Mutagenesis of the conserved residue Glu259 of Gsalpha demonstrates the importance of interactions between switches 2 and 3 for activation

We previously reported that substitution of Arg258 within the switch 3 region of Gsalpha impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976-23983). The adjacent residue (Glu259) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha-E259A), Gln (Gsalpha-E259Q), Asp (Gsalpha-E259D), or Val (Gsalpha-E259V), and the properties of in vitro translation products were examined. The Gsalpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsalpha-E259D stimulated adenylyl cyclase normally in the presence of GTPgammaS but was less efficient with isoproterenol or AlF4-. The other mutants had more severely impaired effector activation, particularly in response to AlF4-. In trypsin protection assays, GTPgammaS was a more effective activator than AlF4- for all mutants, with Gsalpha-E259D being the least severely impaired. For Gsalpha-E259D, the AlF4--induced activation defect was more pronounced at low Mg2+ concentrations. Gsalpha-E259D and Gsalpha-E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GTPgammaS binding). However, for both mutants, the ability of AlF4- to decrease the rate of GTPgammaS binding was impaired, suggesting that they bound AlF4- more poorly. GTPgammaS bound to purified Gsalpha-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis of in vitro translates demonstrated that all mutants except Gsalpha-E259V bind to beta gamma at 0 degreesC and were stable at higher temperatures. In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct.     

Prefrontal control of trace eyeblink conditioning in rabbits: Role in retrieval of the CR?

Rabbits (Oryctolagus cuniculus) were trained on a trace eyeblink (EB) conditioning task to a criterion of 10 consecutive EB conditioned responses (CRs). One week later, ibotenic acid or sham lesions were made in the mPFC centered on the prelimbic region (Brodmann's area 32) or the cingulate cortex (Brodmann's area 24). Following a 1-week postoperative recovery period, all animals were retrained for 4 consecutive days using the same parameters as during acquisition, given 1 week off, and retrained for another 4 days. Mean EB conditioning deficits in the group with area 32 lesions occurred on the first and second days of each retraining period. However, by the third and fourth days of retraining, these lesioned animals were performing at a level comparable to that of the sham group. Lesions of area 24 did not produce deficits at either retesting period. These findings were interpreted to indicate that area 32, but not area 24, is involved in retrieval processes, rather than consolidation or storage, in that the animals were impaired at both retesting times, but were able to relearn the task. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cognitive and affective components of reinforcement.

Responds to B. F. Skinner's (see record 1986-24282-001) article on components of reinforcement and examines why reinforcers strengthen behavior. The present author suggests that (1) the pleasing aspect, (2) the correct or incorrect aspect, and (3) the strengthening aspect are 3 equally important components of reinforcement, referring to the affective, cognitive, and behavioral characteristics of reinforcement, respectively. The occurrence of the 1st 2 characteristics is considered a necessary antecedent for the occurrence of the 3rd. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Greater rate of decline in maximal aerobic capacity with age in physically active vs. sedentary healthy women

Using a meta-analytic approach, we recently reported that the rate of decline in maximal oxygen uptake (VO2 max) with age in healthy women is greatest in the most physically active and smallest in the least active when expressed in milliliters per kilogram per minute per decade. We tested this hypothesis prospectively under well-controlled laboratory conditions by studying 156 healthy, nonobese women (age 20-75 yr): 84 endurance-trained runners (ET) and 72 sedentary subjects (S). ET were matched across the age range for age-adjusted 10-km running performance. Body mass was positively related with age in S but not in ET. Fat-free mass was not different with age in ET or S. Maximal respiratory exchange ratio and rating of perceived exertion were similar across age in ET and S, suggesting equivalent voluntary maximal efforts. There was a significant but modest decline in running mileage, frequency, and speed with advancing age in ET. VO2 max (ml . kg-1 . min-1) was inversely related to age (P < 0.001) in ET (r = -0.82) and S (r = -0.71) and was higher at any age in ET. Consistent with our meta-analysic findings, the absolute rate of decline in VO2 max was greater in ET (-5.7 ml . kg-1 . min-1 . decade-1) compared with S (-3.2 ml . kg-1 . min-1 . decade-1; P < 0. 01), but the relative (%) rate of decline was similar (-9.7 vs -9. 1%/decade; not significant). The greater absolute rate of decline in VO2 max in ET compared with S was not associated with a greater rate of decline in maximal heart rate (-5.6 vs. -6.2 beats . min-1 . decade-1), nor was it related to training factors. The present cross-sectional findings provide additional evidence that the absolute, but not the relative, rate of decline in maximal aerobic capacity with age may be greater in highly physically active women compared with their sedentary healthy peers. This difference does not appear to be related to age-associated changes in maximal heart rate, body composition, or training factors.     

OXIDATION OF HYDROCARBONS BY HEMIN1

A model system for cytochrome P-450 comprised of hemin, thiosalicylic acid, and solvent (acetone and water), oxidized cyclohemne and n-hexane at pH 2.7. Oxidation of cyclohexane yielded more cyclohexanol than cyclohexanone under mild reaction conditions, whereas the oxidation of n-hexane generated more 3- and 2-hexanones than 3- and 2-hemnols. Oxygen was a limiting factor in the reaction, and increased oxygen pressures increased the ratio of cyclohexanone to cyclohexanol formed. Lag time for product formation decreased in a linear fashion with an increase in temperature. In addition, cyclohexanone decreased relative to cyclohexanol as the temperature was increased. Antioxidants increased the lag time for product formation, but had little effect on the final quantity or ratios of the oxidative products generated. 1,3 Diphenylisobenzafiran had no effect on the lag time, whereas Tiron and catechol increased the lag time. In contrast to aliphatic thiols, aromatic thiols were capable of driving the oxidative reaction. The model hemin system was approximately 6% as active as natural cytochrome P-450. Hemin immobilized on glass beads effectively catalyzed the oxidation of cyclohexane.