Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.     

Hotspots of biotic compositional change in lakes along vast latitudinal transects in northern Canada

Ecotones mark zones of rapid change in ecological structure at various spatial scales. They are believed to be particularly susceptible to shifts caused by environmental transformation, making them key regions for studying the effects of global change. Here, we explored the variation in assemblage structure of aquatic primary producer and consumer communities across latitudinal transects in northeastern North America (Québec‐Labrador) to identify spatial patterns in biodiversity that indicated the location of transition zones across the landscape. We analyzed species richness and the cumulative rate of compositional change (expressed as beta‐diversity) of diatoms and chironomids to detect any abrupt shifts in the rate of spatial taxonomic turnover. We used principal coordinates analysis to estimate community turnover with latitude, then applied piecewise linear regression to assess the position of ecotones. Statistically significant changes in assemblage composition occurred at 52 and 55°N, corresponding to the transition between closed‐ and open‐crown forest, and to the southern onset of the forest tundra (i.e., the forest limit), respectively. The spatial distribution of ecotones was most strongly related to air temperature for chironomids and to vegetation‐ and soil‐related chemical attributes of lake water for diatoms, including dissolved organic carbon content and water color. Lakes at mid‐ to high‐latitudes currently face pressures from rapidly rising temperatures, accompanied by large increases in organic carbon inputs from their catchments, often leading to browning and its associated effects. The biota at the base of food webs in lakes located in transition zones are disproportionately affected by the cascading effects of these multi‐factorial changes, concurrent with pronounced terrestrial greening observed in these regions. Similar patterns of biotic shifts have been observed along alpine aquatic transects, indicating the potential for widespread restructuring of cold, high‐altitude and high‐latitude freshwater communities due to global change.     

Chemotherapy regimens induce inhibitory immune checkpoint protein expression on stem-like and senescent-like oesophageal adenocarcinoma cells

Use of immune checkpoint inhibitors (ICIs) with chemotherapy to enhance responses in oesophageal adenocarcinoma (OAC) is an attractive approach. We identified subpopulations of OAC cells expressing inhibitory immune checkpoint (IC) ligands (PD-L1, PD-L2 and CD160) and receptors (PD-1, TIGIT, TIM-3, LAG-3 and A2aR) in vitro and in ex vivo biopsies. Combination chemotherapy regimens FLOT and CROSS promote a more immune-resistant phenotype through upregulation of IC ligands and receptors on OAC cells in vitro. Importantly, this study investigated if OAC cells, enriched for ICs exhibited a more stem-like and senescent-like phentoype. FLOT preferentially upregulates PD-L1 on a stem-like OAC cell phenotype, defined by ALDH activity. Expression of senescence-associated β-galactosidase is induced in a subpopulation of OAC cells following FLOT and CROSS chemotherapy treatment, along with enhanced expression of TIM-3 and A2aR ICs. Blockade of PD-1 signalling in OAC cells induced apoptosis and enhanced FLOT and CROSS chemotherapy toxicity in vitro. Upregulation of ICs on OAC cells following chemotherapy may represent potential mechanisms of chemo-immune resistance. Combination ICIs may be required to enhance the efficacy of chemotherapy and immunotherapy in OAC patients.     

Racism,Gun Ownership and Gun Control: Biased Attitudes in US Whites May Influence Policy Decisions

Objective

Racism is related to policies preferences and behaviors that adversely affect blacks and appear related to a fear of blacks (e.g., increased policing, death penalty). This study examined whether racism is also related to gun ownership and opposition to gun controls in US whites.

Method

The most recent data from the American National Election Study, a large representative US sample, was used to test relationships between racism, gun ownership, and opposition to gun control in US whites. Explanatory variables known to be related to gun ownership and gun control opposition (i.e., age, gender, education, income, conservatism, anti-government sentiment, southern vs. other states, political identification) were entered in logistic regression models, along with measures of racism, and the stereotype of blacks as violent. Outcome variables included; having a gun in the home, opposition to bans on handguns in the home, support for permits to carry concealed handguns.

Results

After accounting for all explanatory variables, logistic regressions found that for each 1 point increase in symbolic racism there was a 50% increase in the odds of having a gun at home. After also accounting for having a gun in the home, there was still a 28% increase in support for permits to carry concealed handguns, for each one point increase in symbolic racism. The relationship between symbolic racism and opposition to banning handguns in the home (OR1.27 CI 1.03,1.58) was reduced to non-significant after accounting for having a gun in the home (OR1.17 CI.94,1.46), which likely represents self-interest in retaining property (guns).

Conclusions

Symbolic racism was related to having a gun in the home and opposition to gun control policies in US whites. The findings help explain US whites’ paradoxical attitudes towards gun ownership and gun control. Such attitudes may adversely influence US gun control policy debates and decisions.     

The pivotal role of hepatocytes in drug discovery

This review promotes the value of isolated hepatocytes in modern Drug Discovery programmes and outlines how increased understanding, particularly in the area of in vitro-in vivo extrapolation (IVIVE), has led to more widespread use. The importance of in vitro metabolic intrinsic clearance data for predicting in vivo clearance has been acknowledged for several years and the greater utility of hepatocytes, compared with hepatic microsomes and liver slices, for this application is discussed. The application of hepatocytes in predicting drug-drug interactions (DDIs) resulting from reversible and irreversible (time-dependent) inhibition is relatively novel but affords the potential to study both phase I and phase II processes together with any impact of drug efflux and/or uptake (cellular accumulation). Progress in this area is reviewed along with current opinions on the comparative use of primary hepatocytes and higher throughput reporter gene-based systems for studying cytochrome P450 (CYP) induction. The appreciation of the role of transporter proteins in drug disposition continues to evolve. The study of hepatic uptake using isolated hepatocytes and the interplay between drug transport and metabolism with respect to both clearance and DDIs and subsequent IVIVE is also considered.     

Development of a biosensor for endocrine disrupting compounds based on tyrosinase entrapped within a poly(thionine) film

Preparation of semiconducting films by electropolymerisation of a monomer which is itself a redox mediator is an attractive and simple method for biosensor fabrication. A polymeric film of the redox dye thionine (phenothiazine) enables the stable immobilisation of polyphenol oxidase (tyrosinase) while acting as mediator for the enzymatic process. The immobilisation method is based on an inner crosslinked tyrosinase layer which contains thionine with an electropolymerised film of poly(thionine) on top. This method gave the most stable redox couple for poly(thionine) and exhibited the greatest response stability. The sensor was tested using a range of synthetic oestrogens and phenolic compounds, which are suspected endocrine disruptors/oestrogen mimics. The device responded well to all compounds tested with limits of detection ranging from 1 to 23 microM (based on three times S/N ratio). The tyrosinase/poly(thionine) electrode response to phenol was 3 orders of magnitude greater than the unmediated response in the absence of poly(thionine).     

Assessing stimulus equivalence with a precursor to the relational evaluation procedure

Previous research has demonstrated that, after being trained on multiple match-to-sample (MTS) tasks (A-B, B-C), most human adults respond in accordance with symmetry (B-A, C-B) and equivalence (C-A) when measured with MTS tests and with a precursor to the Relational Evaluation Procedure (pREP). The latter procedure involves conditional go/no-go discrimination tasks, requiring subjects to press a bar during a 5s interval after the successive presentation of two same-class stimuli, and not to press after the presentation of two different-class stimuli (e.g. Ci -->Ai -->press, Ci -->Aj -->no press). The present study was an effort to replicate these findings. The study consisted of five experiments. Very few subjects evidenced pREP symmetry and equivalence unless they had (a) already demonstrated symmetry and equivalence in a MTS test before, or (b) received pREP pretraining with unrelated stimulus pairs and symmetry was tested before equivalence. Failures to show symmetry were always associated with pressing at or close to 50% of these trials. Failures to show equivalence were associated with pressing or not pressing on (almost) all trials. Current findings are similar to those obtained in equivalence studies involving MTS probes permitting the subjects not to respond to the designated comparisons.