Inhibition of 2-nitropropane-induced rat liver DNA and RNA damage by benzyl selenocyanate

We observed that pretreatment of male F344 rats with benzyl selenocyanate, a versatile organoselenium chemopreventive agent in several animal model systems, decreases the levels of DNA and RNA modifications produced in the liver by the hepatocarcinogen 2- nitropropane. To clarify the mechanisms involved, we pretreated male F344 rats with either benzyl selenocyanate, its sulfur analog benzyl thiocyanate, phenobarbital or cobalt protoporphyrin IX; the latter is a depletor of P450. We then determined (1) the ability of liver microsomes to denitrify 2-nitropropane, (2) effects on 2-nitropropane- induced liver DNA and RNA modifications and (3) amount of nitrate excreted in rat urine following administration of the carcinogen. Pretreatment with benzyl selenocyanate or phenobarbital increased the denitrification activity of liver microsomes by 217 and 765%, respectively, increased liver P4502B1 by 31- and 435-fold, respectively, decreased the levels of 2-nitropropane-induced modifications in liver DNA (29-70% and 17-30%, respectively) and RNA (67-85% and 30-50%, respectively), and increased the 24-h urinary excretion of nitrate by 157 and 209%, respectively. Pretreatment with benzyl thiocyanate had no significant effect on any of these parameters. Pretreatment with cobalt protoporphyrin IX decreased liver P4502B 1 by 87%, decreased the denitrification activity of liver microsomes by 76%, decreased the 24 h urinary excretion of nitrate by 88.5%, but increased the extent of 2-nitropropane-induced liver nucleic acid modifications by 17-67%. These results indicate that the metabolic sequence from 2-nitropropane to the reactive species causing DNA and RNA modifications does not involve the removal of the nitro group. Moreover, they suggest that benzyl selenocyanate inhibits 2-NP-induced liver nucleic acid modifications in part by increasing its detoxication through induction of denitrification, although it is evident that other mechanisms must also be involved.      

Real-time nested multiplex PCR for the detection of herpes simplex virus types 1 and 2 and varicella zoster virus

One hundred forty-nine specimens were tested in a LightCycler nested multiplex polymerase chain reaction (LCnmPCR) for Herpes simplex virus (HSV)1, HSV2, and VZV. Eighty-one were from genitourinary medicine (GUM) patients and the other 68 specimens were from other patients with skin lesions. The results were compared to a conventional multiplex nested PCR (nmPCR) using agarose gel electrophoresis. Twenty-five specimens were positive in both assays for HSV1 and 29 were positive for VZV. For HSV2 there were 27 positive in the LCnmPCR and 26 positive in the nmPCR assay. The melting temperatures (Tms) of each target were different with a mean of 84.75 degrees C for HSV1, 88.57 degrees C for HSV2, and 83.62 degrees C for VZV. The melting curves of positive specimens directly overlaid the melting curves of the positive controls in the assay. The LCnmPCR assay is a convenient alternative to conventional PCR using agarose gel electrophoresis. It improves specimen turnaround time by eliminating the need for gel electrophoresis, transillumination, and gel photography. It also shows increased sensitivity for HSV2 over our standard assay. This LCnmPCR reduces further the possibility of amplicon contamination with nested PCR protocols.     

A simple standardised protocol for the production of monoclonal antibodies against viral and bacterial antigens.

A simple standardised protocol for making monoclonal antibodies against a range of human bacteria and viruses is described. The protocol was designed to reduce the number of steps to a minimum. A one step footpad immunisation was followed by the fusion schedule 10-15 days later. A vital step in the technique was the use of the immunised mouse's spleen to provide a feeder layer post fusion. This simplified the protocol and more importantly greatly accelerated the growth of the hybridomas produced. Immunisation, fusion and clonal expansion of specific antibody secreting hybridomas was complete within 5 weeks. The percentage of hybridomas secreting specific antibody ranged from 6% to 28%, the majority of which were of the IgG isotypes. The method was economical in the use of tissue culture medium and simple to perform.     

A comparison of virus isolation, indirect immunofluorescence and nested multiplex polymerase chain reaction for the diagnosis of primary and recurrent herpes simplex type 1 and type 2 infections

134 swabs in viral transport medium were received from 126 patients with suspected clinical HSV-1 and HSV-2 infections. They were tested by (i) nested multiplex polymerase chain reaction NMPCR (strongly positive specimens had visible bands on both rounds of PCR) without prior extraction, (ii) culture in primary rhesus monkey kidney, E6-Vero, RD and HEp-2 cells and (iii) antigen detection by immunofluorescence (IF). Antigen detection employed four novel pools (A-D) of monoclonal antibodies (Mab): A was HSV-1 specific, B was HSV-2 specific while C and D were generic. In comparison to NMPCR the sensitivity and specificity of (i) culture was 59% (22/37) and 100% (134/134), (ii) IF by Pool A was 59% (16/27) and 100% (117/117), (iii) IF by Pool B was 40% (4/10) and 100% (130/130) and (iv) IF by Pools C and D were 60% (18/30) and 100% (96/96). Specimens positive by culture were more likely to be strongly positive by NMPCR (chi2 P = 0.004). Typing by each method concurred on all occasions. NMPCR was cost effective, easier to perform and was the most sensitive method for HSV detection. It should become the method of choice for HSV diagnosis.