Characterization of CD8+ cytotoxic T-lymphocyte responses after genetic immunization with retrovirus vectors expressing different forms of the hepatitis B virus core and e antigens.

Cytotoxic T-lymphocyte (CTL) activity appears to play an important role in resolving hepatitis B virus (HBV) infection, and the ability to induce such responses remains an important goal for developing effective immunotherapeutics. A panel of recombinant retrovirus vectors expressing different forms of the HBV core antigen (HBcAg) or e antigen (eAg) were found to induce antigen-specific major histocompatibility complex-restricted CTL responses in both mice and macaques. In addition, a novel retrovirus vector expressing an HBcAg-neomycin phosphotransferase II (HBc-Neo) fusion protein [LHBc-NEO(6A3)], which allows the measurement of the anti-Neo antibody response as a means of directly tracking biological activity of the vector, was generated. Doses greater than 10(7) CFU were necessary to induce CTL responses in H-2(k) mice. Intramuscular injections with 10(8) CFU of the LHBc-NEO(6A3) retrovirus vector into rhesus monkeys induced HBc/eAg-specific antibody production and CD8+ CTLs. The CTL response from one of the two responder rhesus monkeys was directed against a 9-residue peptide, GELMTLATW, at positions 63 to 71 of the HBc/eAg sequence. The CTL response is long lived, being detectable as late as 16 weeks after immunization, and can be boosted upon reimmunization. The potent ability of recombinant retrovirus vectors to induce HBcAg- and eAg-specific CTL responses may prove beneficial as a therapeutic treatment for chronic hepatitis B infection.     

The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies

A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.     

Biochemical,immunochemical, and ultrastructural studies of protein storage in poplar(Populus × canadensis ‘robusta’) wood

The seasonal changes in protein content have been followed in the wood of Populus × canadensis Moench robusta, both biochemically and electronmicroscopically at the cellular level. In the storage-parenchyma cells of the twig wood, 4–6 g · mg^–1 DW protein were deposited in the fall, parallel to the yellowing of leaves, and mobilized completely again during the outgrowth of buds in the spring. Environmental impacts on the leaves, e.g. a fungal attack and mechanical injury by a hurricane, were found to affect protein deposition in the wood considerably. Accumulation of protein bodies in the fall and their disappearance from the cells in the spring proceeded parallel to the changes in protein content measured biochemically, proving that these organelles are the main sites of protein storage in the wood parenchyma cells. Using immunogold labelling and an anti-32-kDa poplar storage-protein antibody the protein bodies were shown to be the exclusive sites of storage of a 32-kDa polypeptide. Transient changes in protein content were also observed during fall and winter. Because these changes coincided with changes in protein-body structure and with changes in the population of vesicles and-or tubular membrane cisternae of the cells, an exchange of nitrogen compounds from the storage pool into the structural protein of membranes possibly takes place during these periods. The structural events observed during proteolysis in spring are very similar to those found in seeds. The possible roles of small cytoplasmic vesicles found within protein bodies during proteolysis and of multimembraneous vacuolar compartments during membrane retrieval are discussed.Abbreviations DW dry weight - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Dedicated to Professor Dr. Dr. Hans Marquardt on the occasion of his 80th birthdayThe valuable technical assistance of Miss Sabine Karg and Miss Astrid Diercks is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft.     

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Determination of cholecystokinin tetrapeptide and cholecystokinin octapeptide sulfate in different rat brain regions by high-pressure liquid chromatography with electrochemical detection

A method for the determination of cholecystokinins in biological material, based on high-pressure liquid chromatography with direct electrochemical detection (HPLC-EC), is described. Using this method, the levels of cholecystokinin tetrapeptide and octapeptide sulfate in rat brain cortex, hippocampus, striatum, and brain stem were measured and found to be comparable to those reported using radioimmunoassay methods. We show that HPLC-EC is sensitive enough to accurately determine neuropeptides in brain tissue without prior derivatization and is therefore, due to its simplicity, an attractive alternative to existing methods.     

Methanotrophic communities of Saanich Inlet: a microcosm perspective

Saanich Inlet (British Columbia, Canada) is a seasonally anoxic fjord characterized by high rates of both methane production and consumption. In this study, the diversity of microbial populations residing in intermediate waters, characterized by having a high methane content, was assessed using CH(4)-microcosm experiments coupled with PCR surveys of phylogenetic (16S rRNA gene) and functional gene markers (pmoA and fhcD genes). The experiments revealed that bacteria represented by sequences affiliated with Methylomicrobium within the Methylococcales, Methylophaga and Cycloclasticus within the Thiotrichales, and uncultured Planctomycetes were enriched in response to CH(4) addition.     

Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetaseby adenylate analogs

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. l-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K_i values in the micromolar range (4–27 μM for human mt- and mammalian cyt-AspRSs). 5′-O-[N-(l-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10 nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.     

Role of viral induced vascular endothelial growth factor (VEGF) production in pleural effusion and malignant mesothelioma

Vascular endothelial growth factor (VEGF) plays a key role in formation of pleural effusions and in tumorigenesis and progression of malignant mesothelioma. Mesothelial cells (MC) express the viral receptors Toll-like receptor 3 (TLR3), RIG-I and MDA5. Activation of these receptors by viral RNA exemplified by poly(I:C) RNA leads to a time- and dose-dependent increase of mesothelial VEGF synthesis. To show the specific effect of viral receptors knockdown experiments with siRNA for TLR3, RIG-I and MDA5 were performed. This finding of viral induced mesothelial VEGF synthesis may indicate a novel link between viral infections and formation of pleural effusions and progression of malignant mesothelioma.     

Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents

In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an α-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.