services Association Education Trust. L.L.B was supported by a BBSRC Doctoral Training Grant (BB/F017464/1)

PAX2 Regulates ADAM10 Expression and Mediates Anchorage-Independent Cell Growth of Melanoma Cells

PAX transcription factors play an important role during development and carcinogenesis. In this study, we investigated PAX2 protein levels in melanocytes and melanoma cells by Western Blot and immunofluorescence analysis and characterized the role of PAX2 in the pathogenesis of melanoma. In vitro we found weak PAX2 protein expression in keratinocytes and melanocytes. Compared to melanocytes increased PAX2 protein levels were detectable in melanoma cell lines. Interestingly, in tissue sections of melanoma patients nuclear PAX2 expression strongly correlated with nuclear atypia and the degree of prominent nucleoli, indicating an association of PAX2 with a more atypical cellular phenotype. In addition, with chromatin immunoprecipitation assay, PAX2 overexpression and PAX2 siRNA we present compelling evidence that PAX2 can regulate ADAM10 expression, a metalloproteinase known to play important roles in melanoma metastasis. In human tissue samples we found co-expression of PAX2 and ADAM10 in melanocytes of benign nevi and in melanoma cells of patients with malignant melanoma. Importantly, the downregulation of PAX2 by specific siRNA inhibited the anchorage independent cell growth and decreased the migratory and invasive capacity of melanoma cells. Furthermore, the downregulation of PAX2 abrogated the chemoresistance of melanoma cells against cisplatin, indicating that PAX2 expression mediates cell survival and plays important roles during melanoma progression

Maternal depression and the experience of parenting in the second postnatal year

Cognitive theories of depression maintain that negative thoughts about the self and of the future are central features of depression. In postnatal depression, these negative thoughts may impact upon the mother's experience of parenting. This study investigated the impact of maternal depression during the first postnatal year and beyond and infant gender on parenting stress, on the mother's feelings of attachment to her infant and on her perceptions of her infant's behaviour. One hundred and twelve generally well-educated middle-class mothers who had presented with unsettled infant behaviour to a parent-craft hospital, were interviewed and completed questionnaires when their infants were 4, 12 and 15 months of age. Mothers who had experienced depression reported higher levels of parenting stress at 15 months than never depressed mothers. Only mothers who had experienced chronic depression (12 months duration), however, reported higher parenting stress related to characteristics of their infants. They also reported significantly more negative perceptions of their infant's behaviour and more hostile feelings towards their infants than never depressed mothers. Results are discussed with respect to the impact of the duration of depression on the mothers' reported experiences of parenting and the clinical implications of these findings.12 page(s

Validation of an accelerated high-sensitivity troponin T assay protocol in an Australian cohort with chest pain

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Evaluating rapid rule-out of acute myocardial infarction by use of a high-sensitivity cardiac troponin I assay at presentation

Low concentrations of cardiac troponin (cTn) have been recommended for rapid rule-out of acute myocardial infarction (AMI). We examined the Beckman Coulter Access high-sensitivity cardiac troponin I (hs-cTnI) assay to identify a single test threshold that can safely rule out AMI. This analysis used stored samples collected in 2 prospective observational studies. In all, 1871 patients presenting to a tertiary emergency department with symptoms of acute coronary syndrome had blood taken for measurement of cTnI on presentation. The end point was type 1 myocardial infarction (T1MI). Sensitivity and negative predictive value (NPV) were calculated for hs-cTnI values below the 99th percentile. Ninety-eight patients had T1MI (5.2%), and 638 (34.1%) patients had an hs-cTn