Cigarette tar causes single-strand breaks in DNA

The results of this study demonstrate, for the first time, that cigarette tar causes DNA damage. Incubation in vitro of phage PM2 DNA with aqueous extracts of cigarette tar results in the introduction of DNA single-strand breaks. The effects of protective enzymes and radical scavengers indicate the involvement of active oxygen species. Although the semiquinone components of tar reduce dioxygen forming superoxide radicals and hydrogen peroxide, our results suggest that hydroxyl radicals formed via metal catalyzed decomposition of hydrogen peroxide are ultimately responsible for the DNA lesions. Our results also suggest that the metals in tar are reduced by the semiquinone components of tar and by superoxide at comparable rates.     

Selective effect on punished versus unpunished responding in a conflict test as the criterion for anxiogenic activity

The punished drinking test has been used successfully for identifying and studying anxiolytic agents. By reducing the level of punishment (i.e., decreasing the intensity of shock), it has also been used as a method for measuring anxiogenic activity. Because anxiogenic behavior is a novel and important concept that is not yet fully established, we have reinvestigated the effects of two putative inverse benzodiazepine agonists and pentylenetetrazol in this conflict test. In a series of experiments, using both our version of the procedure and a replication of a previously published method, we were unable to demonstrate a selective reduction in punished responding over unpunished responding caused by CGS 8216 (3 to 40 mg/kg), FG 7142 (2 to 6 mg/kg), and pentylenetetrazol (10 to 20 mg/kg) as reported previously. A careful comparison of the details of our method and the published procedure failed to reveal the source of this discrepancy. If anxiogenic behavior is to be defined as a selective effect of a drug on punished response, the value of this test will depend on identification of its critical variables.     

Ethanol-induced modifications to membrane lipid structure: effect on phospholipase A2-membrane interactions

Chronic ethanol intoxication leads to the development of a resistance to lipid disordering by ethanol, a phenomenon known as "membrane tolerance". In the absence of the added ethanol, the lipid order, as measured by ESR and fluorescence techniques, does not necessarily change as a result of chronic ethanol ingestion (as in liver microsomes, for example). This suggests that the spectroscopic techniques detect tolerance somewhat indirectly, in that the modification responsible may reside in a region distinct from that being probed and also raises the question of whether membrane tolerance is necessarily associated with an alteration in the membrane lipid structure. Here we show that liver microsomes from rats treated chronically with ethanol are rendered relatively resistant to the hydrolytic action of exogenous phospholipase A2, compared to preparations from control animals. This resistance persists in reconstituted lipid vesicles prepared from extracted phospholipids. Since the same substrate (1-palmitoyl-2-N-(4-nitrobenzo-2-oxa-1,3-diazole)amino caproylphosphatidylcholine) was used in both membranes from ethanol-treated animals and controls, the modification appears to reside in the structure and/or organization of the membrane. Further evidence that the lipid structure is modified by chronic ethanol treatment is provided by the observation that perturbance of the membrane structural integrity by increasing levels of oleic acid led to a progressive loss of the ethanol-induced relative resistance to hydrolysis by phospholipase A2. The results of this study support the idea that membrane tolerance involves a modification to lipid structure probably at the bilayer surface. The use of exogenous phospholipase A2 provides a new method for probing the structural modifications induced by chronic ethanol ingestion.     

Aqueous cigarette tar extracts damage human alpha-1-proteinase inhibitor

The elastase inhibitory capacity (EIC) of human alpha-1-proteinase inhibitor (alpha 1PI) is severely compromised by aqueous cigarette tar extract (ACTE). An aqueous extract of the tar from two cigarettes causes a loss of EIC of at least 60% in 24 h at 37 degrees C (pH 7.4) and the damaging capability of the ACTE is retained for many hours. Hydrogen peroxide appears to be an essential component of the mechanism by which ACTE damages alpha 1 PI, since catalase substantially protects alpha 1PI from ACTE-mediated damage. Only mild protection is offered by 10 mM diethylenetriamine pentaacetic acid, indicating only a minor role for transition metal ions in the alpha 1PI-damaging process. Hydroxyl radicals are unlikely agents of alpha 1PI damage in the ACTE system, as judged from hydroxyl radical scavenger studies. Ascorbate and various thiols offer protection to different degrees, dependent on the incubation conditions. Of several amino acids tested, cysteine and methionine (but not methionine sulfoxide) are the only two that protect alpha 1PI. We suggest that components of cigarette smoke particulate matter extracted into the aqueous lung fluid environment may cause local deficiencies in alpha 1PI in smokers' lungs.     

Disease Lesion Mimicry Caused by Mutations in the Rust Resistance Gene rp1

The rp1 locus of maize controls race-specific resistance to the common rust fungus Puccinia sorghi. Four mutant or recombinant Rp1 alleles (rp1-NC3, Rp1-D21, Rp1-MD19, and Rp1-Kr1N) were identified. They condition necrotic phenotypes in the absence of the rust pathogen. These Rp1 lesion mimics fall into three different phenotypic classes: (1) The rp1-NC3 and Rp1-D21 alleles require rust infection or other biotic stimulus to initiate necrotic lesions. These alleles react strongly to all maize rust biotypes tested and also to nonhost rusts. (2) The Rp1-MD19 allele, which has a similar phenotype, also requires a biotic stimulus to initiate lesions. However, Rp1-MD19 shows the race specificity of the Rp1-D gene. (3) The Rp1-Kr1N allele specifies a diffuse necrotic phenotype in the absence of any biotic stimulus and a race-specific reaction when inoculated with maize rust.     

Direct and indirect oxidations by peroxynitrite, neither involving the hydroxyl radical

A new mechanism (Mechanism III) that combines features of mechanisms suggested earlier (Goldstein and Czapski, Inorg. Chem. 34:4041–4048; 1995; Pryor, Jin, and Squadrito Proc. Natl. Acad. Sci. USA 91:11173–11177; 1994) is proposed for oxidations by peroxynitrite. In Mechanism III, oxidations by peroxynitrite can take place either directly by ground-state peroxynitrous acid, ONOOH, or indirectly by ONOOH*, where ONOOH* is an activated form of peroxynitrous acid. In the direct oxidation pathway the reaction is first order in peroxynitrite and first order in substrate, and the oxidation yield approaches 100%. In the indirect oxidation pathway the reaction is first order in peroxynitrite and zero order in substrate. In the presence of sufficient concentrations of a substrate that reacts by the indirect oxidation pathway, about 50–60% of the ONOOH directly isomerizes to nitric acid, and about 40–50% of the ONOOH is converted into ONOOH*. Thus, the oxidation yields by the indirect pathway will not exceed 40–50%, and there will always be a residual yield of nitrate even in the presence of very high concentrations of the substrate. Competitive inhibition studies with various free radical scavengers showed that in some cases these scavengers have no effect on oxidation yields. In others, only partial inhibition was observed, far less than that predicted from to the known rate constants for the reactions of these scavengers with the hydroxyl radical. There are some cases where the extent of inhibition correlates well with the known rate constants of the reactions of these scavengers with hydroxyl radical; nevertheless, even in these cases, the involvement of hydroxyl radicals in indirect oxidations by peroxynitrite is ruled out on the basis of kinetics and oxidation yields. Thus, direct oxidations by peroxynitrite are explained in terms of ONOOH, and indirect oxidations in terms of ONOOH*, and substrates can react by one or both of these pathways.     

Ambient temperature and reproductive success in rodents living at different latitudes

The concern of this research was with the susceptibility of rodent reproduction to latitudinal acclimatization. More specifically, the relationship between ambient temperature and reproductive success was examined in house mice and deer mice whose ancestors had been trapped in northern versus southern climates (Alberta vs. Texas). Third generation adults of each of these four populations were paired and challenged to breed at -6 degrees, 3 degrees, 23 degrees or 34 degrees C. A 50-day period of gradual temperature adaptation preceded pairing. House mice, regardless of their geographic origin, were found to be relatively insensitive to extreme temperatures. Many pairs of both the Alberta and Texas stocks of this species still produced offspring when maintained at the experimental extremes of -6 degrees and 34 degrees C. In marked contrast, both northern and southern deer mice exhibited relatively narrow temperature ranges within which maximum breeding occurred: -6 degrees to 3 degrees for Texas deer mice and only at 23 degrees C for Alberta deer mice. Thus there is no evidence in either species that reproduction is either less sensitive to cold in northern latitudes or less sensitive to heat in southern latitudes; indeed, somewhat the reverse situation exists in deer mice. Evolutionarily, latitudinal considerations seem to have been of secondary rather than of primary importance in shaping the relationship between ambient temperature and reproduction in these two species.