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. 相似文献
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. 相似文献
Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses.
Methods
Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively.
Results
The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains.
Conclusions
In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.
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 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. 相似文献
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. 相似文献
Mammals have experienced a massive decline in their populations and geographic ranges worldwide. The sloth bear, Melursus ursinus (Shaw, 1791), is one of many species facing conservation threats. Despite being endangered in Nepal, decades of inattention to the situation have hindered their conservation and management. We assessed the distribution and patterns of habitat use by sloth bears in Chitwan National Park (CNP), Nepal. We conducted sign surveys from March to June, 2020, in 4 × 4 km grids (n = 45). We collected detection/non‐detection data along a 4‐km trail that was divided into 20 continuous segments of 200 m each. We obtained environmental, ecological, and anthropogenic covariates to understand determinants of sloth bear habitat occupancy. The data were analyzed using the single‐species single‐season occupancy method, with a spatially correlated detection. Using repeated observations, these models accounted for the imperfect detectability of the species to provide robust estimates of habitat occupancy. The model‐averaged occupancy estimate for the sloth bear was 69% and the detection probability was 0.25. The probability of habitat occupancy by sloth bears increased with the presence of termites and fruits and in rugged, dry, open, undisturbed habitats. Our results indicate that the sloth bear is elusive, functionally unique, and widespread in CNP. Future conservation interventions and action plans aimed at sloth bear management must adequately consider their habitat requirements. 相似文献