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.     

Validation of the Applied Biosystems Prism 377 automated sequencer for the forensic short tandem repeat analysis

The Applied Biosystems (ABI) Prism 377 DNA sequencer has been evaluated in an attempt to increase the throughput of samples for short tandem repeat (STR) analysis, in both forensic casework and the UK National Criminal Intelligence DNA Database. The gel system assessed consisted of 0.2 mm, 4% acrylamide 6 M urea gels, with a well-to-read distance of 36 cm. Gels were run at a constant voltage of 3 kV and constant temperature of 51 degrees C. The run time of our second generation multiplex (SGM) STR system was achieved in less than 2 h. Rigorous validation has been performed on the instrument hardware and software. Complete resolution of 1 base differences was obtained, up to and beyond 350 bases; sizing precision across gels was more than 2-fold higher than the 373A and the sensitivity was increased by one third.     

Preferential interactions of fluorescent probe Prodan with cholesterol

The fluorescent probe Prodan has been widely used as a probe of model and biological membranes. Its fluorescent maxima in phospholipid bilayers vary as a function of phase state, with maxima at 485 for the liquid crystal Lalpha, 435 nm for the gel L'beta, and 507 nm for the interdigitated gel LbetaI phase, with excitation at 359 nm. These spectral changes have been used for the detection of phase changes among these phases. In the present study, the fluorescent properties and partition coefficients of Prodan in model membranes of phosphatidylcholines and phosphatidylethanols have been studied as a function of lipid phase state and cholesterol content. It is shown that the Prodan spectrum in the presence of cholesterol no longer reflects the known phase state of the lipid; in each phase state, the presence of cholesterol leads to a spectrum with the maximum at 435 nm, characteristic of the noninterdigitated gel phase. The partition coefficient of Prodan into these lipids also varies with the phase state, giving values of 0.35 x 10(4) in the interdigitated gel, 1.8 x 10(4) in the noninterdigitated gel, and 7. 6 x 10(4) in the liquid crystal phase. In the presence of cholesterol these partition coefficients are increased to 13 x 10(4) for the liquid crystal and the gel phase, and 5.1 x 10(4) in the presence of 100 mg/ml ethanol. These results suggest that Prodan has preferential interactions with cholesterol, and is thus not a randomly distributed fluorescent reporter probe in membranes containing cholesterol. These results suggest that Prodan should be used only with great caution in complex lipid mixtures, particularly biological membranes.     

Immobilization of acrylamide-modified oligonucleotides by co-polymerization

A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5'-terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.     

Molecular requirements for T cell recognition by a major histocompatibility complex class II-restricted T cell receptor: the involvement of the fourth hypervariable loop of the Valpha domain

The role of two central residues (K68, E69) of the fourth hypervariable loop of the Valpha domain (HV4alpha) in antigen recognition by an MHC class II-restricted T cell receptor (TCR) has been analyzed. The TCR recognizes the NH2-terminal peptide of myelin basic protein (Ac1-11, acetylated at NH2 terminus) associated with the class II MHC molecule I-Au. Lysine 68 (K68) and glutamic acid 69 (E69) of HV4alpha have been mutated both individually and simultaneously to alanine (K68A, E69A). The responsiveness of transfectants bearing wild-type and mutated TCRs to Ac1-11-I-Au complexes has been analyzed in the presence and absence of expression of the coreceptor CD4. The data demonstrate that in the absence of CD4 expression, K68 plays a central role in antigen responsiveness. In contrast, the effect of mutating E69 to alanine is less marked. CD4 coexpression can partially compensate for the loss of activity of the K68A mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants, are highly sensitive to anti-CD4 antibody blockade. The observations support models of T cell activation in which both the affinity of the TCR for cognate ligand and the involvement of coreceptors determine the outcome of the T cell-antigen-presenting cell interaction.     

Effects of iminodipropionitrile on cerebral amino acid levels

Iminodipropionitrile (IDPN), a compound that causes dyskinetic symptoms in animals and has possible use as a model for human dyskinesia, was tested in mice and rats for its effect on cerebral amino acids. In mice, 2 h after IDPN administration, the level of total brain alanine was reduced; after 5 h the levels of aspartic acid and glutamic acid were also reduced, and the level of glutamine was increased. In rats, after chronic administration of IDPN, the level of glutamic acid in the total brain tissue was reduced. After acute administration of IDPN using microdialysis, extracellular GABA and extracellular glutamine levels in the striatum were elevated. This study shows that IDPN causes alterations in total and extracellular levels of neurotransmitter amino acids in the brain, which could have a role in IDPN-induced dyskinesia.