Vitamin B12 and hepatitis C: Molecular biology and human pathology

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the H(IV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV), We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host), The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.

Diurnal changes in the photochemical efficiency of the symbiotic dinoflagellates (Dinophyceae) of corals: photoprotection, photoinactivation and the relationship to coral bleaching

The photochemical efficiency of symbiotic dinoflagellates within the tissues of two reef-building corals in response to normal and excess irradiance at wafer temperatures < 30 C were investigated using pulse amplitude modulated (PAM) chlorophyll fluorescence techniques, Dark-adapted F-v/F-m showed clear diurnal changes, decreasing to a low at solar noon and increasing in the afternoon. However, F-v/F-m also drifted downwards at night or in prolonged darkness, and increased rapidly during the early morning twilight. This parameter also increased when the oxygen concentration of the wafer holding the corals was increased. Such changes have not been described previously, and most probably reflect state transition's associated with PQ pool reduction via chlororespiration. These unusual characteristics may be a feature of an endosymbiotic environment, reflective of the well-documented night-time tissue hypoxia that occurs in corals. F-v/F-m decreased to 0.25 in response to full sunlight in shade-acclimated (shade) colonies of Stylophora pistillata, which is considerably lower than in light-acclimated (sun) colonies. In sun colonies, the reversible decrease in F-v/F-m was caused by a lowering of F-m and F-o suggesting photoprotection and no lasting damage. The decrease in F-v/F-m, however, was caused by a decrease in F-m and an increase in F-o in shade colonies suggesting photoinactivation and long-term cumulative photoinhibition. Shade colonies rapidly lost their symbiotic algae (bleached) during exposure to full sunlight. This study is consistent with the hypothesis that excess light leads to chronic damage of symbiotic dinoflagellates and their eventual removal from reef-building corals. It is significant that this can occur with high light conditions alone.

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression

The c fins gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c -fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSS) were identified within mouse intron 2. Sequences of these DHSS were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fins intronic regulatory element (FIRE), which is even more highly conserved than the c-fins proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Spl, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high and low-level c -fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fins gene.

RelB nuclear translocation mediated by C-terminal activator reigons of Epstein Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappaB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IkappaBalpha S32/36A, an NF-kappaB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappaB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

Energy cost of activity assessed by indirect calorimetry and a (CO2)-C-13 breath test

Purpose: The aim of this study was to assess the accuracy of a (CO2)-C-13 breath test for the prediction of short-duration energy expenditure. Methods: Eight healthy volunteers walked at 1.5 km.h(-1) for 60 min followed by 60-min recovery. During this time, the energy cost of physical activity was measured via respiratory calorimetry and a C-13 bicarbonate breath test. A further eight subjects were tested using the same two methods during a 60-min cycle at 0.5 kp. 30 ipm followed by a 60-min recovery. The rate of appearance of (CO2)-C-13, (RaCO2) was measured and the mean ratio, (V) over dot CO2/RaCO2 was used to calculate energy expenditure using the isotopic approach. Results: As would be expected, there was a significant difference in the energy cost of walking and cycling using both methods (P < 0.05). However. no significant differences were observed between respiratory calorimetry and the isotope method for measurement of energy expenditure while walking or cycling. Conclusions: These data suggest that the C-13 breath test is a valid method that can be used to measure the energy cost of short duration physical activity in a field setting.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

The efficiency and condition of oysters and macroalgae used as biological filters of shrimp pond effluent

Current shrimp pond management practices generally result in elevated concentrations of nutrients, suspended solids, bacteria and phytoplankton compared with the influent water. Concerns about adverse environmental impacts caused by discharging pond effluent directly into adjacent waterways have prompted the search for cost-effective methods of effluent treatment. One potential method of effluent treatment is the use of ponds or raceways stocked with plants or animals that act as natural biofilters by removing waste nutrients. In addition to improving effluent water quality prior to discharge, the use of natural biofilters provides a method for capturing otherwise wasted nutrients. This study examined the potential of the native oyster, Saccostrea commercialis (Iredale and Roughley) and macroalgae, Gracilaria edulis (Gmelin) Silva to improve effluent water quality from a commercial Penaeus japonicus (Bate) shrimp farm, A system of raceways was constructed to permit recirculation of the effluent through the oysters to maximize the filtration of bacteria, phytoplankton and total suspended solids. A series of experiments was conducted to test the ability of oysters and macroalgae to improve effluent water quality in a flow-through system compared with a recirculating system. In the flow-through system, oysters reduced the concentration of bacteria to 35% of the initial concentration, chlorophyll a to 39%, total particulates (2.28-35.2 mum) to 29%, total nitrogen to 66% and total phosphorus to 56%. Under the recirculating flow regime, the ability of the oysters to improve water quality was significantly enhanced. After four circuits, total bacterial numbers were reduced to 12%, chlorophyll a to 4%, and total suspended solids to 16%. Efforts to increase biofiltration by adding additional layers of oyster trays and macroalgae-filled mesh bags resulted in fouling of the lower layers causing the death of oysters and senescence of macroalgae. Supplementary laboratory experiments were designed to examine the effects of high effluent concentrations of suspended particulates on the growth and condition of oysters and macroalgae. The results demonstrated that high concentrations of particulates inhibited growth and reduced the condition of oysters and macroalgae. Allowing the effluent to settle before biofiltration improved growth and reduced signs of stress in the oysters and macroalgae. A settling time of 6 h reduced particulates to a level that prevented fouling of the oysters and macroalgae.