Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways.

Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

Acute molecular response of mouse hindlimb muscles to chronic stimulation.

Stimulation of the mouse hindlimb via the sciatic nerve was performed for a 4-h period to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 +/- 0.1 g/g body wt) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon study completion. An immediate-early growth response was present in the extensor digitorum longus (EDL) muscle (FOS, JUN, activating transcription factor 3, and musculoaponeurotic fibrosarcoma oncogene) with a similar but attenuated pattern in the soleus muscle. Transcript profiles showed decreased fast fiber-specific mRNA (myosin heavy chains 2A and 2B, fast troponins T(3) and I, alpha-tropomyosin, muscle creatine kinase, and parvalbumin) and increased slow transcripts (myosin heavy chain-1beta/slow, troponin C slow, and tropomyosin 3y) in the EDL versus soleus muscles. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration in stimulated versus control muscles, whereas ultrastructural analysis showed no evidence of myofiber damage after stimulation. Multiple fiber type-specific transcription factors (tea domain family member 1, nuclear factor of activated T cells 1, peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -beta, circadian locomotor output cycles kaput, and hypoxia-inducible factor-1alpha) increased in the EDL along with transcription factors characteristic of embryogenesis (Kruppel-like factor 4; SRY box containing 17; transcription factor 15; PBX/knotted 1 homeobox 1; and embryonic lethal, abnormal vision). No established in vivo satellite cell markers or genes activated in our parallel experiments of satellite cell proliferation in vitro (cyclins A(2), B(2), C, and E(1) and MyoD) were differentially increased in the stimulated muscles. These results indicated that the molecular onset of fast to slow phenotype conversion occurred in the EDL within 4 h of stimulation without injury or satellite cell recruitment. This conversion was associated with the expression of phenotype-specific transcription factors from resident fiber myonuclei, including the activation of nascent developmental transcriptional programs.

The foot-in-the-door compliance procedure: a multiple-process analysis and review.

Research on the social compliance procedure known as the foot-in-the-door (FITD) technique is reviewed. Several psychological processes that may be set in motion with a FITD manipulation are identified: self-perception, psychological reactance, conformity, consistency, attributions, and commitment. A review of relevant investigations and several meta-analyses support the notion that each of these processes can influence compliance behavior in the FITD situation. I argue that the combined effects of these processes can account for successful FITD demonstrations as well as studies in which the technique was ineffective or led to a decrease in compliance. The experimental conditions most likely to produce an FITD effect are identified.

H2O2-induced increases in cellular F-actin occur without increases in actin nucleation activity.

Previous work has shown that H2O2 causes an increase in polymerized actin (F-actin) inside cells. To test the hypothesis that increased polymerization resulted from a mechanism involving increased actin nucleation activity, we employed methods utilizing pyrene-labeled actin to quantify the actin nucleation activity of cell lysates and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phallacidin binding assays to quantify the amount of F-actin in P388D1 cells. H2O2 increased polymerized actin (NBD-phallacidin assay) in a dose-dependent manner with an effective dose giving 50% response (ED50) approximately 1 mM. Five millimolar H2O2 caused a 1.6-fold increase in NBD-phallacidin staining. In contrast, actin nucleation activity decreased in a dose-dependent manner with a similar ED50. Five millimolar H2O2 caused a 30-40% decrease in actin nucleation activity. The effect was rapid, occurring within 5 min of H2O2 addition. The results indicate that H2O2 causes cytoskeletal changes that enhance NBD-phallacidin binding without increasing actin nucleation activity. Fractionation studies showed that the nucleation activity in H2O2-treated cells and controls sedimented with the Triton X-100-insoluble cytoskeleton, and the cytosolic fraction appeared to contain an inhibitor of actin polymerization.

ATP depletion induces an increase in the assembly of a labile pool of polymerized actin in endothelial cells.

Depletion of cellular ATP is associated with profound effects on the cytoskeleton, particularly disruption of microfilaments. We examined this process in bovine pulmonary artery endothelial cells by inducing differential reductions of cellular ATP using mitochondrial inhibition and variable amounts of glucose. Reduction of cellular ATP to levels < 40% of control produced discrete stages in the visible disruption of microfilaments. Using the deoxyribonuclease I assay, a reversible 11% decrease in monomeric (G) actin occurred in conjunction with microfilament disruption. Polyacrylamide gel electrophoretic (PAGE) analysis of the detergent-insoluble cytoskeleton did not reveal any differences in actin content between normal or ATP-depleted cells. Image analysis of adherent endothelial cells that had been fixed and stained with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin revealed that an increase of F-actin of approximately 20% occurred in cells depleted of ATP. If the cells were lysed with detergent before fixation, the increase in F-actin was lost. PAGE analysis and electron microscopy of detergent-soluble material from the cells obtained by ultracentrifugation directly demonstrated the presence of a labile pool of F-actin within the cells, which increased with ATP loss. These observations suggest that ATP may play an important role in the organization and remodeling of microfilaments within cells.

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.

Actin polymerization in cellular oxidant injury.

Microfilaments undergo an ATP-dependent disruption into shortened bundles following cellular exposure to oxidants. This phenomenon does not require a net change in the amount of polymerized actin. However, increased amounts of polymerized actin have been detected in oxidant-injured cells and it was the purpose of this study to determine the conditions under which the actin polymerization may occur. Utilizing the formation of oxidized glutathione (GSSG) as an indicator of cellular sulfhydryl oxidation, conditions were chosen to accentuate sulfhydryl oxidation within the target P388D1 cell line following exposure to the oxidants, H2O2 and diamide. Using the DNase I and flow cytometric assays of actin polymerization, significant polymerization of actin was detected only under conditions in which sulfhydryl oxidation occurred after exposure to the two oxidizing agents. Greater sulfhydryl oxidation early in the course of injury was associated with a greater rate and extent of actin polymerization in the injured cells. Experiments with cells depleted of glutathione (GSH) demonstrated that neither loss of GSH nor absolute levels of GSSG formed during oxidant exposure were responsible for the polymerization of actin. The data presented are consistent with the hypothesis that oxidizing conditions which induce significant sulfhydryl oxidation in target cells are correlated with assembly of polymerized actin and that this represents a process which is distinct and separate from the ATP-dependent gross disruption of microfilaments.

Protective effect of glutamine on endothelial cell ATP in oxidant injury.

Endothelial cell dysfunction following exposure to H2O2 is associated with rapid inhibition of glucose-dependent pathways of ATP synthesis. The role other substrates for ATP synthesis (e.g., amino acids) may play in the metabolism of H2O2-injured cells is unclear. The effect of glutamine, a precursor of the Kreb's cycle intermediate alpha-ketoglutarate on ATP levels in bovine pulmonary artery endothelial cells exposed to H2O2 was examined. The presence of glutamine during H2O2 injury significantly enhanced ATP levels in the injured cells. Concentrations of glutamine as low as 50 microM produced significant improvement of ATP levels in endothelial cells exposed to 5 mM H2O2. The 2 mM concentration of glutamine produced the greatest benefit, while greater concentrations of glutamine (5-20 mM) were actually associated with progressive decrements of the maximal benefit seen with the 2 mM concentration. The 2 mM concentration of glutamine produced similar enhancement of ATP with 1 and 10 mM H2O2 injury as well. Short-term viability following 5 mM H2O2 injury was significantly improved by the presence of 2 mM glutamine. The most effective concentration of glutamine (2 mM) did not scavenge H2O2 in a fluorometric assay. These observations suggest that mitochondrial substrates, such as glutamine, that bypass glucose-dependent pathways of ATP synthesis may be useful therapeutic agents for maintenance of ATP levels in oxidant-injured cells.

Mechanism of protection of oxidant-injured endothelial cells by glutamine.

Glutamine supplementation before oxidant exposure has recently been shown to significantly enhance adenosine triphosphate (ATP) levels and viability in endothelial cells. The aim of this study was to determine if glutamine can help cells after oxidant injury has been initiated and to demonstrate the mechanism of its action. The activity of glyceraldehyde 3-phosphate dehydrogenase was measured in bovine pulmonary artery endothelial cells exposed to H2O2 (0 to 10 mmol/L). Glyceraldehyde 3-phosphate dehydrogenase activity was completely inhibited by 10 mmol/L H2O2 after 1 minute, resulting in inhibition of glycolysis. The endothelial cells were then exposed to 10 mmol/L H2O2, with glutamine (2 mmol/L) being added at different times in relation to the injury. ATP levels were monitored during a 3-hour time course, and short-term viability was measured 6 hours after addition of the oxidant. Significant improvement of endothelial cell ATP levels and short-term viability was seen with addition of glutamine as late as 15 minutes after addition of H2O2. Mitochondrial inhibition with oligomycin (650 nmol/L) abolished the protective effect of glutamine on ATP levels and short-term viability. Cellular survival at 24 hours was not enhanced by glutamine, which suggests that ATP may not be the only factor determining long-term survival after oxidant injury.

Protamine-induced reductions of endothelial cell ATP.

Protamine, a polycationic protein used to reverse heparin anticoagulation, is frequently associated with decreased oxygen consumption, systemic hypotension, pulmonary artery hypertension, and bradycardia. This investigation examines the hypothesis that these events reflect toxic effects of protamine on endothelial cells. Cultured bovine pulmonary artery endothelium was exposed to protamine (12.5 to 500 micrograms/ml, corresponding to clinical doses 0.75 to 30 mg/kg), either alone (n = 6) or 3 minutes after exposure to heparin, 0.1 IU/microgram protamine (n = 6). ATP was measured 1 to 180 minutes after protamine by a luciferase-luciferin assay and cell viability determined by trypan blue exclusion. Ultrastructure was assessed by transmission electron microscopy. Polylysine, 25 micrograms/ml, a cytotoxic polycationic agent, was also studied. Dose-dependent reductions in ATP (range, -11% to -51%) and ATP per viable cell (up to -41%) occurred. Decreases in ATP did not occur until after 30 minutes with protamine alone, compared with differences as early as 1 minute after protamine with prior heparin. Progressive mitochondrial injury was noted evident by swollen cristae, vacuolization, and eventual disruption. Polylysine caused similar changes. Protamine decreases endothelial cell ATP and prior heparin exposure accelerates this effect. The toxicity may reside in the positive charges on these molecules and mitochondrial damage may account for reductions in cellular ATP and systemic oxygen consumption.

Mechanism of endothelial cell shape change in oxidant injury.

Changes in endothelial cell morphology induced by neutrophil-generated hydrogen peroxide (H2O2) may account for the capillary leak of the adult respiratory distress syndrome (ARDS). The relationship of H2O2 effects on the concentration of intracellular Ca2+ [( Ca2+]i) and ATP to changes in microfilaments and microtubules, important determinants of cell shape, was examined. Bovine pulmonary artery endothelial cells were injured over a 2-hr time course with a range of H2O2 doses (0-20 mM). The higher concentrations of H2O2 consistently produced contraction and rounding of greater than 50-75% of cells by 1-2 hr. The range of 1-20 mM H2O2 produced rapid, significant reductions in endothelial ATP levels over the time course of injury. Although there were significant increases in mean endothelial [Ca2+]i in response to 5, 10, and 20 mM H2O2, 1 mM H2O2 did not affect the [Ca2+]i. Fluorescence microscopy revealed that microfilament disruption occurred as ATP levels fell and preceded depolymerization of microtubules which developed after [Ca2+]i approached 1 X 10(-6) M. H2O2 at 1 mM injury caused microfilament disruption but did not depolymerize microtubules. Microfilament disruption occurred without oxidant exposure, when ATP levels were reduced by glucose depletion and mitochondrial inhibition with oligomycin (650 nM). If a Ca2+ ionophore, ionomycin (5 microM), was then added, [Ca2+]i rose to greater than 1 X 10(-6) M, microtubules fragmented and depolymerized, and cell contraction and rounding very similar to that induced by H2O2 occurred. These results suggest that endothelial cell dysfunction and capillary leak in ARDS may be due to H2O2-mediated changes in cellular ATP and [Ca2+]i.

ATP and microfilaments in cellular oxidant injury.

Oxidant injury produces dramatic changes in cytoskeletal organization and cell shape. ATP synthetic pathways are major targets of oxidant injury resulting in rapid depletion of cellular ATP following oxidant exposure. The relation of ATP depletion to the changes in microfilament organization seen following H2O2 exposure were examined in the P388D1 cell line. Three hours of glucose depletion alone resulted in a decline in cellular ATP levels to less than 10% of controls, which was comparable to ATP levels in cells 30 to 60 minutes after exposure to 5 mM H2O2 in the presence of glucose. Adherent cells stained with rhodamine phalloidin, a probe specific for polymerized (F) actin, revealed a progressive shortening of microfilaments into globular aggregates within cells depleted of glucose over 3 hours, a pattern similar to earlier observations of H2O2-injured cells after 1 hour. The changes in cellular ATP associated with glucose depletion or H2O2 exposure were then correlated with G actin content measured by the DNAse 1 inhibition assay. No real differences in G actin content as a percentage of total actin were seen in P388D1 cells following 3 hours of glucose depletion or 30 to 60 minutes after exposure to 5 mM H2O2. But 2 to 3 hours after exposure to H2O2 there was a progressive decrease in G actin as a percentage of total actin within the cells. Transmission electron microscopy of cells depleted of glucose for 3 h or 1 hour after exposure to H2O2 revealed the presence of side-to-side aggregates or bundles of microfilaments within the cells. These observations suggest that declining levels of ATP either from metabolic inhibition or H2O2 injury are correlated with the fragmentation and shortening of microfilaments into aggregates. No net change in monomeric or polymeric actin was necessary for this to occur. However, at later time points after H2O2 exposure some actin assembly did occur.

Predicting contraceptive behavior among college students: the role of communication, knowledge, sexual anxiety, and self-esteem.

Undergraduate students were surveyed about their sexual behavior and contraceptive behavior. In addition, measures of their self-esteem, knowledge about contraception, communication with their dating partners, communication about sexual matters with their sexual partners, and sexual anxiety were taken. Consistent with Byrne's (1983) model of effective contraception, it was found that general and sexual communication with one's partner were significant predictors of contraception use. Directional, but statistically weak, support was obtained for the predictions that knowledge about contraception and sexual anxiety would be related to contraception use. No support was found for the prediction that general self-esteem would be associated with contraceptive behavior.

PIP: This paper reports findings of a survey of the sexual and contraceptive behavior of US male and female undergraduate students; measures of self-esteem, knowledge about contraception, communication with dating partners, communication about sexual matters with their sexual partners, and sexual anxiety are also compiled. Byrne's 5 step model outlining the effective contraception behavior sequence includes: 1) acquisition of accurate contraceptive information, 2) acknowledgment of likely sexual intercourse, 3) obtaining contraceptive devices, 4) communication with one's partner about contraception, and 5) using the chosen method of contraception. Results of the survey measured sexual behavior by 4 measures: 1) age at 1st intercourse, 2) number of partners, 3) total number of conditions for intercourse, and 4) total number of sexual acts participated in. The 2 measures of contraception calculated were: 1) percentage of time subjects estimated that they used contraception when they engaged in sexual intercourse, and 2) percentage of times subjects used each contraceptive method, multiplied by the average effectiveness rating for that method. The general communication level with the partner is the best predictor of contraceptive behavior for male subjects. For females, sexual communication with one's partner is the best predictor of contraceptive behavior. Overall, sexual communication and sexual anxiety are related to many of the sexual behavior measures for both sexes. Results provide partial support for some of the Byrne model steps for understanding effective cotnraception behavior among college students. The 4th step, communicating with one's partner, received the greatest amount of support. Discussing contraception and low sexual anxiety are positively related to contraception use, but these correlations were weak. Self-esteem was found to be a poor predictor of contraceptive behavior. Sexual behavior, including contraceptive behavior is influenced by many divergent sources; this may help explain the problem of increasing numbers of unwanted pregnancies.

The influence of parents, church, and peers on the sexual attitudes and behaviors of college students.

Male and female undergraduate students were surveyed concerning their sexual attitudes, sexual behaviors, and contraceptive behavior. In addition, the general attitudes about sexuality the students perceived as communicated to them by their parents, their church, and their peers were assessed. It was found for female students that general attitudes about sexuality, as defined on an erotophilia-erotophobia dimension, and sexual behaviors were correlated with the perceived attitudes of peers, rather than those of parents and church. However, male students' attitudes and some sexual behaviors were correlated with the perceived attitudes of their parents, rather than the views of their peers and church. Church attitudes were not found to be related to any of the measures. None of the sources of influence, parents, peers, or church attitudes, or erotophilia-erotophobia was related to contraceptive behavior.

Desire for control, locus of control, and proneness to depression.

Two personality constructs, desire for control and locus of control, were related to depression among college students. Measures of levels of depression, desire for control, and locus of control were taken from subjects. Approximately six months later 71% of these subjects returned a questionnaire concerning their experiences with depression during that six-month period. It was found that locus of control scores, particularly the extent to which subjects perceived that their lives were controlled by chance, were significantly related to the depression levels. It was also found that high desire for control subjects who held external perceptions of control were most likely to seek nonprofessional help for depression. In addition, high desire for control subjects who perceived their lives as generally controlled by chance were most likely to have suicidal thoughts. The results are interpreted in terms of a general style that may promote a proneness to depression for certain individuals.

Social anxiety, self-presentation, and the self-serving bias in causal attribution.

Two experiments were conducted to provide evidence concerning the contribution of self-presentation concerns to the self-serving bias in causal attribution (individuals' tendency to assume more personal responsibility for a success than for a failure outcome) and its occasional, but systematic, reversal. In Experiment 1 high- but not low-social-anxiety participants presented themselves in a far more modest light when a committee of high prestige others was to join the experimenter in evaluating their behavior than when the committee evaluation was canceled. In Experiment 2 this reversal of the self-serving bias among high-social-anxiety subjects (in the evaluative context) was replicated, and it was also found that both high- and low-social-anxiety participants portrayed the causes of their behavior in a more modest fashion when they responded via the "bogus pipeline," a measurement technique designed to reduce distortion and dissimulation in verbal responses, than when they responded in the traditional paper-and-pencil format (although the influence of the bogus pipeline above and beyond the committee evaluation in eliciting "honest" responses from subjects only reached significance for low-social-anxiety subjects). These findings are discussed in terms of the varying self-presentation strategies and differing self-concepts of individuals high and low in social anxiety, as well as the self-presentation component to apparently self-enhancing and self-effacing causal attributions for performance.