The effects of elevated summer temperature and sublethal pollutants (ammonia, low pH) on protein turnover in the gill and liver of rainbow trout (Oncorhynchus mykiss) on a limited food ration.

Protein synthesis, degradation and growth of the liver and gills were determined in juvenile rainbow trout (Oncorhynchus mykiss) fed a limited ration and exposed for 90 days to normal or elevated summer temperatures (+2 degrees above ambient) and either low pH (5.2) in softwater or 70 microM total ammonia in hardwater. The limited ration resulted in low rates of growth (< 0.80% per day) and protein synthesis in all fish. In softwater, whole-body growth was significantly inhibited by elevated temperature but stimulated by low pH, although tissue protein metabolism was generally unaffected by these treatments. There was no significant difference in final size between the groups of fish in hardwater, but liver protein synthesis and degradation were significantly lower at +2 degrees C, the reduction in synthesis being due to an inhibition of both the capacity for protein synthesis, Cs and the RNA translational efficiency, kRNA. Gill protein metabolism was unaffected by the experimental treatments in trout in hardwater. The authors conclude that a global warming scenario would be detrimental to protein synthesis and growth in freshwater fish under conditions of food limitation in summer, and when late summer temperatures approached the upper thermal limit of the species, regardless of food availability.     

Clathrin Functions in the Absence of the Terminal Domain Binding Site for Adaptor-associated Clathrin-Box Motifs

Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and during endocytosis. Clathrin recruitment to membranes is mediated by the clathrin heavy chain (HC) N-terminal domain (TD), which forms a seven-bladed β-propeller. TD binds membrane-associated adaptors, which have short peptide motifs, either the clathrin-box (CBM) and/or the W-box; however, the importance of the TD binding sites for these motifs has not been tested in vivo. We investigated the importance of the TD in clathrin function by generating 1) mutations in the yeast HC gene (CHC1) to disrupt the binding sites for the CBM and W-box (chc1-box), and 2) four TD-specific temperature-sensitive alleles of CHC1. We found that TD is important for the retention of resident TGN enzymes and endocytosis of α-factor; however, the known adaptor binding sites are not necessary, because chc1-box caused little to no effect on trafficking pathways involving clathrin. The Chc1-box TD was able to interact with the endocytic adaptor Ent2 in a CBM-dependent manner, and HCs encoded by chc1-box formed clathrin-coated vesicles. These data suggest that additional or alternative binding sites exist on the TD propeller to help facilitate the recruitment of clathrin to sites of vesicle formation.     

Molecular structures of coat and coat-associated proteins: function follows form

Endocytic clathrin-coated vesicles arise through the deformation of a small region of plasma membrane encapsulated by a cytosol-oriented clathrin lattice. The coat assembles from soluble protomers in a rapid and highly cooperative process, and invagination is tightly linked to the selective enrichment of cargo molecules within the nascent bud. Recent structural and functional studies demonstrate that coat assembly, membrane deformation, local actin dynamics and the final scission event are intricately coupled, and begin to reveal how key multifunctional, modular proteins are responsible for this linkage. An emerging mechanistic theme is how sequential engagement of common interaction surfaces or network hubs can evict prior binding partners from the assembly zone to ensure vectorial progression of the coat assembly process.     

The recycling of apolipoprotein E and its amino-terminal 22 kDa fragment: evidence for multiple redundant pathways

A portion of apolipoprotein E (apoE) internalized by hepatocytes is spared degradation and is recycled. To investigate the intracellular routing of recycling apoE, primary hepatocyte cultures from LDL receptor-deficient mice and mice deficient in receptor-associated protein [a model of depressed expression of LDL receptor-related protein (LRP)] were incubated with human VLDL containing 125I-labeled human recombinant apoE3. Approximately 30% of the internalized intact apoE was recycled after 4 h. The N-terminal 22 kDa fragment of apoE was also resecreted, demonstrating that this apoE domain contains sufficient sequence to recycle. The 22 kDa fragment has reduced affinity for lipoproteins, suggesting that apoE recycling is linked to the ability of apoE to bind directly to a recycling receptor. Finally, apoE was found to recycle equally well in the presence of brefeldin A, a drug that blocks transport from the endoplasmic reticulum and leads to collapse of the Golgi stacks. Our studies demonstrate that apoE recycling occurs 1) in the absence of the LDL receptor or under conditions of markedly reduced LRP expression; 2) when apoE lacks the carboxyl-terminal domain, which allows binding to the lipoprotein; and 3) in the absence of an intact Golgi apparatus. We conclude that apoE recycling occurs through multiple redundant pathways.     

The Anopheles maculipennis complex (Diptera: Culicidae) in Iran: molecular characterization and recognition of a new species

Mosquitoes of the Anopheles maculipennis complex were collected in nine provinces of Iran (Esfahan, Fars, Gilan, Golestan, Kohkiluyeh va Boyerahmad, Mazandaran, Tehran, Azarbaijan-e Gharbi and Zanjan) between June 1983 and September 2002. The nuclear rDNA ITS2 sequences of 86 specimens were compared with those of seven species of the complex available in GenBank. Three genetically distinct species of the complex were distinguished: A. maculipennis Meigen, A. sacharovi Favre and a previously unrecognized species. The last species is most similar to, but clearly distinct from, A. martinius Shingarev and A. sacharovi. The taxonomy of A. martinius and A. sacharovi is critically reviewed, and justification is provided for formally recognizing the third species as Anopheles persiensis sp.n. The new species is the first culicid to be characterized and named principally on the basis of DNA evidence. Anopheles persiensis was collected only in the northern Caspian Sea littoral provinces of Gilan and Mazandaran, and it seems likely that this species could be responsible for malaria transmission in this region that was previously attributed to A. maculipennis. A species-specific RFLP-PCR assay based on ITS2 sequences was developed to facilitate further studies of the three species in Iran.     

Recycling of apolipoprotein E in mouse liver

Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoE-containing beta-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d = 1.019-1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoE-containing remnant lipoproteins.     

Vector prime/protein boost vaccine that overcomes defects acquired during aging and cancer

We showed that the Ad-sig-TAA/ecdCD40L vaccine induces a tumor suppressive immune response to the hMUC-1 and rH2N tumor-associated self Ags (TAA) and to the Annexin A1 tumor vascular Ag, even in mice in which anergy exists to these Ags. When the TAA/ecdCD40L protein is given s.c. as a boost following the Ad-sig-TAA/ecdCD40L vector, the levels of the TAA-specific CD8 T cells and Abs increase dramatically over that seen with vector alone, in young (2-mo-old) as well as old (18-mo-old) mice. The Abs induced against hMUC-1 react with human breast cancer. This vaccine also induces a 4-fold decrement of negative regulatory CD4CD25FOXP3-T cells in the tumor tissue of 18-mo-old mice. These results suggest that the Ad-sig-TAA/ecdCD40L vector prime-TAA/ecdCD40L protein boost vaccine platform may be valuable in reducing postsurgery recurrence in a variety of epithelial neoplasms.     

Molecular phylogeny of Pholadoidea Lamarck, 1809 supports a single origin for xylotrophy (wood feeding) and xylotrophic bacterial endosymbiosis in Bivalvia

The ability to consume wood as food (xylotrophy) is unusual among animals. In terrestrial environments, termites and other xylotrophic insects are the principle wood consumers while in marine environments wood-boring bivalves fulfill this role. However, the evolutionary origin of wood feeding in bivalves has remained largely unexplored. Here we provide data indicating that xylotrophy has arisen just once in Bivalvia in a single wood-feeding bivalve lineage that subsequently diversified into distinct shallow- and deep-water branches, both of which have been broadly successful in colonizing the world’s oceans. These data also suggest that the appearance of this remarkable life habit was approximately coincident with the acquisition of bacterial endosymbionts. Here we generate a robust phylogeny for xylotrophic bivalves and related species based on sequences of small and large subunit nuclear rRNA genes. We then trace the distribution among the modern taxa of morphological characters and character states associated with xylotrophy and xylotrepesis (wood-boring) and use a parsimony-based method to infer their ancestral states. Based on these ancestral state reconstructions we propose a set of plausible hypotheses describing the evolution of symbiotic xylotrophy in Bivalvia. Within this context, we reinterpret one of the most remarkable progressions in bivalve evolution, the transformation of the “typical” myoid body plan to create a unique lineage of worm-like, tube-forming, wood-feeding clams. The well-supported phylogeny presented here is inconsistent with most taxonomic treatments for xylotrophic bivalves, indicating that the bivalve family Pholadidae and the subfamilies Teredininae and Bankiinae of the family Teredinidae are non-monophyletic, and that the principle traits used for their taxonomic diagnosis are phylogenetically misleading.     

Cytoplasmic fatty acid-binding proteins: emerging roles in metabolism and atherosclerosis

Cytoplasmic fatty acid-binding proteins (FABPs) are a family of proteins, expressed in a tissue-specific manner, that bind fatty acid ligands and are involved in shuttling fatty acids to cellular compartments, modulating intracellular lipid metabolism, and regulating gene expression. Several members of the FABP family have been shown to have important roles in regulating metabolism and have links to the development of insulin resistance and the metabolic syndrome. Recent studies demonstrate a role for intestinal FABP in the control of dietary fatty acid absorption and chylomicron secretion. Heart FABP is essential for normal myocardial fatty acid oxidation and modulates fatty acid uptake in skeletal muscle. Liver FABP is directly involved in fatty acid ligand signaling to the nucleus and interacts with peroxisome proliferator-activated receptors in hepatocytes. The adipocyte FABP (aP2) has been shown to affect insulin sensitivity, lipid metabolism and lipolysis, and has recently been shown to play an important role in atherosclerosis. Interestingly, expression of aP2 by the macrophage promotes atherogenesis, thus providing a link between insulin resistance, intracellular fatty acid disposition, and foam cell formation. The FABPs are promising targets for the treatment of dyslipidemia, insulin resistance, and atherosclerosis in humans.