Phylogeny and expression of carbonic anhydrase-related proteins

Background  

Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR.     

The role of phosphatidylinositol-3-kinases P85/P110 and hVPS34 in endocytosis of EGF-receptor complexes

The previous data (Zheleznova et al., 2001) did not enable the authors to conclude which particular wortmannin sensitive PI-3-kinase--p85/p110 (I class PI-3-K) or hVPS34 (III class PI-3-K)--may be involved in the regulation of EGF-receptor endocytosis. In the present work, we have shown that upon stimulation of EGF-receptor endocytosis additional structures stained with antibody against p85 appear in A431 cells, but the p85-positive compartment never co-localized with EGF-receptor-containing compartments either in control or in wortmannin-treated cells. At the same time, wortmannin treatment prevented association of hVPS34 with endosomal membranes. We have also found that early endosomal markers--Rab5 and EEA1 (membrane association of the latter depends on Rab5 and hVPS34)--co-localized with EGF-receptor in the juxtranuclear region during late stages of endocytosis, both in control and upon wortmannin treatment. These observations favor our suggestions that the transition of EGF-receptors from early to late endosomes may occur directly in this juxtranuclear region and be tightly associated with the formation of so called multivesicular bodies (MVB), which are late endosomes per se. We suggest that wortmannin may have no effect on early EEA1-dependent stage of the receptor endocytosis but blocks a transition of EGF-receptor complexes into the late endosomes by inhibiting activity of hVPS34 and removing it from membranes. The hVPS34 product PI-3-K, according to the known data, is involved in the formation of internal vesicles of MVB. Accumulation of EGF-receptors in these vesicles is believed to be necessary for the receptor degradation.     

Peroxisomal targeting signal-1 receptor protein PEX5 from Leishmania donovani. Molecular, biochemical, and immunocytochemical characterization

The human pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other key metabolic pathways in unique subcellular microbodies called glycosomes, organelles related to the peroxisomes of mammals and yeast. The molecular machinery that carries out the specific targeting of glycosomal proteins to the organelle has not been characterized, although the bulk of glycosomal proteins contain the COOH-terminal tripeptide glycosomal peroxisomal targeting signal-1 (PTS-1) similar to the mammalian and fungal peroxisomal targeting signal. To characterize the mechanisms of glycosomal targeting, the gene encoding PEX5, designated LdPEX5, has been isolated from Leishmania donovani. LdPEX5 encodes a 625-amino acid protein with a molecular mass of 69.7 kDa. Like its homologs in yeast and humans, LdPEX5 predicts a protein with seven copies of a tetratricopeptide repeat in its COOH-terminal half proposed to mediate PTS-1 binding and three copies of a WXXX(Y/F) motif in its NH(2) terminus conjectured to be essential for protein translocation into the organelle. LdPEX5 was overexpressed in Escherichia coli and purified to homogeneity for binding experiments and generation of antibodies. Recombinant LdPEX5 bound xanthine phosphoribosyltransferase (XPRT), a PTS-1 containing glycosomal protein with a K(D) of 4.2 nm, but did not bind an XPRT in which the PTS-1 had been deleted. Moreover, binding studies with the COOH-terminal half of the LdPEX5 confirmed that this portion of the PEX5 protein was capable of binding the XPRT PTS-1 with an affinity of 17.3 nm. Confocal microsocopy revealed that LdPEX5 was predominantly in the cytosolic milieu, and genetic analysis implied that LdPEX5 was an essential gene.     

c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY(418))-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY(418)-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α(2)β(1)-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.     

Detoxification of ochratoxin A,a food contaminant: Prevention of growth of Aspergillus ochraceus and its production of ochratoxin A

The toxicity of ochratoxin A (OTA), a mycotoxin produced by fungi ofAspergillus orPenicillium genera is now well documented. Its nephrotoxicity, immunosuppression, teratogenicity, and carcinogenicity have been widely studied. Physical and biochemical methods have been studied to prevent these toxinogenicAspergillus andPenicillium from producing OTA, and/or to destroy the mycotoxin when already produced in a liquid or a solid medium. Repeated freezing at ? 20?C and thawing at + 26?C aleatory reduce OTA production in a liquid medium. Exposure to UV B for different periods of time is efficient in preventing OTA production in a liquid medium. Gamma-irradiation from 2 to 5 kGy gives good results in preventing the production of OTA or destroying it when already produced. Carboxypeptidase is very efficient at 5 units/50 ml in a liquid medium for cleaving the OTA already produced.     

Autumn Retranslocation of Heavy Metals from Leaves of Woody Plants in Forest Ecosystems

Biology Bulletin - This paper examines the autumn retranslocation of heavy metals (HMs), namely Cu, Zn, and Cd, from the photosynthetic organs of woody plants. This study was carried out in the...