In situ localization and in vitro induction of plant COPI-coated vesicles

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.     

Relationship between dental morphology, sex, body length and age in Pontoporia blainvillei and Sotalia fluviatilis (Cetacea) in Northern Rio de Janeiro, Brazil

The relationship between dental morphology, sex, body length and age of small cetaceans can be used to determine ontogeny, sexual dimorphism and geographical variation. The objective of this study was to determine the relationship between dental morphology, sex, body size and age. A total of 91 specimens of P. blainvillei and 80 specimens of S. fluviatilis accidentally captured in fisheries or stranded in northern Rio de Janeiro (21 masculine37'-22 masculine25'S), from September 1988 to November 1996 were analysed. The teeth root diameter in P. blainvillei was significantly different between the sex; the values for females were larger than males. In neither species aid we observed significant in variations dimension and number of teeth, thickness of dentine and cemental layers and in the maximum width of cement as a function of body size. Age was related to increases in tooth length, root and cingulum diameters, and maximum width of cement in individuals of P. blainvillei, and tooth and crown lengths and maximum width of cement in individuals of S. fluviatilis. The observation of a linear growth between maximum width of cement and age in both species indicates that the equations obtained can be used to estimate relative age in P. blainvillei and S. fluviatilis in northern of Rio de Janeiro.     

Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.     

Gefitinib-mediated Reactive Oxygen Specie (ROS) Instigates Mitochondrial Dysfunction and Drug Resistance in Lung Cancer Cells

Therapeutic benefits offered by tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa) and erlotinib (Tarceva), are limited due to the development of resistance, which contributes to treatment failure and cancer-related mortality. The aim of this study was to elucidate mechanistic insight into cellular perturbations that accompany acquired gefitinib resistance in lung cancer cells. Several lung adenocarcinoma (LAD) cell lines were screened to characterize epidermal growth factor receptor (EGFR) expression and mutation profile. To circumvent intrinsic variations between cell lines with respect to response to drug treatments, we generated gefitinib-resistant H1650 clone by long-term, chronic culture under gefitinib selection of parental cell line. Isogenic cells were analyzed by microarray, Western blot, flow cytometry, and confocal and transmission electron microscope. We observed that although chronic gefitinib treatment provided effective action against its primary target (aberrant EGFR activity), secondary effects resulted in increased cellular reactive oxygen species (ROS). Gefitinib-mediated ROS correlated with epithelial-mesenchymal transition, as well as striking perturbation of mitochondrial morphology and function. However, gefitinib treatment in the presence of ROS scavenger provided a partial rescue of mitochondrial aberrations. Furthermore, withdrawal of gefitinib from previously resistant clones correlated with normalized expression of epithelial-mesenchymal transition genes. These findings demonstrate that chronic gefitinib treatment promotes ROS and mitochondrial dysfunction in lung cancer cells. Antioxidants may alleviate ROS-mediated resistance.     

Trophic flexibility and opportunism in pike Esox lucius

The first comprehensive investigation of pike Esox lucius trophic ecology in a region (Ireland) where they have long been thought to be a non‐native species is presented. Diet was investigated across habitat types (lake, river and canal) through the combined methods of stable‐isotope and stomach content analyses. Variations in niche size, specialization and the timing of the ontogenetic dietary switch were examined, revealing pronounced opportunism and feeding plasticity in E. lucius, along with a high occurrence of invertivory (up to 60 cm fork length, L_F) and a concomitant delayed switch to piscivory. Furthermore, E. lucius were found to primarily prey upon the highly available non‐native roach Rutilus rutilus, which may alleviate predation pressure on brown trout Salmo trutta, highlighting the complexity of dynamic systems and the essential role of research in informing effective management.     

Properties of the thermostable glutamate dehydrogenase of the mesophilic anaerobe Peptostreptoccus asaccharolyticus purified by a novel method after over-expression in an Escherichia coli host

Peptostreptococcus asaccharolyticus glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating); EC 1.4.1.2) overexpressed in Escherichia coli has been purified by two new methods. Enzyme made by the first method showed remarkable thermophilicity, with a temperature optimum of 60 degrees C, and also thermostability, which suggested the second, simpler method, incorporating a heat step. This produced 94 mg of homogeneous protein per litre culture medium. The basic kinetic parameters for P. asaccharolyticus glutamate dehydrogenase with all substrates are revealed at pH 7.0. The enzyme is highly specific for NAD+, with values for kcat/Km 405 times greater than for NADP+. In the reverse direction of reaction, the kcat/Km value for NADH is almost 1000-fold greater than for NADPH.     

Degradation of mutated bovine pancreatic trypsin inhibitor in the yeast vacuole suggests post-endoplasmic reticulum protein quality control

The rate-limiting step in protein secretion is folding, which occurs in the endoplasmic reticulum (ER) lumen, and almost all secreted proteins contain disulfide bonds that form in the ER and stabilize the native state. Secreted proteins unable to fold may aggregate or they may be subject to ER-associated protein degradation. To examine the fate of aberrant forms of a well characterized, disulfide-bonded secreted protein, we expressed bovine pancreatic trypsin inhibitor in yeast. Bovine pancreatic trypsin inhibitor is a single domain, 58-amino acid polypeptide containing three disulfide bonds, and yeast cells secrete the wild type protein. In contrast, the Y35L mutant, which folds rapidly but is unstable, remains soluble and is not secreted. Surprisingly, the proteolysis of Y35L is unaffected in yeast containing mutations in genes encoding factors required for ER-associated protein degradation and is stable if artificially retained in the ER. Rather, Y35L is diverted from the Golgi to the vacuole and degraded. Because only the mutant protein is quantitatively proteolyzed these data suggest that a post-ER quality control check-point diverts unstable proteins to the vacuole for degradation.     

Application of a rapid method of SNP analysis (glycosylase mediated polymorphism detection) to mtDNA and nuclear DNA of cod Gadus morhua

A novel method of single nucleotide polymorphism (SNP) analysis in fishes [glycosylase mediated polymorphism detection (GMPD)] was applied, for the first time in any fish species, to polymorphisms present in the Atlantic cod Gadus morhua mtDNA D-loop and nuclear transferrin genes. The utility of the GMPD technique for population genetics of Atlantic cod is highlighted, since significant haplotype frequency differences were observed between Baltic and Atlantic samples of cod at a D-loop SNP and allele frequencies differences between west and east Atlantic samples at an SNP located in the transferrin gene.