Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(o)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(o)/G(1), blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(o)/G(1), and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEG-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta -dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(o)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms. (C) 2001 Elsevier Science Inc. All rights reserved.

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.

Lucerne biology and genetic improvement - an analysis of past activities and future goals in Australia

Breeding methodologies for cultivated lucerne (Medicago sativa L.), an autotetraploid, have changed little over the last 50 years, with reliance on polycross methods and recurrent phenotypic selection. There has been, however, an increase in our understanding of lucerne biology, in particular the genetic relationships between members of the M. sativa complex, as deduced by DNA analysis. Also, the differences in breeding behaviour and vigour of diploids versus autotetraploids, and the underlying genetic causes, are discussed in relation to lucerne improvement. Medicago falcata, a member of the M. sativa complex, has contributed substantially to lucerne improvement in North America, and its diverse genetics would appear to have been under-utilised in Australian programs over the last two decades, despite the reduced need for tolerance to freezing injury in Australian environments. Breeding of lucerne in Australia only commenced on a large scale in 1977, driven by an urgent need to introgress aphid resistance into adapted backgrounds. The release in the early 1980s of lucernes with multiple pest and disease resistance (aphids, Phytophthora, Colletotrichum) had a significant effect on increasing lucerne productivity and persistence in eastern Australia, with yield increases under high disease pressure of up to 300% being recorded over the predominant Australian cultivar, up to 1977, Hunter River. Since that period, irrigated lucerne yields have plateaued, highlighting the need to identify breeding objectives, technologies, and the germplasm that will create new opportunities for increasing performance. This review discusses major goals for lucerne improvement programs in Australia, and provides indications of the germplasm sources and technologies that are likely to deliver the desired outcomes.

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.

Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3

Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.

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.

Woodstoves uncovered: a paediatric problem

Objectives: To document and describe the effects of woodstove burns in children. To identify how these accidents occur so that a prevention strategy can be devised. Design, Patients and Setting: Retrospective departmental database and case note review of all children with woodstove burns seen at the Burns Unit of a Tertiary Referral Children's Hospital between January 1997 and September 2001. Main outcome measures: Number and ages of children burned: circumstances of the accidents; injuries-sustained, treatment-required and long-term sequelae. Results. Eleven children, median age 1.0 year, sustained burns, usually to the hands, of varying thickness. Two children required skin grafting and five required scar therapy. Seven children intentionally placed their hands onto the Outside of the stove. In all children, burns occurred despite adult supervision Conclusions: Woodstoves area cause of burns in children. These injuries are associated with significant morbidity and financial costs. Through public education, woodstove burns can easily be prevented utilising simple safety measures. (C) 2002 Elsevier Science Ltd and ISBI All rights reserved.