Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex

To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9- epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB- binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB- binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.      

New hepatitis B virus mutant form in a blood donor that is undetectable in several hepatitis B surface antigen screening assays

BACKGROUND: Envelope mutant forms of hepatitis B virus (HBV), impairing HBV antibody recognition, have been reported with mutations in single or multiple sites of the hepatitis B surface antigen (HBsAg) group- specific "a" determinant. Blood donors infected with such an HBsAg mutant form of HBV may escape detection by HBsAg screening assays and therefore may affect the safety of the blood supply. CASE REPORT: A repeat blood donor became HBsAg-reactive in an enzyme immunoassay. Confirmatory testing yielded negative results for HBsAg in a radioimmunoassay and in four enzyme immunoassays used in blood donor screening. The specificity of the HBsAg reactivity in the first enzyme immunoassay was confirmed by HBsAg neutralization with antibody to HBsAg. Additional HBV confirmatory test results were positive for antibody to hepatitis B core antigen and antibody to hepatitis B e antigen; negative for antibody to HBsAg and for hepatitis B e antigen; and positive for HBV DNA. DNA sequence analysis of the "a" determinant region of HBsAg revealed amino acid substitutions from Q (Gln) to R (Arg) at codon 129 and from M (Met) to T (Thr) at codon 133. CONCLUSION: This case illustrates the presence of HBsAg mutant forms of HBV in a West European blood donor population that were undetected by several HBsAg screening assays. Adaptation of HBsAg screening is indicated to overcome deficiencies in sensitivity in detecting HBsAg mutant forms of HBV. Screening for antibody to hepatitis B core antigen or HBV DNA may also detect blood donors infected with HBsAg mutant forms of HBV     

The periodontal health of homosexual men with HIV infection: a controlled study

OBJECTIVE Identify types, prevalence and severity of periodontal changes associated with HIV infection. DESIGN: Cross-sectional controlled blinded study. SETTING: Open access genito-urinary medicine clinic. PARTICIPANTS: Convenience sample of 794 homosexual men aged 18–65.
OUTCOME MEASURES: Prevalence, extent and severity of probing attachment loss (PAL), pocketing, gingival ulceration, gingivitis, bleeding on probing (BOP), gingival red bands and diffuse and punctate erythema of the attached gingiva (selected a priori ).
RESULTS: Prevalences in men with (n = 312) and without HIV (n = 260) were: PAL (≥l site ≥4 mm), 59.6% and 28.5% respectively (P < 0.001. x²); pocketing (≥1 site ≥4 mm) 51.0% and 31.9% (P < 0.001); BOP, 96.5% and 92.3% (P = 0.038); gingival ulceration. 3.2% and 1.0% (P = 0.031), red banding, 12.2% and 10.0% (P = 0.410); diffuse erythema, 12.5% and 3.1% (P < 0.001) and punctate erythema, 9.6% and 1.1% (P < 0.001). Decreased CD4 lymphocyte counts predicted the presence, extent and severity of PAL (P = 0.023, 0.027 and 0.060) but not pocketing. Oral candidiasis predicted the extent and severity of gingivitis and the presence of diffuse and punctate erythema (P = 0.037, 0.011, 0.002 and <0.001).
CONCLUSIONS: Destruction of periodontal attachment is associated with progression of HIV disease whereas pocketing is associated with HIV infection but not disease progression. Gingival ulceration is associated with HIV but gingivitis and erythema of the attached gingiva are most strongly associated with oral candidiasis. Gingival red bands were not associated with HIV infection.