The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb

Geostatistical analysis of the spatial distribution of mycotoxin concentration in bulk cereals

Deoxynivalenol (DON) and ochratoxin A (OTA) in agricultural commodities present hazards to human and animal health. Bulk lots are routinely sampled for their presence, but it is widely acknowledged that designing sampling plans is particularly problematical because of the heterogeneous distribution of the mycotoxins. Previous studies have not take samples from bulk. Sampling plans are therefore designed on the assumption of random distributions. The objective of this study was to analyse the spatial distribution of DON and OTA in bulk commodities with geostatistics. This study was the first application of geostatistical analysis to data on mycotoxins contamination of bulk commodities. Data sets for DON and OTA in bulk storage were collected from the literature and personal communications, of which only one contained data suitable for geostatistical analysis. This data set represented a 26-tonne truck of wheat with a total of 100 sampled points. The mean concentrations of DON and OTA were 1342 and 0.59 mu g kg(-1), respectively. The results showed that DON presented spatial structure, whilst OTA was randomly distributed in space. This difference between DON and OTA probably reflected the fact that DON is produced in the field, whereas OTA is produced in storage. The presence of spatial structure for DON implies that sampling plans need to consider the location of sample points in addition to the number of points sampled in order to obtain reliable estimates of quantities such as the mean contamination

A bi-directional HPTLC development method for the detection of low levels of aflatoxin in maize extracts

A bi-directional high performance thin layer chromatography (HPTLC) method for the determination of low levels of aflatoxin in extracts of maize has been evaluated. The method, using diethylether and chloroform:xylene:acetone (6∶3∶1) as the two developing solvents was found to have a limit of detection of 0.8 (B1), 0.4 (B2), 1.7 (G1) and 0.4 (G2) μg/kg respectivety, with CVs equal or less than 1.7% (B1), 2.5% (B2), 3.1% (G1), and 3.1% (G2). Inter-plate variation was significantly greater than intra-plate variation. Compared with two-dimensional HPTLC methods, a 15 to 30 fold improvement in the sample capacity per plate is achieved and positioning errors are much reduced during densitometry

Evaluation of non-polar bonded-phases for the clean-up of maize extracts prior to aflatoxin assay by HPTLC

The ability of a PH non-polar bonded phase to extract aflatoxin from aqueous solutions (1% v/v) containing 5, 10, 20 and 30% acetone, acetonitrile or methanol was determined. The amount of aflatoxin extracted by the cartridge from aqueous solutions remained almost constant (97–102%) as the concentration of methanol increased, but decreased rapidly when the amount of acetone or acetonitrile increased beyond 10%. Examination of the ability of a number of bonded phases (C2, C8, C18, CH and PH) to extract aflatoxin from aqueous methanol extracts of maize revealed that the PH bonded phase was the most efficient. Aflatoxin is eluted from the cartridge by chloroform (7 ml). An additional clean-up step involving bi-directional development was required when quantifying low levels of aflatoxin in maize by HPTLC

Rapid method for the clean-up of extracts of cottonseed prior to aflatoxin determination by bi-directional HPTLC

A PH non-polar bonded-phase clean-up procedure has been developed for determining aflatoxin concentrations in aqueous acetone extracts of cottonseed by bi-directional HPTLC. It involves the addition of lead acetate solution to the extract prior to its passage through the bonded-phase cartridge. The accuracy and precision of the method have been evaluated for a range of aflatoxin concentrations between 4.7–806.1 μg/kg (B1), 2.8–419.8 μg/kg (B2), 5.1–820.3 μg/kg (G1) and 3.3–396.9 μg/kg (G2). The coefficients of variation varied between 0.8 and 12%, with mean recoveries between 83% and 99%. Correlation coefficients of 1.0 for each aflatoxin showed the closeness of fit of the weighted regression curves. Small systematic errors were detected for aflatoxins B1, B2 and G1 as the 95% confidence intervals of the regression intercepts failed to include the expected value of zero. The limits of detection were found to be between 1.1 μg/kg (B2 and G2) and 2.7 μg/kg (B1). Comparison of the method with the first action AOAC method for cottonseed showed that the PH cartridge method recovered 80% more aflatoxin B1 and 52% more B2 from a sample naturally contaminated with aflatoxin. It was also more precise for aflatoxin B1 as well as being more rapid and cost effective