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.     

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.     

Projections of the nucleus of the basal optic root in pigeons (Columba livia) revealed with biotinylated dextran amine

The nucleus of the basal optic root (nBOR) of the accessory optic system is known to be involved in the analysis of the visual consequences of self-motion. Previous studies have shown that the nBOR in pigeons projects bilaterally to the vestibulocerebellum, the inferior olive, the interstitial nucleus of Cajal, and the oculomotor complex and projects unilaterally to the ipsilateral pretectal nucleus lentiformis mesencephali and the contralateral nBOR. By using the anterograde tracer biotinylated dextran amine, we confirmed these projections and found (previously unreported) projections to the nucleus Darkshewitsch, the nucleus ruber, the mesencephalic reticular formation, and the area ventralis of Tsai as well as ipsilateral projections to the central gray, the pontine nuclei, the cerebellar nuclei, the vestibular nuclei, the processus cerebellovestibularis, and the dorsolateral thalamus. In addition to previous studies, which showed a projection to the dorsomedial subdivision of the contralateral oculomotor complex, we found terminal labelling in the ventral and dorsolateral subdivisions. Individual fibers were reconstructed from serial sections, and collaterals to various nuclei were demonstrated. For example, collaterals of fibers projecting to the vestibulocerebellum terminated in the vestibular or cerebellar nuclei; collaterals of fibers to the inferior olive terminated in the pontine nuclei; many individual neurons projected to the interstitial nucleus of Cajal, the nucleus Darkshewitsch, and the central gray and also projected to the nucleus ruber and the mesencephalic reticular formation; collaterals of fibers to the contralateral nucleus of the basal optic root terminated in the mesencephalic reticular formation and/or the area ventralis of Tsai; neurons projecting to the nucleus lentiformis mesencephali also terminated in the dorsolateral thalamus. The consequences of these data for understanding the visual control of eye movements, neck movements, posture, locomotion, and visual perception are discussed.     

How the Wastewater Discharge Fee Might Become a Tool of River-Basin Management

The Federal German Water Law of 1976 postulated for the first time that a licence for the discharge of wastewater into public watercourses might only be granted when the wastewater, prior to discharge, is treated according to laid-down standards. In 1976 it was also decided that for the residual pollution load in sewage-treatment plant effluents a pollution fee has to be paid.
At present three pollution units have to be paid per capita and annually for untreated domestic wastewater. One pollution unit is assessed at 17/annum, but the price will be increased to 30/annum in future.
Since the pollution units encompass COD, nitrogen, phosphorus, heavy metals, and chlorinated hydrocarbon compounds, the pollution unit is a good tool to compare the effectiveness of treatment. For different drainage systems the residual pollution units (per litre and second) can also be calculated for low-flow conditions.     

Bacillus subtilis N-acetylmuramic acid L-alanine amidase

The N-acetymuramic acid L-alanine amidase from Bacillus subtilis (ATCC 6051) has been purified to homogeneity. It is a monomeric protein of molecular weight 50,000. The enzyme has a high affinity for homologous cell walls and once attached to a cell wall will hydrolyze the wall completely before initiating the hydrolysis of a new cell wall. The affinity of the enzyme for cell walls devoid of teichoic acid or for cell walls of Bacillus megaterium is much lower than that for B. subtilis cell walls. A second homogenous protein has been isolated from B. subtilis which specifically combines with the amidase in a 1:1 molar ratio and stimulates enzyme activity. This modifier protein has no intrinsic cell wall lytic activity. The binding of enzyme and modifier protein has a dissociation constant of 8.5 times 10-9 M in 0.1 M LiCl, pH 8.0, but the two proteins can be completely dissociated in 3 M LiCl at pH 8.0.     

Sensitivities of ocular tissues to acute pressure-induced ischemia

Intraocular pressure was artificially elevated for eight hours in eight owl monkeys. The first permanent effect (produced at a perfusion pressure of plus 15 mm Hg) was partial necrosis of iris stroma and ciliary processes, associated with microscopic lesions in the photoreceptors and retina pigment epithelium around the disc and in the retinal periphery. At a slightly higher pressure, visual nerve fibers in the retina and optic nerve and their ganglion cells were affected. Simultaneously, the outer retinal layers showed damage to the pigment epithelium, photoreceptors, and other nuclear layers. At even higher pressures, nearly all the other intraocular tissues were affected except for Müller cells, astroglia in the optic nerve head, epithelium of the pars plana, and the pigment cells of the choroid. The possibility is raised of a nonischemic pressure-induced mechanism for destruction of disc astrocytes in human chronic glaucoma.