Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest.

The tumor suppressor p53 contributes to maintaining genome stability by inducing a cell cycle arrest or apoptosis in response to conditions that generate DNA damage. Nuclear injection of linearized plasmid DNA, circular DNA with a large gap, or single-stranded circular phagemid is sufficient to induce a p53-dependent arrest. Supercoiled and nicked plasmid DNA, and circular DNA with a small gap were ineffective. Titration experiments indicate that the arrest mechanism in normal human fibroblasts can be activated by very few double strand breaks, and only one may be sufficient. Polymerase chain reaction assays showed that end-joining activity is low in serum-arrested human fibroblasts, and that higher joining activity occurs as cells proceed through G1 or into S phase. We propose that the exquisite sensitivity of the p53-dependent G1 arrest is partly due to inefficient repair of certain types of DNA damage in early G1.

G protein subunits and the stimulation of phospholipase C by Gs-and Gi-coupled receptors: Lack of receptor selectivity of Galpha(16) and evidence for a synergic interaction between Gbeta gamma and the alpha subunit of a receptor activated G protein.

Stimulatory guanine nucleotide binding protein (Gs)-coupled receptors activated by luteinizing hormone, vasopressin, and the catecholamine isoproterenol (luteinizing hormone receptor, type 2 vasopressin receptor, and types 1 and 2 beta-adrenergic receptors) and the Gi-coupled M2 muscarinic receptor (M2R) were expressed transiently in COS cells, alone and in combination with Gbeta gamma dimers, their corresponding Galphas (Galpha(s), or Galpha(i3)) and either Galpha(q) or Galpha(16). Phospholipase C (PLC) activity, assessed by inositol phosphate production from preincorporated myo[3H]inositol, was then determined to gain insight into differential coupling preferences among receptors and G proteins. The following were observed: (i) All receptors tested were able to stimulate PLC activity in response to agonist occupation. The effect of the M2R was pertussis toxin sensitive. (ii) While, as expected, expression of Galpha(q) facilitated an agonist-induced activation of PLC that varied widely from receptor to receptor (400% with type 2 vasopressin receptor and only 30% with M2R), expression of Galpha(16) facilitated about equally well the activation of PLC by any of the tested receptors and thus showed little if any discrimination for one receptor over another. (iii) Gbeta gamma elevated basal (agonist independent) PLC activity between 2- and 4-fold, confirming the proven ability of Gbeta gamma to stimulate PLCbeta. (iv) Activation of expressed receptors by their respective ligands in cells coexpressing excess Gbeta gamma elicited agonist stimulated PLC activities, which, in the case of the M2R, was not blocked by pertussis toxin (PTX), suggesting mediation by a PTX-insensitive PLC-stimulating Galpha subunit, presumably, but not necessarily, of the Gq family. (v) The effects of Gbeta gamma and the PTX-insensitive Galpha elicited by M2R were synergistic, suggesting the possibility that one or more forms of PLC are under conditional or dual regulation of G protein subunits such that stimulation by one sensitizes to the stimulation by the other.

Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.

Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding.

Neighboring base composition and transversion/transition bias in a comparison of rice and maize chloroplast noncoding regions.

The correspondence between the transversion/transition ratio and the neighboring base composition in chloroplast DNA is examined. For 18 noncoding regions of the chloroplast genome, alignments between rice (Oryza sativa) and maize (Zea mays) were generated by two different methods. Difficulties of aligning noncoding DNA are discussed, and the alignments are analyzed in a manner that reduces alignment artifacts. Sequence divergence is < 10%, so multiple substitutions at a site are assumed to be rare. Observed substitutions were analyzed with respect to the A+T content of the two immediately flanking bases. It is shown that as this content increases, the proportion of transversions also increases. When both the 5'- and 3'-flanking nucleotides are G or C (A+T content of 0), only 25% of the observed substitutions are transversions. However, when both the 5'- and 3'-flanking nucleotides are A or T (A+T content of 2), 57% of the observed substitutions are transversions. Therefore, the influence of flanking base composition on substitutions, previously reported for a single noncoding region, is a general feature of the chloroplast genome.

Fusogenic selectivity of the envelope glycoprotein is a major determinant of human immunodeficiency virus type 1 tropism for CD4+ T-cell lines vs. primary macrophages.

We investigated the relationship between the fusion selectivity of the envelope glycoprotein (env) and the tropism of different human immunodeficiency virus type 1 (HIV-1) isolates for CD4+ human T-cell lines vs. primary macrophages. Recombinant vaccinia viruses were prepared encoding the envs from several well-characterized HIV-1 isolates with distinct cytotropisms. Cells expressing the recombinant envs were mixed with various CD4+ partner cell types; cell fusion was monitored by a quantitative reporter gene assay and by syncytia formation. With CD4+ continuous cell lines as partners (T-cell lines, HeLa cells expressing recombinant CD4), efficient fusion occurred with the envs from T-cell line-tropic isolates (IIIB, LAV, SF2, and RF) but not with the envs from macrophage-tropic isolates (JR-FL, SF162, ADA, and Ba-L). The opposite selectivity pattern was observed with primary macrophages as cell partners; stronger fusion occurred with the envs from the macrophage-tropic than from the T-cell line-tropic isolates. All the envs showed fusion activity with peripheral blood mononuclear cells as partners, consistent with the ability of this cell population to support replication of all the corresponding HIV-1 isolates. These fusion selectivities were maintained irrespective of the cell type used to express env, thereby excluding a role for differential host cell modification. We conclude that the intrinsic fusion selectivity of env plays a major role in the tropism of a HIV-1 isolate for infection of CD4+ T-cell lines vs. primary macrophages, presumably by determining the selectivity of virus entry and cell fusion.

Combinatorial association and abundance of components of interferon-stimulated gene factor 3 dictate the selectivity of interferon responses.

Genes containing the interferon-stimulated response element (ISRE) enhancer have been characterized as transcriptionally responsive primarily to type I interferons (IFN alpha/beta). Induction is due to activation of a multimeric transcription factor, interferon-stimulated gene factor 3 (ISGF3), which is activated by IFN alpha/beta but not by IFN gamma. We found that ISRE-containing genes were induced by IFN gamma as well as by IFN alpha in Vero cells. The IFN gamma response was dependent on the ISRE and was accentuated by preexposure of cells to IFN alpha, a treatment that increases the abundance of ISGF3 components. Overexpression of ISGF3 polypeptides showed that the IFN gamma response depended on the DNA-binding protein ISGF3 gamma (p48) as well as on the 91-kDa protein STAT91 (Stat1 alpha). The transcriptional response to IFN alpha required the 113-kDa protein STAT113 (Stat2) in addition to STAT91 and p48. Mutant fibrosarcoma cells deficient in each component of ISGF3 were used to confirm that IFN gamma induction of an ISRE reporter required p48 and STAT91, but not STAT113. A complex containing p48 and phosphorylated STAT91 but lacking STAT113 bound the ISRE in vitro. IFN gamma-induced activation of this complex, preferentially formed at high concentrations of p48 and STAT91, may explain some of the overlapping responses to IFN alpha and IFN gamma.

Codominance and toxins: a path to drugs of nearly unlimited selectivity.

The effectiveness of drugs is often limited by their insufficient selectivity. I propose designs of therapeutic agents that address this problem. The key feature of these reagents, termed comtoxins (codominance-mediated toxins), is their ability to utilize codominance, a property characteristic of many signals in proteins, including degradation signals (degrons) and nuclear localization signals. A comtoxin designed to kill cells that express intracellular proteins P1 and P2 but to spare cells that lack P1 and/or P2 is a multidomain fusion containing a cytotoxic domain and two degrons placed within or near two domains P1* and P2* that bind, respectively, to P1 and P2. In a cell containing both P1 and P2, these proteins would bind to the P1* and P2* domains of the comtoxin and sterically mask the nearby (appropriately positioned) degrons, resulting in a long-lived and therefore toxic drug. By contrast, in a cell lacking P1 and/or P2, at least one of the comtoxin's degrons would be active (unobstructed), yielding a short-lived and therefore nontoxic drug. A comtoxin containing both a degron and a nuclear localization signal can be designed to kill exclusively cells that contain P1 but lack P2. Analogous strategies yield comtoxins sensitive to the presence (or absence) of more than two proteins in a cell. Also considered is a class of comtoxins in which a toxic domain is split by a flexible insert containing binding sites for the target proteins. The potentially unlimited, combinatorial selectivity of comtoxins may help solve the problem of side effects that bedevils present-day therapies, for even nonselective delivery of a comtoxin would not affect cells whose protein "signatures" differ from the targeted one.

Interval bias in 2AFC detection tasks: sorting out the artifacts

Proportion correct in two-alternative forcedchoice (2AFC) detection tasks often varies when the stimulus is presented in the first or in the second interval.Reanalysis of published data reveals that these order effects (or interval bias) are strong and prevalent, refuting the standard difference model of signal detection theory. Order effects are commonly regarded as evidence that observers use an off-center criterion under the difference model with bias. We consider an alternative difference model with indecision whereby observers are occasionally undecided and guess with some bias toward one of the response options. Whether or not the data show order effects, the two models fit 2AFC data indistinguishably, but they yield meaningfully different estimates of sensory parameters. Under indeterminacy as to which model governs 2AFC performance, parameter estimates are suspect and potentially misleading. The indeterminacy can be circumvented by modifying the response format so that observers can express indecision when needed. Reanalysis of published data collected in this way lends support to the indecision model. We illustrate alternative approaches to fitting psychometric functions under the indecision model and discuss designs for 2AFC experiments that improve the accuracy of parameter estimates, whether or not order effects are apparent in the data.

Amino Acid Residues Implicated in the Interaction of Melanocortin Ligands and their Receptors: A Study of MC2R Selectivity

Author: Kristopher D. Veo Title: Amino acid residues implicated in the interaction of Melanocortin ligands and their receptors: A study of MC2R selectivity Advisor: Dr. Robert M. Dores Degree Date: August 2009 ABSTRACT Melanocortin receptor ligand selectivity has been a question not easily answered. The inability to functionally express melanocortin 2 receptor (MC2R) has inhibited the study of why MC2R is only stimulated by ACTH, a melanocortin hormone. With the recent discovery of the MC2R accessory protein (MRAP), creating a heterologous system is now feasible. Using a general cell line like CHO-K1 cells, which do not express endogenous MCRs, we were able to create a heterologous expression system and test the selectivity of MC2R using analog variants of ACTH(1-24). Our results indicate an amino acid requirement in the C-terminal portion of ACTH(1-24) for activation, which supports the 2-step method of activation hypothesized for MC2R. This site, the tetra basic cleavage site, when altered does not stimulate cAMP production and does not compete with ACTH(1-24) for binding. We also demonstrate the potential for a non-mammalian MC2R system in cloning full length Silurana tropicalis MC2R and completed localization studies with this system with MRAP using CHO-K1 cells.

What We Don't Know Can Hurt Us: Clinical Inference Bias and Constructed Memories of Sexual Abuse.

The false memory/recovered memory debate, research regarding the malleability of memory, and the current lack of methods for validating recovered memories all support the view that heightened care is required of therapists dealing with clients whom they suspect have been sexually abused. The judgmental heuristics that underlie the major clinical inference biases of confirmatory bias, biased covariation, base rate fallacies, and schematic processing errors are all relevant to the processes leading to therapist-client constructions of memories of sexual abuse. Suggestions for minimizing each of these biases are offered. Personal motivations of the client and client suggestibility are factors that may contribute to the construction of memories of sexual abuse, and suggestions for minimizing the impact of these motivations are offered. In conclusion, general suggestions for minimizing the impact of clinical inference biases within the sexual abuse treatment context are summarized.

Difference Blindness vs. Bias Awareness: Why Law Firms with the Best of Intentions Have Failed to Create Diverse Partnerships

This Article uses the example of BigLaw firms to explore the challenges that many elite organizations face in providing equal opportunity to their workers. Despite good intentions and the investment of significant resources, large law firms have been consistently unable to deliver diverse partnership structures - especially in more senior positions of power. Building on implicit and institutional bias scholarship and on successful approaches described in the organizational behavior literature, we argue that a significant barrier to systemic diversity at the law firm partnership level has been, paradoxically, the insistence on difference blindness standards that seek to evaluate each person on their individual merit. While powerful in dismantling intentional discrimination, these standards rely on an assumption that lawyers are, and have the power to act as, atomistic individuals - a dangerous assumption that has been disproven consistently by the literature establishing the continuing and powerful influence of implicit and institutional bias. Accordingly, difference blindness, which holds all lawyers accountable to seemingly neutral standards, disproportionately disadvantages diverse populations and normalizes the dominance of certain actors - here, white men - by creating the illusion that success or failure depends upon individual rather than structural constraints. In contrast, we argue that a bias awareness approach that encourages identity awareness and a relational framework is a more promising way to promote equality, equity, and inclusion.

Examining the relationship among genes, attention bias to emotion, and depression in youth

The investigation of biologically initiated pathways to psychological disorder is critical to advance our understanding of mental illness. Research has suggested that attention bias to emotion may be an intermediate trait for depression associated with biologically plausible candidate genes, such as the serotonin transporter (5-HTTLPR) and catechol-o-methyl-transferase (COMT) genes, yet there have been mixed findings in regards to the precise direction of effects. The experience of recent stressful life events (SLEs) may be an important, yet currently unstudied, moderator of the relationship between genes and attention bias as SLEs have been associated with both gene expression and attention to emotion. Additionally, although attention biases to emotion have been studied as a possible intermediate trait associated with depression, no study has examined whether attention biases within the context of measured genetic risk lead to increased risk for clinical depressive episodes over time. Therefore, this research investigated both whether SLEs moderate the link between genetic risk (5-HTTLPR and COMT) and attention bias to emotion and whether 5-HTTLPR and COMT moderated the relationship between attention biases to emotional faces and clinical depression onset prospectively across 18 months within a large community sample of youth (n= 467). Analyses revealed a differential effect of gene. Youth who were homozygous for the low expressing allele of 5-HTTLPR (S/S) and had experienced more recent SLEs within the last three months demonstrated preferential attention toward negative emotional faces (angry and sad). However, youth who were homozygous for the high expressing COMT genotype (Val/Val) and had experienced more recent SLEs showed attentional avoidance of positive facial expressions (happy). Additionally, youth who avoided negative emotion (i.e., anger) and were homozygous for the S allele of the 5-HTTLPR gene were at greater risk for prospective depressive episode onset. Increased risk for depression onset was specific to the 5-HTTLPR gene and was not found when examining moderation by COMT. These findings highlight the importance of examining risk for depression across multiple levels of analysis, such as combined genetic, environmental, and cognitive risk, and is the first study to demonstrate clear evidence of attention biases to emotion functioning as an intermediate trait predicting depression.

Quality of pharmaceutical advertising and gender bias in medical journals (1998-2008): a review of the scientific literature

Objective: To review the scientific literature on pharmaceutical advertising aimed at health professionals in order to determine whether gender bias has decreased and the quality of information in pharmaceutical advertising has improved over time. Methods: We performed a content analysis of original articles dealing with medical drug promotion (1998-2008), according to quality criteria such as (a) the number, validity and accessibility of bibliographic references provided in pharmaceutical advertising and (b) the extent to which gender representations were consistent with the prevalence of the diseases. Databases: PUBMED, Medline, Scopus, Sociological Abstract, Eric and LILACS. Results: We reviewed 31 articles that analyzed advertising in medical journals from 1975-2005 and were published between 1998 and 2008. We found that the number of references used to support pharmaceutical advertising claims increased from 1975 but that 50% of these references were not valid. There was a tendency to depict men in paid productive roles, while women appeared inside the home or in non-occupational social contexts. Advertisements for psychotropic and cardiovascular drugs overrepresented women and men respectively. Conclusions: The use of bibliographic references increased between 1998 and 2008. However, representation of traditional male-female roles was similar in 1975 and 2005. Pharmaceutical advertisements may contribute to reinforcing the perception that certain diseases are associated with the most frequently portrayed sex.

The high-bias stability of monatomic chains

For the metals Au, Pt and Ir it is possible to form freely suspended monatomic chains between bulk electrodes. The atomic chains sustain very large current densities, but finally fail at high bias. We investigate the breaking mechanism, that involves current-induced heating of the atomic wires and electromigration forces. We find good agreement of the observations for Au based on models due to Todorov and co-workers. The high-bias breaking of atomic chains for Pt can also be described by the models, although here the parameters have not been obtained independently. In the limit of long chains the breaking voltage decreases inversely proportional to the length.

High selectivity of TiC-CDC for CO2/N2 separation

A series of carbide-derived carbons (CDC) have been prepared starting from TiC and using different chlorine treatment temperatures (500–1200 °C). Contrary to N2 adsorption measurements at −196 °C, CO2 adsorption measurements at room temperature and high pressure (up to 1 MPa) together with immersion calorimetry measurements into dichloromethane suggest that the synthesized CDC exhibit a similar porous structure, in terms of narrow pore volume, independently of the temperature of the reactive extraction treatment used (samples synthesized below 1000 °C). Apparently, these carbide-derived carbons exhibit narrow constrictions were CO2 adsorption under standard conditions (0 °C and atmospheric pressure) is kinetically restricted. The same accounts for a slightly larger molecule as N2 at a lower adsorption temperature (−196 °C), i.e. textural parameters obtained from N2 adsorption measurements on CDC must be underestimated. Furthermore, here we show experimentally that nitrogen exhibits an unusual behavior, poor affinity, on these carbide-derived carbons. CH4 with a slightly larger diameter (0.39 nm) is able to partially access the inner porous structure whereas N2, with a slightly smaller diameter (0.36 nm), does not. Consequently, these CDC can be envisaged as excellent sorbent for selective CO2 capture in flue-gas streams.