Mechanisms of Genotoxin-Induced Transcription and Hypermutation in p53

It is widely assumed that genotoxin-induced damage (e.g., G-to-T transversions) to the tumor suppressor gene, p53, is a direct cause of cancer. However, genotoxins also induce the stress response, which upregulates p53 transcription and the formation of secondary structures from ssDNA. Since unpaired bases are thermodynamically unstable and intrinsically mutable, increased transcription could be the cause of hypermutation, and thus cancer. Support for this hypothesis has been obtained by analyzing 6662 mutations in all types of cancer compared to lung and colon cancers, using the p53 mutation database. The data suggest that genotoxins have two independent effects: first, they induce p53 transcription, which increases the number of mutable bases that determine the incidence of cancer. Second, genotoxins may alter the fate, or ultimate mutation of a mutable base, for example, by causing more of the available mutable Gs to mutate to T, leaving fewer to mutate to A. Such effects on the fate of mutable bases have no impact on the incidence of cancer, as both types of mutations lead to cancer

Investigations of the Australian bovine lentivirus

The bovine lentiviruses (BL), Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are two relatively unknown lentiviruses that produce remarkably different disease syndromes. BIV typically causes a mild and transient lymphocytosis with associated lymphadenopathy in Bos taurus cattle with some suggested mild immune suppression. JDV, on the other hand, is an acutely pathogenic lentivirus causing marked leucopoenia with associated lymphadenopathy and splenomegaly and has an experimental mortality rate of approximately 20%. Recombinant antigens of both bovine lentivirus including the immunodominant viral proteins, capsid (CA or p26) and transmembrane (TM or p42) envelope proteins were developed using a glutathione-s-transferase fusion protein expressed m a heterologous Escherichia coli expression system. The recombinant proteins were recognised by antisera from experimentally infected animals of the respective viruses but also showed significant cross-reactivity. The recombinant proteins were used as antigens to develop western immunoblot and ELISA serological assays to identify cattle with antibody to bovine lentivirus with high specificity and sensitivity. An antigenically-related bovine lentivirus was identified in Australian cattle including the states of Queensland, South Australia and Western Austnilia. Sera from random cattle provided by the local agricultural department (AgWA) showed reactivity to BIV and JDV CA antigens with only slight reactivity to TM proteins. Interestingly, serum samples varied in specificity for the BIV or JDV antigens indicating exposure to antigenically variable viruses. Of the 690 Dairy cattle in Western Australia surveyed, approximately 16% by western immunoblot (WIB) and 4% by ELISA were found to have been exposed to an antigenically-related bovine lentivirus. A high percentage (74% by ELISA and 61% by WIB) of WA dairy properties were also found to have at least one animal with bovine lentivirus antibody. Subsequent investigation concluded that bovine lentivirus infection is endemic to Australian cattle herds. Several herds in Queensland, South Australia and Western Australia with a history of immune suppressive disorders of unknown aetiology were also investigated for the seroprevalence of bovine lentivirus. There was high level of correlation of bovine lentivirus antigen reactivity and the presence of the immune suppressive disease in IX these problem herds. However, the role of bovine lentivirus infection in the cause of the condition was unclear but unlikely to be the cause of such diseases. A PCR assay was developed that could identify .bovine lentiviruses. Many sets of published PCR primers for the identification of BIV were examined for specificity and sensitivity. Specific primer sets that could identify both of the known bovine lentiviruses and also differentiate between JDV and BIV were determined. The sensitivity of the PCR assay was adequate but showed high specificity to the target. Attempts to further investigate the Australian bovine lentivirus by in vitro culture and nucleotide sequencing from antibody-positive cattle were unsuccessful. There was an indication that a very low number of lymphocytes contain bovine lentivirus genetic material and most probably contains a divergent nucleotide sequence to that known for the US isolate. In addition, co-culture of peripheral blood mononuclear cells from antibody-positive animals with primary foetal bovine lung cells did not show any significant evidence of BIV replication. For these reasons, it concluded that the PCR assay for BIV based upon US isolates was not applicable to the Australian bovine lentivirus, possibly due to low specificity and sensitivity. Experimental infection with blood from seropositive cattle commonly caused a mild transient lymphocytosis and mild lymphadenopathy but had no association with apparent clinical disease. Individual animals responded differently to experimental infection ranging from a marked persistent lymphocytosis to a mild and transient lymphocytosis. As a direct result of this research, there is an increased understanding of the extent of bovine lentivirus infection in Australia and a possible association with immune suppressive disorders present. This study has produced information about the Australian bovine lentivirus that will be the foundation of bovine lentivirus research to determine the effect it may have on the cattle industry in Australia and Indonesia. This study has been valuable for the reagents developed that will be used for the specific surveillance of JDV in Indonesia and vaccination for JDV in Indonesian cattle

Compartmentalization of HIV-1 in the Central Nervous System: Role of the Choroid Plexus

Objectives: To determine the genetic and phenotypic composition of HIV-1 found in the choroid plexus (CPx) and its relationship to virus in the brain and peripheral lymphoid tissue. Design: Phenotypic and molecular comparisons of HIV-1 found in autopsy brain, CPx, and spleen tissues. Methods: HIV-1 was co-cultured from matched postmortem brain (basal ganglia), CPx, and spleen tissues of AIDS patients with and without HIV-associated encephalitis and dementia. Viral phenotypes were determined by infection of monocyte-derived macrophages, MT-2 or co-receptor-specific cell lines. Viral env and pol sequences were determined from genomic DNA isolated directly from tissues or co-cultures, and phylogenetic comparisons were performed. Results: CCR5-utilization was the most prevalent viral tropism found in all tissues, although spleen isolates also displayed CXCR4 usage. Viruses isolated from CPx consisted of both peripheral and brain-like virus, but were more related phenotypically and genetically to those found in the brain. Mutations found in the pol gene that could confer drug resistance to brain and CPx isolates were similar to those found in the periphery. Conclusion: The CPx contained replication-competent virus that was most similar, although distinct, from that found in the brain. It also contained some viruses with high similarity to those of peripheral origin. Compartmentalization of viral env and pol sequences indicated that differential selective pressures exist in each tissue examined. These studies suggest that the CPx may provide an environment that promotes the evolution of drug-resistant strains with central nervous system tropism, although it is unlikely to be a reservoir for archival HIV-1 variants

Serological evidence of an Australian bovine lentivirus

Recombinant 26kDa capsid (CA) proteins of bovine lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), were expressed in Escherichia coli and utilised as antigens for an enzyme-linked immunosorbent assay (ELISA) and a western immunoblot (WIB) procedure for the detection of antibody in dairy cattle in Western Australia. A total of 690 serum samples, 30 from each of 23 farms, were tested by ELISA with a JDV CA protein antigen, and antibody was detected in 3.8% (p<0.05) of the sera. Nine sera from each farm were also tested by WIB with JDV CA protein antigens and antibody was detected in 15.9% of these samples. All ELISA-positive results were also WIB-positive, and all sera antibody-positive by WIB with JDV CA protein antigens were also antibody-positive by the WIB using recombinant BIV CA antigens. This study showed that recombinant protein antigens can be used for serological tests to detect bovine lentivirus infection in Australia

Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses

Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia. The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV). Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV. The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione. The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV. The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera. The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera. The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses. The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle

Tracking the acetate threshold using DO-transient control during medium and high cell density cultivation of recombinant Escherichia coli in complex media

DO-transient nutrient controllers use the dissolved oxygen signal to attempt acetate threshold tracking during fed-batch cultivation of recombinant E coli. Here we apply DO-transient control to the production of Jembrana disease virus protein in complex Super Luria medium and compare performance against a high-limit pH-stat controller. For induction at medium cell density (harvest between 31 and 32.5 g dcw L) a total productivity of 0.27 g L h was achieved as compared to 0.24 g L h with the high-limit pH-stat. For induction at high cell density (harvest at 60 g dcw L), decreased productivity (0.12 g L h) was attributed to the effect of acetate accumulation on recombinant protein formation and a concomitant lowering of the critical growth rate. Our results suggest that complex media provides a difficult environment for the application of acetate threshold tracking DO-transient control because of difficulties in re-oxidizing acetate, and apparent localized production of acetate below the production threshold (as detected by the DO-transient controller as SPOURcrit). Configuring the DO-transient controller to avoid aggressive threshold probing is suggested as a means to improve performance and reduce acetate accumulation in complex media. (C) 2003 Wiley Periodicals, Inc