Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits

The subunits of phosphorylase kinase are separated and isolated in high yield by gel filtration chromatography in pH 3.3 phosphate buffer containing 8 M urea. Three protein peaks are obtained: the alpha and beta subunits coelute in the first, whereas the gamma and delta subunits are separate peaks. Upon dilution of the denaturant, catalytic activity reappears, associated only with the gamma subunit. As has been previously observed (Kee, S.M., and Graves, D.J. (1986) J. Biol. Chem. 261, 4732-4737), addition of calmodulin dramatically stimulates the reactivation of gamma. Inclusion of increasing amounts of the alpha/beta subunit mixture in the renaturation progressively decreases the activity of the renatured gamma or gamma-calmodulin. This inhibition by alpha/beta is likely due to specific interactions with the gamma subunit because the inhibition is less at pH 8.2 than at pH 6.8 and less when equivalent amounts of phosphorylated alpha/beta subunits are used (both alkaline pH and phosphorylation are known to stimulate the activity of the holoenzyme). These results suggest that the role of either the alpha or beta subunits, or perhaps both, in the nonactivated (alpha 2 beta 2 gamma 2 delta 2)2 complex of phosphorylase kinase is to suppress the activity of the gamma subunit and that activation of the enzyme, by phosphorylation for instance, is due to deinhibition caused by release of this quaternary constraint by alpha and/or beta upon gamma.     

The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin.

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, beta- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, beta- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only beta-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.     

Linking responses of native and invasive plants to hurricane disturbances: implications for coastal plant community structure

Hurricane disturbances produce significant changes in forest microclimates, creating opportunities for seedling regeneration of native and invasive plant species alike. However, there is limited information on how changes in microclimates and pre-existing forest conditions affect native and invasive plants responses to hurricane disturbances. In this manipulative study, we examined the responses of three common shrub/small stature tree species, two of which are native to the coastal region of the southeastern USA (Baccharis halimifolia and Morella cerifera) and one that is invasive (Triadica sebifera), to two key components of hurricane disturbance (canopy damage and saline storm surge). In a greenhouse, we grew seedlings of these species under a range of shade levels that mimicked pre-and post-hurricane canopy conditions for wet pine forest and mixed hardwood forest, two forest communities common in coastal areas of the southeastern USA. Seedlings were subjected to saline storm surges equivalent to full strength sea water for 3 days. Seedling responses (mortality and growth) to the treatments were monitored for 16 months. All species benefitted from higher canopy openness. Storm surge effects were short-lived and seedlings readily recovered under high light conditions. The storm surge had stronger negative effects on survival and growth of all species when coupled with high shade, suggesting storm surge has greater negative impacts on seedlings where hurricane winds cause minimal or no canopy damage. The invasive T. sebifera was by far more shade tolerant than the natives. Survival of T. sebifera seedlings under highly shaded conditions may provide it a competitive edge over native species during community reassembly following tropical storms. Differential responses of native and invasive species to hurricane disturbances will have profound consequences on community structure across coastal forest stands, and may be regulated by legacies of prior disturbances, community structure, extent of canopy damage, and species’ tolerance to specific microclimates.

     

A new fused tetracyclic heterocyclic antioxidant from Serratia sp. PAMC 25557

A new fused tetracyclic heterocyclic compound, (4bR,10bR)-4b-hydroxy-10b,12-dihydrodibenzo[c,h][2,6]naphthyridine-5,11(4bH,6H)-dione (1), and a known compound, butyl 2-[(benzoyloxy)methyl]benzoate, spatozoate 2, were isolated from the broth culture of Serratia sp. PAMC 25557. The structure of 1 was determined by analyzing spectroscopic data. Compound 1 did not exhibit antimicrobial activity against Escherichia coli, Staphylococcus aureus, or Candida albicans. In addition, up to 100 μg/ml compound 1 did not show any toxicity against Artemia salina larvae. However, compound 1 showed DPPH free radical scavenging activity (IC₅₀ = 16.7 ± 0.34 μg/ml). This was the first report of spatozoate isolation from bacterial sources.     

Red panda fine‐scale habitat selection along a Central Himalayan longitudinal gradient

Red panda Ailurus fulgens, an endangered habitat specialist, inhabits a narrow distribution range in bamboo abundance forests along mountain slopes in the Himalaya and Hengduan Mountains. However, their habitat use may be different in places with different longitudinal environmental gradients, climatic regimes, and microclimate. This study aimed to determine the habitat variables affecting red panda distribution across different longitudinal gradients through a multivariate analysis. We studied habitat selection patterns along the longitudinal gradient in Nepal's Himalaya which is grouped into the eastern, central, and western complexes. We collected data on red panda presence and habitat variables (e.g., tree richness, canopy cover, bamboo abundance, water availability, tree diameter, tree height) by surveys along transects throughout the species’ potential range. We used a multimodal inference approach with a generalized linear model to test the relative importance of environmental variables. Although the study showed that bamboo abundance had a major influence, habitat selection was different across longitudinal zones. Both canopy cover and species richness were unimportant in eastern Nepal, but their influence increased progressively toward the west. Conversely, tree height showed a decreasing influence on habitat selection from Eastern to Western Nepal. Red panda's habitat selection revealed in this study corresponds to the uneven distribution of vegetation assemblages and the dry climatic gradient along the eastern‐western Himalayas which could be related to a need to conserve energy and thermoregulate. This study has further highlighted the need of importance of bamboo conservation and site‐specific conservation planning to ensure long‐term red panda conservation.     

14-3-3 connects glycogen synthase kinase-3 beta to tau within a brain microtubule-associated tau phosphorylation complex

In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3beta and tau are parts of an approximately 400-500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 zeta co-elutes with the tau phosphorylation complex components tau and GSK3 beta. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3 zeta, GSK3 beta, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3 beta, 14-3-3 zeta co-precipitates with GSK3 beta, indicating that GSK3 beta binds to 14-3-3 zeta. From HEK-293 cells transfected with tau, GSK3 beta, and 14-3-3 zeta in different combinations, tau co-immunoprecipitates with GSK3 beta only in the presence of 14-3-3 zeta. In vitro, approximately 10-fold more tau binds to GSK3 beta in the presence of than in the absence of 14-3-3 zeta. In transfected HEK-293 cells, 14-3-3 zeta stimulates GSK3 beta-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 zeta dimer simultaneously binds and bridges tau and GSK3 beta and stimulates GSK3 beta-catalyzed tau phosphorylation.     

Management scenario evaluation for a large treatment wetland using a spatio-temporal phosphorus transport and cycling model

This paper describes the development of a two-dimensional, spatially distributed model to simulate coupled hydrologic and phosphorus (P) biogeochemical processes in a 147-ha cell of a 1544-ha stormwater treatment wetland designed to help protect the greater Everglades, FL, USA. The model was used to assess the effects of a suite of feasible management alternatives on the long-term ability of the wetland to sustain total P (TP) removal. The spatial and temporal dynamics of TP retention were simulated under historical (1995–2000) conditions, and under assumptions of removal of short-circuiting channels and ditches, changes in external hydraulic and TP loading, and long-term (>20 years) impacts on soil and water column TP dynamics under current and reduced load conditions. Internal hydrology and transport processes were calibrated against measured tracer concentrations, and subsequently validated against outflow discharge and spatial chloride concentration data. Cycling of P was simulated as first-order uptake and release, with different uptake coefficients for open water/sparse submerged aquatic vegetation (SAV) areas (0.2 day^?1) and dense SAV areas (0.4 day^?1), and a much lower, uniform release coefficient (1.97 × 10^?4 day^?1). The calibration and validation of the P model showed good agreement with field measurements of water column TP concentrations measured at the wetland outlet (calibration RMSE = 10.5 μg L^?1; validation RMSE = 15.6 μg L^?1). Under simulated conditions of preferential channels eliminated, average annual TP treatment effectiveness increased by 25%. When inflow TP loads were assumed to be eliminated after 6 years of loading, the release of accumulated soil P sustained predicted annual average outlet concentrations above 6.7 μg L^?1 for 18 years, decreasing at a rate of 0.16 μg L^?1 yr^?1. Sensitivity analyses indicate that the most critical model input factors include flow resistance parameters, initial soil TP content, and P cycling parameters compared to initial water level, initial TP concentration in water column, ET and transport parameters.     

Glucose sensors made of novel carbon nanotube-gold nanoparticle composites

Carbon nanotube and metal particle composites have been exploited to fabricate high performance electrochemical devices. However, the physical and chemical procedures to synthesize the composites are labor intensive and inefficient. Our study reveals an one-step wet chemistry method to accomplish fast and controllable production of gold nanoparticle (AuNP) and carbon naotube (CNT) composites. Such a process is sensitive to the surface charge. Especially, when functionalized with carboxyl groups, the CNTs carried negative charges and showed low level association with negatively charged AuNPs. Thermal treatment was employed to decompose the carboxyl groups and render each CNT a charge-free surface thereby achieving a high level AuNP-CNT association. The fabricated glucose sensors demonstrated dependence of their sensitivities to the amount of AuNPs on the CNTs. The enhancement of sensitivity can be attributed to an accelerated electron transfer rate from glucose oxidase Gox to the electrode. The Michaelis-Menten kinetics also indicated improved performance in the glucose sensor made of AuNP-CNTs. Therefore, our research revealed a novel approach to produce metallic nanoparticle and CNT composite for fabricating high performance electrochemical sensors.     

Population status and habitat occupancy of endangered river dolphins in the Karnali River system of Nepal during low water season

Ganges river dolphin abundance has undergone a predominant decline across its range since monitoring began. In Nepal, disappearance from some of the rivers it once used has already occurred. Today this species can only be found in three river systems in Nepal, the Karnali, Sapta Koshi, and Narayani, but numbers are low in these locations. To determine the abundance of dolphins remaining in the Karnali system (which includes the Karnali, Geruwa, and Mohana), and factors affecting dolphin habitat use, we conducted surveys where we recorded dolphin presence. Dolphins within this river system were sighted only in the Karnali and an abundance estimate of 5.04 ± 0.753 SE was calculated. This pattern of ranging differed from that previously reported (from previous sightings only in the Geruwa to current sightings only in the Karnali). River depth likely contributed to the presence or absence of dolphins. Shifts in available habitat between the Geruwa and Karnali have resulted from changes in the course of the main stream Karnali following construction of the Chisapani irrigation intake. Because of the low numbers of dolphins reported, there is great concern that loss of this species in Nepal is likely in the near future.