An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.     

Antigenic and functional organization of human parainfluenza virus type 3 fusion glycoprotein.

Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 nonneutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) glycoprotein of human type 3 parainfluenza virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which participate in neutralization. Competitive binding assays indicated that the 14 neutralization epitopes are organized into three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB) and that the 6 nonneutralization epitopes form four sites (D, E, F, and G). Most of the neutralizing MAbs were involved in nonreciprocal competitive binding reactions, suggesting that they induce conformational changes in other neutralization epitopes. Fusion-inhibition and complemented-enhanced neutralization assays indicated that antigenic sites AB, B, and C may correspond to functional domains of the F molecule. Our results indicated that antibody binding alone is not sufficient for virus neutralization and that many anti-F MAbs neutralize by mechanisms not involving fusion-inhibition. The degree of antigenic variation in the F epitopes of clinical strains was examined by binding and neutralization tests. It appears that PIV3 frequently develops mutations that produce F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies.     

The association of bacterial mutagenicity of hydrocarbon-derived 'bay-region' dihydrodiols with the Iball indices for carcinogenicity and with the extents of DNA-binding on mouse skin of the parent hydrocarbons.

The mutagenic activities of benz[alpha]anthracene, 7-methylbenz[alpha]anthracene, 7,12-dimethylbenz[alpha]anthracene, 3-methylcholanthrene and benzo[alpha]pyrene, together with those of the trans-dihydrodiols derived from these hydrocarbons that would be expected to yield 'bay-region' vicinal diolepoxides on further metabolism have been examined in assays with S. typhimurium TA100 using post-mitochondrial supernatant fractions prepared from the livers of 3-methylcholanthrene-treated rats. Mutagenic activities obtained have been compared with: (a) the extents of reaction with DNA that occur in mouse skin following treatment with these hydrocarbons; (b) the carcinogenicities of the hydrocarbons expressed as Iball indices; (c) their activities as tumour-initiating agents on mouse skin. Close positive associations were found between the microsome-mediated mutagenicities of the dihydrodiols that could yield "bay-region" diol-epoxides and: (a) the extents of reaction with DNA in hydrocarbon-treated mouse skin; (b) the carcinogenic potencies of the parent hydrocarbons; although these correlations are not perfect, the mutagenic activities of the hydrocarbons themselves in microsome-mediated assays with S. typhimurium show no correlation with their extents of DNA binding on mouse skin and a poor correlation with their activities as initiating agents. These comparisons also indicated a statistically-significant positive correlation between carcinogenicity and the in vivo DNA binding on mouse skin treated with the hydrocarbons. Differences in the metabolic pathways by which polycyclic hydrocarbons are activated in vivo and in vitro are discussed in relation to the improved correlations found with the dihydrodiols.     

Activation of solubilized liver membrane adenylate cyclase by guanyl nucleotides.

Liver plasma membranes of hypophysectomized rats were purified, treated with 0.1 m Lubrol-PX and centrifuged at 165,000g for 1 h. The detergent solubilized 50% of the membrane protein; adenylate cyclase activity was present in the supernatant fraction. Optimal substrate concentration of the soluble enzyme was 0.32 mm ATP. Basal activity of 25 preparations of the solubilized enzyme ranged from 124 to 39 pmol cyclic AMP/mg protein/10 min. The solubilized enzyme retained the same sensitivity to activation by guanyl nucleotides as was present in the membrane preparation from which it was derived. Relative sensitivity of the solubilized enzyme with 0.1 mm nucleotides or -side was GDP > GTP > GMP > guanosine; GMP-PNP = GMP-PCP > ITP > GTP. GTP, GMP-PCP, GMP-PNP and other nucleotides were hydrolyzed by phosphohydrolases present in liver membranes that were solubilized with Lubrol-PX along with adenylate cyclase. The presence of the ATP regenerating system in the adenylate cyclase assay also aided in maintaining guanyl nucleotide concentrations. The degree of adenylate cyclase activation by guanyl nucleotides was not related to the sparing effects of nucleotides on substrate ATP hydrolysis. These findings demonstrate that activation of adenylate cyclase by nucleotides is a consequence of a nucleotide-enzyme interaction that is independent of membrane integrity.     

Insights into the biophysical properties of GABA(A) ion channels: modulation of ion permeation by drugs and protein interactions

The fundamental properties of ion channels assure their selectivity for a particular ion, its rapid permeation through a central pore and that such electrical activity is modulated by factors that control the opening and closing (gating) of the channel. All cell types possess ion channels and their regulated flux of ions across the membrane play critical roles in all steps of life. An ion channel does not act alone to control cell excitability but rather forms part of larger protein complexes. The identification of protein interaction partners of ion channels and their influence on both the fundamental biophysical properties of the channel and its expression in the membrane are revealing the many ways in which electrical activity may be regulated. Highlighted here is the novel use of the patch clamp method to dissect out the influence of protein interactions on the activity of individual GABA(A) receptors. The studies demonstrate that ion conduction is a dynamic property of a channel and that protein interactions in a cytoplasmic domain underlie the channel's ability to alter ion permeation. A structural model describing a reorganisation of the conserved cytoplasmic gondola domain and the influence of drugs on this process are presented.     

Snow depth manipulation and its influence on soil frost and water dynamics in a northern hardwood forest

Climate change will likelyresult in warmer winter temperatures leading toless snowfall in temperate forests. Thesechanges may lead to increases in soil freezingbecause of lack of an insulating snow cover andchanges in soil water dynamics during theimportant snowmelt period. In this study, wemanipulated snow depth by removing snow for twowinters, simulating the late development of thesnowpack as may occur with global warming, toexplore the relationships between snow depth,soil freezing, soil moisture, and infiltration.We established four sites, each with two pairedplots, at the Hubbard Brook Experimental Forest(HBEF) in New Hampshire, U.S.A. and instrumentedall eight plots with soil and snow thermistors,frost tubes, soil moisture probes, and soillysimeters. For two winters, we removed snowfrom the designated treatment plots untilFebruary. Snow in the reference plots wasundisturbed. The treatment winters (1997/1998 and1998/1999) were relatively mild, withtemperatures above the seasonal norm and snowdepths below average. Results show the treatedplots accumulated significantly less snow andhad more extensive soil frost than referenceplots. Snow depth was a strong regulator ofsoil temperature and frost depth at all sites.Soil moisture measured by time domainreflectometry probes and leaching volumescollected in lysimeters were lower in thetreatment plots in March and April compared tothe rest of the year. The ratio of leachatevolumes collected in the treatment plots tothat in the reference plots decreased as thesnow ablation seasons progressed. Our data showthat even mild winters with low snowfall,simulated by snow removal, will result inincreased soil freezing in the forests at theHBEF. Our results suggest that a climate shifttoward less snowfall or a shorter duration ofsnow on the ground will produce increases insoil freezing in northern hardwood forests.Increases in soil freezing will haveimplications for changes in soil biogeochemicalprocesses.     

Health Professionals “Make Their Choice”: Pharmaceutical Industry Leaders’ Understandings of Conflict of Interest

Conflicts of interest, stemming from relationships between health professionals and the pharmaceutical industry, remain a highly divisive and inflammatory issue in healthcare. Given that most jurisdictions rely on industry to self-regulate with respect to its interactions with health professionals, it is surprising that little research has explored industry leaders’ understandings of conflicts of interest. Drawing from in-depth interviews with ten pharmaceutical industry leaders based in Australia, we explore the normalized and structural management of conflicts of interest within pharmaceutical companies. We contrast this with participants’ unanimous belief that the antidote to conflicts of interest with health professionals were “informed consumers.” It is, thus, unlikely that a self-regulatory approach will be successful in ensuring ethical interactions with health professionals. However, the pharmaceutical industry’s routine and accepted practices for disclosing and managing employees’ conflicts of interest could, paradoxically, serve as an excellent model for healthcare.