Analysis of MEMO1 Binding Specificity for ErbB2 Using Fluorescence Polarization and Molecular Dynamics Simulations.

ErbB2 signaling pathways are linked to breast cancer formation, growth, and aggression; therefore, understanding the behavior of proteins associated with these pathways as well as regulatory factors that influence ErbB2 function is essential. MEMO1 is a redox active protein that is shown to associate with phosphorylated ErbB2 and mediate cell motility. We have developed a fluorescence polarization assay to probe the interaction between MEMO1 and an ErbB2-derived peptide containing a phosphorylated tyrosine residue. This interaction is shown to be pH-dependent and stronger with longer peptides as would be expected for protein-protein interactions. We have quantitatively mapped the binding interface of MEMO1 to the peptide using the fluorescence polarization assay and molecular dynamics simulations. We have confirmed that phosphorylation of the peptide is essential for binding and through mutagenesis have identified residues that contribute to favorable interactions. Our results highlight the importance of the protein-protein interactions of MEMO1 that complement the oxidase activity. In the future, these studies will provide a method for screening for selective modulators of MEMO1, which will allow for additional biological investigations.

Cooperation in rats playing the iterated Prisoner's Dilemma game.

Humans and animals show cooperative behaviour, but our understanding of cooperation among unrelated laboratory animals is limited. A classic test of cooperation is the iterated Prisoner's Dilemma (IPD) game, where two players receive varying payoffs for cooperation or defection in repeated trials. To determine whether unrelated rats cooperate in the IPD, we tested pairs of rats making operant responses to earn food reward in 25 trials/day. The operant chamber was bisected by a metal screen with a retractable lever and pellet dispenser on each side. When levers extended, rats had 2 s to respond. Mutual cooperation (Reward) delivered three pellets each, mutual defection (Punishment) provided no pellets, and unilateral defection (Temptation) gave five pellets to the defector, while the partner (Sucker) received none. In eight pairs of males (RM-) and females (RF-), cooperation was defined by withholding a response. In seven pairs of RM+ males, cooperation was defined by responding on the lever. In males, food restriction significantly inhibited both cooperation and pellets received. There was no effect of dominance status. Males and females made similar numbers of responses under ad libitum feeding. However, neither food restriction nor dominance status affected responses in females. Rats were subsequently tested for reciprocity in 24 alternating trials/day. A response on the lever within 5 s delivered three pellets to the partner. Females made significantly more responses for their cage-mate than males. Responses within pairs were significantly correlated for males, but not for females. For both sexes, responses declined significantly when paired with an unfamiliar partner who never reciprocated ('bad stooge'). These results demonstrate that rats working for food show cooperation in IPD and direct reciprocity. Their responses depend on food availability and responses of their partner.

Anabolic-androgenic steroids and appetitive sexual behavior in male rats.

Anabolic-androgenic steroids (AAS) increase libido and sexual behavior, but the underlying behavioral mechanisms are unclear. One way AAS may enhance expression of sexual behavior is by increasing the willingness to work for sex. In the present study, sexually-experienced male rats received daily injections of testosterone at supraphysiologic doses (7.5 mg/kg in water with 13% cyclodextrin) or vehicle and were tested for appetitive sexual behavior measured by operant responding for access to an estrous female. Initially, rats were trained in their home cage to respond on a nose-poke under a 10-min fixed-interval schedule for food reward. Once rats achieved stable response rates, the food was replaced by a female, followed by mating for 10 min. There was no effect of testosterone on operant responding for food (28.1 ± 4.4 responses/10 min for testosterone, 30.6 ± 4.3 for vehicle) or sex (35.0 ± 4.0 responses/10 min for testosterone, 37.3 ± 5.2 for vehicle). However, rats made significantly more responses for sex than for food (p < 0.05), and responses for food and sex were positively correlated among individuals (R(2) = 0.6). Additional groups of rats were trained to respond on a lever for the female under a 2nd-order schedule of reinforcement, where 5 responses opened a door to show the female for 5s. After 15 door openings, the male gained access to the female. There was no effect of testosterone on time to complete 75 responses: 38.4 ± 7.8 min for vehicle controls vs 43.3 ± 6.6 min for testosterone-treated rats (p > 0.05). These findings suggest that chronic high-dose testosterone does not enhance appetitive drive for sexual behavior.

Testosterone enhances risk tolerance without altering motor impulsivity in male rats.

Anabolic-androgenic steroids (AAS) increase impulsive and uncontrolled aggressive ('roid rage) in humans and enhance agonistic behavior in animals. However, the underlying mechanisms for AAS-induced aggression remain unclear. Potential contributing elements include an increase risk-taking and/or motor impulsivity due to AAS. This study addressed the effects of chronic high-dose testosterone on risk tolerance using a risky decision-making task (RDT) and motor impulsivity with a go/no-go task in operant chambers. Male Long-Evans rats were treated for at least 4 weeks with testosterone (7.5mg/kg) or vehicle beginning in late adolescence. Testosterone was used because it is popular among human AAS users. In RDT testing, one lever was paired with delivery of a small "safe" food reward, while the other was paired with a large "risky" reward associated with an increasing risk of footshock (0%, 25%, 50%, 75%, 100%) in successive test blocks. Three shock intensities were used: 1.0, 1.2, and 1.4mA/kg. As shock intensity and risk of shock increased, preference for the lever signifying a large reward significantly declined for both vehicle- and testosterone-treated rats (p<0.05). There was also a significant effect of drug (p<0.05), where testosterone-treated rats showed greater preference for the large reward, compared to vehicle-treated controls. Increased preference for the large reward, despite risk of footshock, is consistent with increased risk tolerance. In go/no-go testing, rats were trained to press a single lever if the go cue was presented (stimulus light) or to refrain from pressing during the no-go cue (tone). There was no effect of testosterone on pre-cue responses, or performance in go and no-go trials. These results suggest that AAS may increase risk-tolerance without altering motor impulsivity.