Murine mCLCA6 is an integral apical membrane protein of non-goblet cell enterocytes and co-localizes with the cystic fibrosis transmembrane conductance regulator.

The CLCA family of proteins consists of a growing number of structurally and functionally diverse members with distinct expression patterns in different tissues. Several CLCA homologs have been implicated in diseases with secretory dysfunctions in the respiratory and intestinal tracts. Here we present biochemical protein characterization and details on the cellular and subcellular expression pattern of the murine mCLCA6 using specific antibodies directed against the amino- and carboxy-terminal cleavage products of mCLCA6. Computational and biochemical characterizations revealed protein processing and structural elements shared with hCLCA2 including anchorage in the apical cell membrane by a transmembrane domain in the carboxy-terminal subunit. A systematic light- and electron-microscopic immunolocalization found mCLCA6 to be associated with the microvilli of non-goblet cell enterocytes in the murine small and large intestine but in no other tissues. The expression pattern was confirmed by quantitative RT-PCR following laser-capture microdissection of relevant tissues. Confocal laser scanning microscopy colocalized the mCLCA6 protein with the cystic fibrosis transmembrane conductance regulator CFTR at the apical surface of colonic crypt cells. Together with previously published functional data, the results support a direct or indirect role of mCLCA6 in transepithelial anion conductance in the mouse intestine.     

Feature-based,automated segmentation of cerebral infarct patterns using T2- and diffusion-weighted imaging

Diffusion-weighted imaging enables the diagnosis of cerebral ischemias very early, thus supporting therapies such as thrombolysis. However, morphology and tissue-characterizing parameters (e.g. relaxation times or water diffusion) may vary strongly in ischemic regions, indicating different underlying pathologic processes. As the determination of the parameters by a supervised segmentation is very time consuming, we evaluated whether different infarct patterns may be segmented by an automated, multidimensional feature-based method using a unified segmentation procedure. Ischemias were classified into 5 characteristic patterns. For each class, a 3D histogram based on T(2)- and diffusion-weighted images as well as calculated apparent diffusion coefficients (ADC) was generated from a representative data set. Healthy and pathologic tissue classes were segmented in the histogram as separate, local density maxima with freely shaped borders. Segmentation control parameters were optimized in a 3-step procedure. The method was evaluated using synthetic images as well as results of a supervised segmentation. For the analysis of cerebral ischemias, the optimal control parameter set led to sensitivities and specificities between 1.0 and 0.9.     

Monthly stem increment in relation to climatic variables during 7 years in an East African rainforest

Monthly stem increment of 766 trees was assessed for 7 years in Kakamega Forest, Kenya and related to monthly climatic variables. Mean stem increment of all tree individuals correlated negatively with maximum temperature but not with mean and minimum temperatures. For the precipitation variables sum of precipitation and number of rainy days we found positive correlations. Stem increment of the trees in the early-, mid-, and late-successional groups correlated positively with the number of rainy days. For late-successional trees increment correlated negatively with mean and maximum temperature and positively with all other precipitation variables. For mid-successional trees we found a negative correlation with mean temperature. In addition, the stem increment of most species related positively to precipitation variables and negatively to mean and maximum temperature. In view of the expected increasing temperatures and fewer but heavier rain events, our results suggest that climate change will lead to a reduction in stem increment. The results appertaining to the successional groups imply that early and mid-successional species are better equipped to perform well under the expected future climatic conditions than the late-successional species. This could reduce the role of this East African forest as a carbon store. As the responses to climatic variables were highly group- and species-specific it is likely that climate change will result in a species composition shift, presumably in favour of drought-resistant and heat-tolerating species.     

Hyphal walls of isolated lichen fungi

The hyphal walls of three mycobionts, isolated from the lichens Xanthoria parietina, Tornabenia intricata and Sarcogyne sp. were investigated by two techniques: microautoradiography of fungal colonies exposed to radioactive carbohydrate precursors; and binding, in vivo, of fluorescein conjugated lectins to hyphal walls of such colonies.N-[³H] acetylglucosamine was readily incorporated into tips, young hyphal walls and septa of the three mycobionts and the free-living fungus Trichoderma viride, but not into Phytophthora citrophthora, indicating that chitin is a major component of the mycobionts' hyphal walls. All three mycobionts, but neither of the free-living fungi, incorporated [³H] mannose and [³H] mannitol into their hyphal walls.Fluorescein-conjugated wheat germ agglutinin was bound to the hyphal walls of the three mycobionts and T. viride, but not to the walls of P. citrophthora; the binding pattern was similar to the grain pattern obtained in autoradiographs after short N-[³H] acetylglucosamine labelling. As wheat germ agglutinin binds specifically to chitin oligomers, the lectin binding tests further confirmed that chitin is a mycobiont hyphal wall component.Binding characteristics of several fluorescein-conjugated lectins to the three mycobionts indicated that this technique can yield useful information concerning the chemical composition of hyphal wall surfaces.List of abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - TCA trichloroacetic acid - PNA peanut agglutinin - LA lotus agglutinin - Glc NAc N-acetylglucosamine - ConA concanavalin A - SBA soybean agglutinin - WBA waxbean agglutinin Part of an M.Sc. thesis submitted by A. Braun to the Department of Botany, Tel Aviv University.     

Maurer's clefts: a novel multi-functional organelle in the cytoplasm of Plasmodium falciparum-infected erythrocytes

Discovered in 1902 by Georg Maurer as a peculiar dotted staining pattern observable by light microscopy in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum, the function of Maurer's clefts have remained obscure for more than a century. The growing interest in protein sorting and trafficking processes in malarial parasites has recently aroused the Maurer's clefts from their deep slumber. Mounting evidence suggests that Maurer's clefts are a secretory organelle, which the parasite establishes within its host erythrocyte, but outside its own confines, to route parasite proteins across the host cell cytoplasm to the erythrocyte surface where they play a role in nutrient uptake and immune evasion processes. Moreover, Maurer's clefts seem to play a role in cell signaling, merozoite egress, phospholipid biosynthesis and, possibly, other biochemical pathways. Here, we review our current knowledge of the ultrastructure of Maurer's clefts, their proteinaceous composition and their function in protein trafficking.     

Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.

The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called ''der'' for ''degradation in the ER''. DER1 was cloned by complementation of the der1-2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1-deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.     

Probing FhuA''-''PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12

Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.     

Regulation and solubilization of Candida albicans chitin synthetase.

A cytoplasmic component which inhibited the activation of chitin synthetase was studied in the dimorphic fungus Candida albicans. The inhibitor was found to be heat stable and trypsin sensitive and was only effective when incubated with a vacuolar protease, an activator of chitin synthetase, before the activation of chitin synthetase. In addition, the particulate chitin synthetase from the yeast form of C. albicans was solubilized by a sodium cholate-digitonin extraction and subsequently was purified approximately 30-fold by Sepharose column chromatography and Amicon XM 100 filtration. Activity of the soluble enzyme was increased by the addition of trypsin or phosphatidyl serine. The molecular weight of the enzyme was estimated to be 400,000.     

Association between Gestational Weight Gain and Postpartum Diabetes: Evidence from a Community Based Large Cohort Study

We have investigated the prospective association between excess gestational weight gain (GWG) and development of diabetes by 21 years post-partum using a community-based large prospective cohort study in Brisbane, Australia. There were 3386 mothers for whom complete data were available on GWG, pre-pregnancy BMI and self-reported diabetes 21 years post-partum. We used The Institute of Medicine (IOM) definition to categorize GWG as inadequate, adequate and excessive. We found 839 (25.78%) mothers gained inadequate weight, 1,353 (39.96%) had adequate weight gain and 1,194 (35.26%) had gained excessive weight during pregnancy. At 21 years post-partum, 8.40% of mothers self-reported a diagnosis of diabetes made by their doctor. In the age adjusted model, we found mothers who gained excess weight during pregnancy were 1.47(1.11,1.94) times more likely to experience diabetes at 21 years post-partum compared to the mothers who gained adequate weight. This association was not explained by the potential confounders including maternal age, parity, education, race, smoking, TV watching and exercise. However, this association was mediated by the current BMI. There was no association for the women who had normal BMI before pregnancy and gained excess weight during pregnancy. The findings of this study suggest that women who gain excess weight during pregnancy are at greater risk of being diagnosed with diabetes in later life. This relationship is likely mediated through the pathway of post-partum weight-retention and obesity. This study adds evidence to the argument that excessive GWG during pregnancy for overweight mothers has long term maternal health implications.