Biomedical subjects

Robert Jordan

Publications and source records attributed to Robert Jordan.

26 records · Page 2Linked to original sources

PubMed2003

Monitoring tumor cell proliferation by targeting DNA synthetic processes with thymidine and thymidine analogs.

UNLABELLED: The use of radiolabeled thymidine (TdR) and thymidine analogs as PET-based tracers of tumor growth rate is based on the assumption that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK(1)) activity, provides an accurate measure of active cell proliferation in tumors. The goal of this study was to test this hypothesis and determine how well these tracers track changes in proliferation of tumor cells. METHODS: TK(1) activity; S-phase fraction; and uptake of TdR, 3'-deoxy-3'-fluorothymidine (FLT), and 2'-fluoro-5-methyl-1-(beta-D-2-arabino-furanosyl) uracil (FMAU) were determined in plateau-phase and exponentially growing cultures of 3 human and 3 murine tumor cell lines. RESULTS: TK(1) activity and S-phase fraction increased in all cell lines as cells moved from plateau-phase conditions to exponential growth. Some cell lines had relatively large TK(1) activities and S-phase fractions under plateau-phase conditions, consistent with a loss of normal cell cycle checkpoint control in these cells. There were also 2 cell lines in which TK(1) activity changed little as cells moved from the plateau phase to exponential growth, suggesting that in these cell lines, de novo nucleotide synthesis pathways predominate over salvage pathways. Both TdR and FLT detected changes in TK(1) activity. The slope of the relationship between TdR uptake and TK(1) activity was nearly twice that for FLT and more than 40-fold that for FMAU. CONCLUSION: Although not all tumors show a strong TK(1) dependence of proliferation, in all cell lines for which proliferation is highly TK(1) dependent, phosphorylation of TdR or FLT accurately reflects changes in TK(1) enzyme activity.

Animals↗

PubMed2002

Inhibition of host ER glucosidase activity prevents Golgi processing of virion-associated bovine viral diarrhea virus E2 glycoproteins and reduces infectivity of secreted virions.

Recently, it was shown that replication of bovine viral diarrhea virus (BVDV) is sensitive to inhibitors of host ER glucosidases. Consistent with these findings, we report that incubation of BVDV-infected MDBK cells with the glucosidase inhibitor n-butyl-deoxynojirimycin (nB-DNJ) reduced BVDV yields by 70- to 100-fold (n = 27), while having no effect on MDBK cell viability. However, the 70- to 100-fold reduction in infectious virus was associated with only a 2-fold reduction in genomic RNA synthesis and secretion of enveloped virus particles. Analysis of secreted virions showed that in the absence of glucosidase inhibitor, approximately 50% of the virion-associated BVDV E2 glycoprotein was resistant to endoglycosidase H (endo H) digestion, whereas intracellular E2 was completely sensitive to endo H digestion. In the presence of glucosidase inhibitor, virion-associated E2 and intracellular E2 were completely sensitive to endo H digestion. Taken together, these results suggest that BVDV is secreted through a Golgi-mediated pathway and that host ER glucosidase activity is required for production of infectious virions and Golgi processing of envelope E2 protein during virus egress.

1-Deoxynojirimycin↗

PubMed2002

Effects of 2 different anti-tumor necrosis factor-alpha agents in a primate model of subcutaneous abscess formation.

Tumor necrosis factor (TNF)-alpha exerts both physiologic and pathologic effects in response to infection, conferring the benefit of host defense against infection at the risk of eliciting severe pathology if the response is excessive or inappropriate. In the present study, the effects of an anti-TNF-alpha monoclonal antibody (MAb) and a TNF-alpha receptor construct (p75-Fc) were compared with that of saline in a primate model of subcutaneous abscess induced with Staphylococcus aureus. Intravenous administration of anti-TNF-alpha MAb delayed the onset and reduced the incidence and the severity of abscess formation in response to inoculation with S. aureus at concentrations of 10(9) and 10(10) cfu/mL, compared with administration of saline. In contrast, no improvement in abscess formation was observed in animals treated with p75-Fc. These results supply initial evidence that anti-TNF-alpha MAb, unlike p75-Fc, provides a beneficial effect in this abscess model.

Abscess↗

PubMed2002

The combination of interferon alpha-2b and n-butyl deoxynojirimycin has a greater than additive antiviral effect upon production of infectious bovine viral diarrhea virus (BVDV) in vitro: implications for hepatitis C virus (HCV) therapy.

Interferon alpha-2b (IFN) alone or in combination with Ribavirin is approved in the United States for the treatment of chronic hepatitis C virus (HCV) infection. We have previously reported that the glucosidase inhibitor, n-butyl deoxynojirimycin (nB-DNJ) inhibits the production of infectious bovine diarrhea virus (BVDV) (Proc. Natl. Acad. Sci. 96 (1999) 11878). Since BVDV has been used as a model for HCV and grows productively in tissue culture, and IFN and glucosidase inhibitors are thought to act at different steps in the virus life cycle, it was of interest to determine the antiviral impact of combining nB-DNJ with IFN. Using plaque reduction and single-step growth analyses of the cytopathic BVDV strain NADL, data are presented that shows human IFN inhibited BVDV production in a dose dependent manner, with 3 IU/ml inhibiting 50% of the yield of virus (IC50) when added within 1 h post infection. Under the same conditions, the glucosidase inhibitors nB-DNJ and castanospermine (CST) also prevented BVDV production in a dose dependent manner with IC50s of 226 microM and 47 microM, respectively. In combination with 138 microM nB-DNJ the apparent IC50 for IFN was 0.056 IU/ml. This 54-fold increase in IFN potency suggests that nB-DNJ can synergize with IFN. Two additional independent analyses were performed to measure combination effects which demonstrated that the combined antiviral effect of nB-DNJ and IFN were greater than would be expected for a simple additivity. These data are consistent with an interpretation that glucosidase inhibitors and IFN have a synergistic antiviral effect in tissue culture. The relevance of these finding to treatment of HCV infection is discussed.

1-Deoxynojirimycin↗

PubMed2002

Stability and circularization of herpes simplex virus type 1 genomes in quiescently infected PC12 cultures.

Herpes simplex virus type 1 (HSV-1) DNA has been shown to exist as a linear, double-stranded molecule in the virion and as a non-linear (endless), episomal, nucleosomal form in latently infected trigeminal ganglia. The kinetics of the formation and appearance of endless viral genomes and the stability of linear genomes in neuronal cells are not well understood. Nerve growth factor (NGF)-differentiated PC12 cells can sustain long-term, quiescent infections with HSV-1. In this report, the structure and stability of HSV-1 viral DNA in NGF-differentiated PC12 cells was studied as a function of time following infection using both wild-type and replication-defective virus. Unexpectedly, unencapsidated linear genomes were stable in the nucleus of NGF-differentiated PC12 cells for up to 2-3 weeks following infection, beyond the period at which there is no detectable viral gene expression. However, following infection with wild-type HSV, the majority of quiescent viral genomes were in an endless form after 3-4 weeks. These data suggest that the stability and fate of HSV-1 DNA in non-mitotic neuronal-like cells is different from that in productively infected cells and thus there is a significant cellular role in this process. The relevance to the virus life-cycle in neurones in vivo is discussed.

Animals↗

PubMed2002

Replication of a cytopathic strain of bovine viral diarrhea virus activates PERK and induces endoplasmic reticulum stress-mediated apoptosis of MDBK cells.

Endoplasmic reticulum (ER) stress signaling is an adaptive cellular response to the loss of ER Ca(2+) homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen. ER stress-activated signaling pathways regulate protein synthesis initiation and can also trigger apoptosis through the ER-associated caspase 12. Viruses that utilize the host cell ER as an integral part of their life cycle would be predicted to cause some level of ER stress. Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus of the Flaviviridae family. BVDV and related flaviviruses use the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication, and particle assembly. We are using a cytopathic strain of BVDV (cpBVDV) that causes cellular apoptosis as a model system to determine how virus-induced ER stress contributes to pathogenesis. We show that, in a natural infection of MDBK cells, cpBVDV activates the ER transmembrane kinase PERK (PKR-like ER kinase) and causes hyperphosphorylation of the translation initiation factor eIF2 alpha, consistent with the induction of an ER stress response. Additionally, we show that initiation of cellular apoptosis correlates with downregulation of the antiapoptotic Bcl-2 protein, induced expression of caspase 12, and a decrease in intracellular glutathione levels. Defining the molecular stress pathways leading to cpBVDV-induced apoptosis provides the basis to study how other ER-tropic viruses, such as hepatitis C and B viruses, modulate the host cell ER stress response during the course of persistent infection.

Animals↗

PubMed2002

Imino sugars that are less toxic but more potent as antivirals, in vitro, compared with N-n-nonyl DNJ.

Imino sugar glucosidase inhibitors have selective antiviral activity against certain enveloped, mammalian viruses. Deoxynojirimycins (DNJs) modified by N-alkylation to contain a nine carbon atom side chain (N-n-nonyl-deoxynojirimycin; N-nonyl-DNJ, NN-DNJ) were shown to be, for example, at least 20 times more potent in inhibiting hepatitis B virus (HBV) and bovine viral diarrhoea virus (BVDV) in cell based assays than the non-alkylated DNJ. These data suggested that modification of the alkyl side chain could influence antiviral activity. Previous work has focused on varying side chain length. In this report, the influence of side chain branching and cyclization upon toxicity and antiviral activity was explored. Briefly, using a virus secretion assay for HBV and a single step growth (yield reduction) assay for BVDV, 14 different DNJ-based sugars, possessing various N-alkyl substitutions, were tested for antiviral activity. Of the series, N-methoxy-nonyl-DNJ and N-butyl-cyclohexyl DNJ were determined to have the best selectivity index against BVDV and HBV, with the N-methoxy analogue being the most potent with micromolar antiviral activity. The results of this antiviral survey and the implications for the mechanism of action and ultimate therapeutic potential of the DNJ-based imino sugars is provided and discussed.

1-Deoxynojirimycin↗

PubMed2002

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions.

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.

Cell Division↗