ABCG5 and ABCG8 require MDR2 for secretion of cholesterol into bile

The major pathway for the removal of cholesterol from the body is via secretion into the bile. Three members of the ATP binding cassette (ABC) family, ABCG5 (G5), ABCG8 (G8), and ABCB4 (MDR2), are required for the efficient biliary export of sterols. Here, we examined the interdependence of these three ABC transporters for biliary sterol secretion. Biliary lipid levels in mice expressing no MDR2 (Mdr2-/- mice) were compared with those of Mdr2-/- mice expressing 14 copies of a human G5 (hG5) and hG8 transgene (Mdr2-/-;hG5G8Tg mice). Mdr2-/- mice had only trace amounts of biliary cholesterol and phospholipids. The Mdr2-/-;hG5G8Tg mice had biliary cholesterol levels as low as those of Mdr2-/- mice. Thus, MDR2 expression is required for G5G8-mediated biliary sterol secretion. To determine whether the reduction in fractional absorption of dietary sterols associated with G5G8 overexpression is secondary to the associated increase in biliary cholesterol, we compared the fractional absorption of sterols in Mdr2-/-;hG5G8Tg and hG5G8Tg animals. Inactivation of MDR2 markedly attenuated the reduction in fractional sterol absorption associated with G5G8 overexpression. These results are consistent with the notion that increased biliary cholesterol secretion contributes to the reduction in fractional sterol absorption associated with G5G8 overexpression.     

Factors associated with whole carcass condemnation rates in provincially-inspected abattoirs in Ontario 2001-2007: implications for food animal syndromic surveillance

Background  

Ontario provincial abattoirs have the potential to be important sources of syndromic surveillance data for emerging diseases of concern to animal health, public health and food safety. The objectives of this study were to: (1) describe provincially inspected abattoirs processing cattle in Ontario in terms of the number of abattoirs, the number of weeks abattoirs process cattle, geographical distribution, types of whole carcass condemnations reported, and the distance animals are shipped for slaughter; and (2) identify various seasonal, secular, disease and non-disease factors that might bias the results of quantitative methods, such as cluster detection methods, used for food animal syndromic surveillance.     

Substrate recognition of type III secretion machines--testing the RNA signal hypothesis

Secretion by the type III pathway of Gram-negative microbes transports polypeptides into the extracellular medium or into the cytoplasm of host cells during infection. In pathogenic Yersinia spp., type III machines recognize 14 different Yop protein substrates via discrete signals genetically encoded in 7-15 codons at the 5' portion of yop genes. Although the signals necessary and sufficient for substrate recognition of Yop proteins have been mapped, a clear mechanism on how proteins are recognized by the machinery and then initiated into the transport pathway has not yet emerged. As synonymous substitutions, mutations that alter mRNA sequence but not codon specificity, affect the function of some secretion signals, recent work with several different microbes tested the hypothesis of an RNA-encoded secretion signal for polypeptides that travel the type III pathway. This review summarizes experimental observations and mechanistic models for substrate recognition in this field.     

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.     

Novel versus unsupported clades: assessing the qualitative support for clades in MRP supertrees

Matrix representation with parsimony (MRP) supertree construction has been criticized because the supertree may specify clades that are contradicted by every source tree contributing to it. Such unsupported clades may also occur using other supertree methods; however, their incidence is largely unknown. In this study, I investigated the frequency of unsupported clades in both simulated and empirical MRP supertrees. Here, I propose a new index, QS, to quantify the qualitative support for a supertree and its clades among the set of source trees. Results show that unsupported clades are very rare in MRP supertrees, occurring most often when there are few source trees that all possess the same set of taxa. However, even under these conditions the frequency of unsupported clades was <0.2%. Unsupported clades were absent from both the Carnivora and Lagomorpha supertrees, reflecting the use of large numbers of source trees for both. The proposed QS indices are correlated broadly with another measure of quantitative clade support (bootstrap frequencies, as derived from resampling of the MRP matrix) but appear to be more sensitive. More importantly, they sample at the level of the source trees and thus, unlike the bootstrap, are suitable for summarizing the support of MRP supertree clades.     

Proenkephalin and risk of developing chronic kidney disease: the Prevention of Renal and Vascular End-stage Disease study

Background: Proenkephalin (pro-ENK) was recently found to be associated with low estimated glomerular filtration rate (eGFR). The association of pro-ENK with urinary albumin excretion (UAE), another marker for chronic kidney disease (CKD), has not been investigated. We examined the association of pro-ENK with eGFR and UAE as markers of CKD.

Methods: We included 4375 subjects of the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study. CKD_eGFR was defined as development of eGFR?<60?ml/min/1.73 m² and CKD_UAE as albuminuria?>30?mg/24?h.

Results: Baseline median pro-ENK was 52.2 (IQR: 44.9–60.5) pmol/L. After a median follow-up of 8.4 (IQR: 7.9–8.9) years, 183 subjects developed CKD_eGFR and 371 developed CKD_UAE. The association of pro-ENK with CKD_eGFR was modified by sex (P_interaction?SD increase in ¹⁰log-transformed pro-ENK, 1.65; 95% CI: 1.15–2.36) and not in women (0.83; 0.58–1.20). No significant association was observed between pro-ENK and CKD_UAE risk (0.83; 0.58–1.20).

Conclusions: High pro-ENK is associated with increased risk of CKD_eGFR in men, but not in women. No association of pro-ENK with CKD_UAE was observed. These results should be interpreted with caution, since residual confounding and potential overfitting of models could have influenced the results.     

Localization of fibroblast growth factor 2 (FGF-2) protein and the receptors FGFR 1–4 in normal human seminiferous epithelium

 Fibroblast growth factor 2 (FGF-2), which occurs in various isoforms both species and tissue specifically, regulates cell proliferation and differentiation via a dual receptor system consisting of heparan sulphate proteoglycans and receptor tyrosine kinases (FGFRs). This study demonstrates for the first time the distribution pattern of FGF-2 and the receptors FGFR 1–4 in the normal seminiferous epithelium of adult men. In western blot analyses, the polyclonal antibody, anti-FGF-2, shows two immunoreactive bands at 18 and 24 kDa. On paraffin sections, positive immunoreaction occurs within the cytoplasm of spermatogonia. The distribution pattern of the polyclonal anti-FGFR 1–4 antibodies is as follows: anti-FGFR-1 (one 68-kDa band) stains nuclei and cytoplasm of spermatogonia; anti-FGFR-3 (five bands at 68, 78, 105, 125 and 145 kDa) stains the nuclei of all germ cells except those of elongated spermatids; and anti-FGFR-4 (one 48-kDa band) stains the cytoplasm of primary pachytene spermatocytes. We were unable to demonstrate FGFR-2 immunoreactivity either in western blot analysis or on paraffin sections. This distribution pattern suggests that FGF-2 in spermatogonia is involved in the autocrine and paracrine regulation of the proliferation and differentiation of spermatogonia and spermatocytes via the receptors FGFR-1, FGFR-3 and FGFR-4. Accepted: 23 December 1997     

Editorial

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.     

The role of gelatinases in colorectal cancer progression and metastasis

Various proteases are involved in cancer progression and metastasis. In particular, gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, have been implicated to play a role in colon cancer progression and metastasis in animal models and patients. In the present review, the clinical relevance and the prognostic value of messenger ribonucleic acid (mRNA) and protein expression and proenzyme activation of MMP-2 and MMP-9 are evaluated in relation to colorectal cancer. Expression of tissue inhibitors of MMPs (TIMPs) in relation with MMP expression in cancer tissues and the relevance of detection of plasma or serum levels of MMP-2 and/or MMP-9 and TIMPs for prognosis are also discussed. Furthermore, involvement of MMP-2 and MMP-9 in experimental models of colorectal cancer is reviewed. In vitro studies have suggested that gelatinase is expressed in cancer cells but animal models indicated that gelatinase expression in non-cancer cells in tumors contributes to cancer progression. In fact, interactions between cancer cells and host tissues have been shown to modulate gelatinase expression in host cells. Inhibition of gelatinases by synthetic MMP inhibitors has been considered to be an attractive approach to block cancer progression. However, despite promising results in animal models, clinical trials with MMP inhibitors have been disappointing so far. To obtain more insight in the (patho)physiological functions of gelatinases, regulation of MMP-2 and MMP-9 expression is discussed. Mitogen activated protein kinase (MAPK) signalling has been shown to be involved in regulation of gelatinase expression in both cancer cells and non-cancer cells. Expression can be triggered by a variety of stimuli including growth factors, cytokines and extracellular matrix (ECM) components. On the other hand, MMP-2 and MMP-9 activity regulates bioavailability and activity of growth factors and cytokines, affects the immune response and is involved in angiogenesis. Because of the multifunctionality of gelatinases, it is unpredictable at what stage of cancer development and in which processes gelatinase activity is involved. Therefore, it is concluded that the use of MMP inhibitors to treat cancer should be considered carefully.     

The TORNADO1 and TORNADO2 genes function in several patterning processes during early leaf development in Arabidopsis thaliana

In multicellular organisms, patterning is a process that generates axes in the primary body plan, creates domains upon organ formation, and finally leads to differentiation into tissues and cell types. We identified the Arabidopsis thaliana TORNADO1 (TRN1) and TRN2 genes and their role in leaf patterning processes such as lamina venation, symmetry, and lateral growth. In trn mutants, the leaf venation network had a severely reduced complexity: incomplete loops, no tertiary or quaternary veins, and vascular islands. The leaf laminas were asymmetric and narrow because of a severely reduced cell number. We postulate that the imbalance between cell proliferation and cell differentiation and the altered auxin distribution in both trn mutants cause asymmetric leaf growth and aberrant venation patterning. TRN1 and TRN2 were epistatic to ASYMMETRIC LEAVES1 with respect to leaf asymmetry, consistent with their expression in the shoot apical meristem and leaf primordia. TRN1 codes for a large plant-specific protein with conserved domains also found in a variety of signaling proteins, whereas TRN2 encodes a transmembrane protein of the tetraspanin family whose phylogenetic tree is presented. Double mutant analysis showed that TRN1 and TRN2 act in the same pathway.