Membrane position of a basic aromatic peptide that sequesters phosphatidylinositol 4,5 bisphosphate determined by site-directed spin labeling and high-resolution NMR

The membrane interactions and position of a positively charged and highly aromatic peptide derived from a secretory carrier membrane protein (SCAMP) are examined using magnetic resonance spectroscopy and several biochemical methods. This peptide (SCAMP-E) is shown to bind to membranes containing phosphatidylinositol 4,5-bisphosphate, PI(4,5)P2, and sequester PI(4,5)P2 within the plane of the membrane. Site-directed spin labeling of the SCAMP-E peptide indicates that the position and structure of membrane bound SCAMP-E are not altered by the presence of PI(4,5)P2, and that the peptide backbone is positioned within the lipid interface below the level of the lipid phosphates. A second approach using high-resolution NMR was used to generate a model for SCAMP-E bound to bicelles. This approach combined oxygen enhancements of nuclear relaxation with a computational method to dock the SCAMP-E peptide at the lipid interface. The model for SCAMP generated by NMR is consistent with the results of site-directed spin labeling and places the peptide backbone in the bilayer interfacial region and the aromatic side chains within the lipid hydrocarbon region. The charged side chains of SCAMP-E lie well within the interface with two arginine residues lying deeper than a plane defined by the position of the lipid phosphates. These data suggest that SCAMP-E interacts with PI(4,5)P2 through an electrostatic mechanism that does not involve specific lipid-peptide contacts. This interaction may be facilitated by the position of the positively charged side chains on SCAMP-E within a low-dielectric region of the bilayer interface.     

The importance of physiological ecology in conservation biology

Many of the threats to the persistence of populations of sensitivespecies have physiological or pathological mechanisms, and thosemechanisms are best understood through the inherently integrativediscipline of physiological ecology. The desert tortoise waslisted under the Endangered Species Act largely due to a newlyrecognized upper respiratory disease thought to cause mortalityin individuals and severe declines in populations. Numeroushypotheses about the threats to the persistence of desert tortoisepopulations involve acquisition of nutrients, and its connectionto stress and disease. The nutritional wisdom hypothesis positsthat animals should forage not for particular food items, butinstead, for particular nutrients such as calcium and phosphorusused in building bones. The optimal foraging hypothesis suggeststhat, in circumstances of resource abundance, tortoises shouldforage as dietary specialists as a means of maximizing intakeof resources. The optimal digestion hypothesis suggests thattortoises should process ingesta in ways that regulate assimilationrate. Finally, the cost-of-switching hypothesis suggests thatherbivores, like the desert tortoise, should avoid switchingfood types to avoid negatively affecting the microbe communityresponsible for fermenting plants into energy and nutrients.Combining hypotheses into a resource acquisition theory leadsto novel predictions that are generally supported by data presentedhere. Testing hypotheses, and synthesizing test results intoa theory, provides a robust scientific alternative to the popularuse of untested hypotheses and unanalyzed data to assert theneeds of species. The scientific approach should focus on hypothesesconcerning anthropogenic modifications of the environment thatimpact physiological processes ultimately important to populationphenomena. We show how measurements of such impacts as nutrientstarvation, can cause physiological stress, and that the endocrinemechanisms involved with stress can result in disease. Finally,our new syntheses evince a new hypothesis. Free molecules ofthe stress hormone corticosterone can inhibit immunity, andthe abundance of "free corticosterone" in the blood (thoughtto be the active form of the hormone) is regulated when thecorticosterone molecules combine with binding globulins. Thesex hormone, testosterone, combines with the same binding globulin.High levels of testosterone, naturally occurring in the breedingseason, may be further enhanced in populations at high densities,and the resulting excess testosterone may compete with bindingglobulins, thereby releasing corticosterone and reducing immunityto disease. This sequence could result in physiological andpathological phenomena leading to population cycles with a periodthat would be essentially impossible to observe in desert tortoise.Such cycles could obscure population fluctuations of anthropogenicorigin.     

Characterization of osteoblastic properties of 7F2 and UMR-106 cultures after acclimation to reduced levels of fetal bovine serum

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated the development of a culturing environment reduced in estrogenic compounds. The selected medium (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red, a suspected estrogenic agent. This protocol reduced the concentration of FBS supplementation to 0% through successive 24 h incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast-like phenotype of 7F2 and UMR-106 cell lines when compared with control cells grown in various concentrations of FBS. Although the rate of mitotic divisions declined, the 7F2 and UMR-106 cultures continued to express osteoblast-specific markers and exhibited estrogen responsiveness. These experimental findings demonstrate that the culture protocol developed did not alter the osteoblast nature of the cell lines and provides a model system to study estrogen's antiresorptive role on skeletal turnover.     

Design and performance of a pilot-scale constructed wetland treatment system for natural gas storage produced water

To test the hypothesis that water produced from natural gas storage wells could be treated effectively by constructed wetland treatment systems, a modular pilot-scale system was designed, built, and used for treating gas storage produced waters. Four simulated waters representing the range of contaminant concentrations typical of actual produced waters were treated, and the system's performance was monitored. Freshwater wetland cells planted with Schoenoplectus californicus and Typha latifolia were used to treat fresh and brackish waters. Saline and hypersaline waters were treated by saltwater wetland cells planted with Spartina alterniflora and by reverse osmosis. Effective removal of cadmium, copper, lead, and zinc was achieved by the pilot-scale system. Results suggest that use of specifically designed constructed wetland treatment systems provides a flexible and effective approach for treating gas storage produced waters over a wide range of compositions.     

A Gradualist Theory of Discovery in Ecology

The distinction between the context ofdiscovery and the context of justificationrestricts philosophy of science to the rationalreconstruction of theories, and characterizesscientific discovery as rare, theoreticalupheavals that defy rational reconstruction. Kuhnian challenges to the two contextsdistinction show that non-rational elementspersist in the justification of theories, butgo no further to provide a positive account ofdiscovery. A gradualist theory of discoverydeveloped in this paper shows, with supportfrom ecological cases, that discoveries areroutinely made in ecology by extending modelsto new domains, or by making additions toearlier models. The logic of discovery isphilosophically accessible once it isappreciated that model truth is presumed, evenif counterfactually, in ecologists' applicationof models. A gradualist view shows thatmodels' heuristic power routinely leads todiscoveries.     

TRAF6 Mediates Basal Activation of NF-κB Necessary for Hematopoietic Stem Cell Homeostasis

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THE PHOTOTROPIC EFFECT OF POLARIZED LIGHT

For the growing cell of Phycomyces, a difference in the phototropic effect of light is described depending on its plane of polarization with reference to the axis of the cell. The difference which is found is primarily due to differences in the reflection losses at the cell surface. The magnitude of the effect approximates that deduced from the theory of phototropism suggested for this system. No specific effect of plane polarized light on the growth processes of the cell need be postulated.     

Secretory carrier membrane protein SCAMP2 and phosphatidylinositol 4,5-bisphosphate interactions in the regulation of dense core vesicle exocytosis

Secretory carrier membrane protein 2 (SCAMP2) functions in late steps of membrane fusion in calcium-dependent granule exocytosis. A basic/hydrophobic peptide segment within SCAMP2 (SCAMP2 E: CWYRPIYKAFR) has been implicated in this function and shown to bind and sequester phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 or PIP2] within membranes through an electrostatic mechanism. We now show that alanine substitution of tryptophan W2 within SCAMP2 E substantially weakens peptide binding to negatively charged liposomes; other substitutions for arginine R4 and lysine K8 have only limited effects on binding. Electron paramagnetic resonance analysis of liposomes containing spin-labeled PIP2 shows that R4 but not K8 is critical for SCAMP E binding to PIP2. The interfacial locations of SCAMP E and its structural variants within lipid bicelles measured by oxygen enhancement of nuclear relaxation are all similar. Corresponding point mutations within full-length SCAMP2 (SC2-R204A, SC2-K208A, and SC2-W202A) have been analyzed for biological effects on dense core vesicle exocytosis in neuroendocrine PC12 cells. With the same level of overexpression, SC2-R204A but not SC2-K208A inhibited secretion of cotransfected human growth hormone and of noradrenalin. Inhibition by SC2-R204A was the same as or greater than previously observed for SC2-W202A. Analysis of noradrenalin secretion by amperometry showed that inhibitory mutants of SCAMP2 decrease the probability of fusion pore opening and the stability of initially opened but not yet expanded fusion pores. The strong correlation between SCAMP2 E interactions with PIP2 and inhibition of exocytosis, particularly by SC2-R204A, led us to propose that SCAMP2 interaction with PIP2 within the membrane interface regulates fusion pore formation during exocytosis.     

EMF genes regulate Arabidopsis inflorescence development.

Mutations in EMBRYONIC FLOWER (EMF) genes EMF1 and EMF2 abolish rosette development, and the mutants produce either a much reduced inflorescence or a transformed flower. These mutant characteristics suggest a repressive effect of EMF activities on reproductive development. To investigate the role of EMF genes in regulating reproductive development, we studied the relationship between EMF genes and the genes regulating inflorescence and flower development. We found that APETALA1 and AGAMOUS promoters were activated in germinating emf seedlings, suggesting that these genes may normally be suppressed in wild-type seedlings in which EMF activities are high. The phenotype of double mutants combining emf1-2 and apetala1, apetala2, leafy1, apetala1 cauliflower, and terminal flower1 showed that emf1-2 is epistatic in all cases, suggesting that EMF genes act downstream from these genes in mediating the inflorescence-to-flower transition. Constitutive expression of LEAFY in weak emf1, but not emf2, mutants increased the severity of the emf phenotype, indicating an inhibition of EMF activity by LEAFY, as was deduced from double mutant analysis. These results suggest that a mechanism involving a reciprocal negative regulation between the EMF genes and the floral genes regulates Arabidopsis inflorescence development.