[show abstract][hide abstract] ABSTRACT: The hypoxia responsive region (HRR) of the VEGF promoter plays a key role in regulating VEGF expression. We found that the cold shock domain (Y-box) repressor proteins, dbpA and dbpB/YB-1, bind distinct strands of the human VEGF HRR. We find both dbpA and dbpB are phosphorylated by ERK2 and GSK3beta in vitro, and the binding of dbpB to single-strand VEGF HRR DNA is regulated by this phosphorylation. These findings suggest the ERK/MAPK and PI3K pathways may regulate VEGF expression in part through regulating the action of these repressor proteins.
[show abstract][hide abstract] ABSTRACT: A real time RT-PCR, using the LightCycler, was developed and compared with rapid antigen enzyme immunoassay (AgEIA) and enhanced virus culture for rapid detection of influenza A viruses in stored and prospectively collected respiratory specimens. Specific hybridization probes were used for simultaneous detection and differentiation between H1N1 and H3N2 subtypes. The sensitivity of the RT-PCR for influenza A H1N1 was 120 copies and H3N2 350 copies of in vitro transcribed RNA. A specimen was considered positive for influenza A when it was culture positive or at least two methods yielded a positive test result. Using these criteria, with stored samples, the RT-PCR sensitivity, specificity, positive and negative predictive values were 82.9, 95.5, 98.9 and 52.5%, respectively. In specimens collected prospectively the RT-PCR sensitivity, specificity, positive and negative predictive values were 100, 87.9, 82.8 and 100%, respectively. There was complete concordance with subtype differentiation by hybridization probe melting temperature analysis and haemagglutination inhibition assay.
Journal of Virological Methods 06/2004; 117(2):103-12. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Overexpression of vascular endothelial growth factor (VEGF) is implicated in a number of diseases. It is therefore critical that mechanisms exist to strictly regulate VEGF expression. A hypoxia-responsive (HR) region of the VEGF promoter which binds the HIF-1 transcription factor is a target for many signals that up-regulate VEGF transcription. Repressors targeting the HIF-1 transcription factor have been identified but no repressors directly binding the HR promoter region had been reported. We now report a novel mechanism of repression of the VEGF HR region involving DNA binding. We find that single strand DNA-specific cold shock domain (CSD or Y-box) proteins repress the HR region via a binding site downstream of the HIF-1 site. The repressor site is functional in unstimulated, normoxic fibroblasts and represents a novel means to prevent expression of VEGF in the absence of appropriate stimuli. We characterized complexes forming on the VEGF repressor site and identified a previously unreported nuclear CSD protein complex containing dbpA. Nuclear dbpA appears to bind as a dimer and we determined a means by which nuclear CSD proteins may enter double strand DNA to bind to their single strand sites to bring about repression of the VEGF HR region.
Nucleic Acids Research 12/2002; 30(22):4845-54. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV.
The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant.
This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days).