Kinetic and fluorescence studies of the interaction of p21(ras) with guanine nucleotides and the GTPase activating proteins, p120-GAP and Nfi-GAP

The protein products of the ras proto-oncogenes, p21ras, are 21 KDa guanine nucleotide binding proteins which possess a slow intrinsic GTPase activity. These proteins are thought to be involved in the regulation of cell growth and differentiation, and some single point mutations in the ras genes lead to cell transformation. The biological signal mediated by p21ras is determined by the in vivo concentration of the p21ras.GTP complex since only when bound to GTP are these proteins thought to be biologically active. Nucleotide exchange of bound GDP for cytoplasmic GTP leads to the reformation of the p21ras.GTP complex. Two proteins (GAPs) have been identified which accelerate the rate of GTP hydrolysis by wild p21ras but not by oncogenic p21ras mutants. The kinetic mechanism of the p21ras.GTPase in the presence of the catalytic-domains of two GAPs, p120-GAP and neurofibromin, has been investigated. The studies are based primarily on the use of a fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl)-GTP (mantGTP), which shows changes in fluorescence intensity during several elementary steps in the GTPase mechanism. The experimental results are compatible with a mechanism where the intrinsic hydrolysis of GTP by p21ras is preceded, and controlled, by a protein conformational change in the p21ras.GTP complex. p120-GAP accelerates the overall rate of GTP cleavage by promoting this rate limiting conformational change. The binding of p120-GAP to p21ras.GTP is a rapidly reversible reaction (equilibrium dissociation constant, Kd = 20 μM at l ≈ 20 mM). p120-GAP accelerates the rate of the subsequent conformational change in p21ras.GTP by a factor of 105 compared to the rate of the equivalent reaction in the absence of p120-GAP. A similar extent of activation was observed with neurofibromin although the affinity for p21N-ras.GTP was 20-fold higher than that of p120-GAP. In contrast to p120-GAP, the binding reaction with neurofibromin may not be rapidly reversible. For both proteins, increasing ionic strength lead to a marked increase in the Kd; the rate constant of the conformational change being essentially unaltered. The binding of p120-GAP and neurofibromin to the p21ras.mantGTP complex is associated with an increase in fluorescence intensity, anisotropy and energy transfer (from tryptophan residues in the GAP proteins). These fluorescence signals can be used to determine the equilibrium dissociation constants for the binding of GAP proteins to p21ras. The intrinsic GTPase activity of the Gly 12→ Pro mutant of p21ras is only weakly accelerated by either p120-GAP or neurofibromin although both GAPs bind with an affinity similar to that with the wild type p21ras protein. An explanation for the weakly transforming phenotype of this mutant compared to other Gly 12 mutants is offered. One consequence of this interpretation is that the in vivo rate of GAP-activated GTP hydrolysis and nucleotide exchange may be slower than generally assumed. Finally, the hydrodynamic properties of p21ras proteins have been investigated using time resolved fluorescence techniques combined with a range of fluorescent guanine nucleotide analogues. In all cases, the rotational correlation time of p21ras is consistent with a dimeric structure for these proteins in solution

Remote sensing observations of ocean physical and biological properties in the region of the Southern Ocean Iron Experiment (SOFeX)

Author Posting. © American Geophysical Union, 2006. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 111 (2006): C06026, doi:10.1029/2005JC003289.Satellite remote sensing estimates of surface chlorophyll, temperature, wind speed, and sea ice cover are examined in the region of the Southern Ocean Iron Experiment (SOFeX). Our objectives are to place SOFeX into a regional context and highlight regional mesoscale spatial and monthly temporal variability. SOFeX fertilized two patches with iron, one south of the Antarctic Polar front (PF) and one north of the PF but south of the Subantarctic Front (SAF). Satellite observable phytoplankton blooms developed in both patches. The spring sea-ice retreat near the south patch site was delayed in the 2001-2002 season, in turn delaying the naturally occurring, modest spring bloom in this region. Ambient surface chlorophyll concentrations for the area surrounding the southern patch during January 2002 are low (mean 0.26 mg/m3) compared with climatological January values (0.42 mg/m3). Regions east and west at similar latitudes exhibited higher mean chlorophyll concentrations (0.79 and 0.74 mg/m3, respectively). These modest phytoplankton blooms were likely stimulated by melting sea-ice via changes in the light-mixing regime and release of iron, and were smaller in magnitude than the iron-induced bloom within the SOFeX southern patch (> 3 mg/m3). Iron inputs from melting ice may drive much of the natural spatial and temporal variability within the seasonal ice zone. Mean chlorophyll concentrations surrounding the SOFeX northern patch site were similar to climatological values during the SOFeX season. The northern patch was stretched into a long, thin filament along the southern boundary of the SAF, likely increasing the mixing/dilution rate with surrounding waters.S. Doney and K. Moore were supported by NASA grant NAG5-12520 from the NASA Ocean Biogeochemistry Program

Syndemics and the PrEP Cascade: Results from a Sample of Young Latino Men Who Have Sex with Men.

Young Latino men who have sex with men (MSM) are a highly vulnerable population for HIV infection. Pre-exposure prophylaxis (PrEP) is a novel biomedical HIV prevention tool that may aid in reducing the disparity in HIV incidence among Latino MSM. However, PrEP use is disproportionally low among Latino MSM and, therefore, identifying barriers along the PrEP continuum of care (the "PrEP cascade") would provide insight into how to best deploy PrEP interventions. Syndemics theory is a prominent framework employed in HIV prevention; however, to date, no known studies have applied this theory to PrEP. Thus, the aim of the current study was to explore the association between syndemics and the PrEP cascade, including the degree to which psychosocial and structural syndemic constructs are related to the PrEP cascade. Participants were 151 young Latino MSM (M age = 24 years; SD = 3) residing in San Diego, California, who completed a battery of online self-report measures. Results indicated high levels of syndemic indicators and varying levels of engagement across the PrEP cascade. As syndemic indicators increased, the odds of engagement across the PrEP cascade were significantly lowered. Psychosocial and structural syndemic factors accounted for unique variance in the PrEP cascade. Results highlight the need for combination interventions that address both psychosocial and structural barriers to PrEP use and persistence among young Latino MSM

Brief communication: Understanding solar geoengineering's potential to limit sea level rise requires attention from cryosphere experts

Stratospheric aerosol geoengineering, a form of solar geoengineering, is a proposal to add a reflective layer of aerosol to the stratosphere to reduce net radiative forcing and so to reduce the risks of climate change. The efficacy of solar geoengineering at reducing changes to the cryosphere is uncertain; solar geoengineering could reduce temperatures and so slow melt, but its ability to reverse ice sheet collapse once initiated may be limited. Here we review the literature on solar geoengineering and the cryosphere and identify the key uncertainties that research could address. Solar geoengineering may be more effective at reducing surface melt than a reduction in greenhouse forcing that produces the same global-average temperature response. Studies of natural analogues and model simulations support this conclusion. However, changes below the surfaces of the ocean and ice sheets may strongly limit the potential of solar geoengineering to reduce the retreat of marine glaciers. High-quality process model studies may illuminate these issues. Solar geoengineering is a contentious emerging issue in climate policy and it is critical that the potential, limits, and risks of these proposals are made clear for policy makers.</p

TB174: Maine Wild Blueberries Field Winnowing Systems

The objective of this study was to determine if there were differences in berry quality between the two winnowing systems currently used in the Maine wild blueberry industry. The following experiment was performed three times during the 1997 field season.https://digitalcommons.library.umaine.edu/aes_techbulletin/1034/thumbnail.jp

Wisdom of the Crowd within Enterprises: Practices and Challenges

The Wisdom of the Crowd advocates that decisions collectively made by a diverse crowd could be better than those made by an elite group of experts. The Wisdom of the Crowd puts preconditions on this to work correctly. This concerns the di- versity of the crowd, their independence from each other, their decentralisation, and the methods of aggregating their distributed knowledge and forming collec- tive decisions. Although the concept is inspiring, its interpretation and conduct differ significantly amongst enterprises, especially with regard to the culture and style of management. In addition, we still lack reflections on how the Wisdom of the Crowd worked in the practice of modern enterprises. To address this lack of knowledge, this paper conducts an empirical study following a mixed method approach involving 35 senior managers coming from 33 different industries in the UK. In the first phase we interview eight managers and, in the second, we con- firm and enhance the results by a survey consisting of open-ended questions and involving 27 other managers. The results shed light on the current practice of the Wisdom of the Crowd in several UK enterprises, which can inform the analysis and design of future software tools meant to aid this emerging decision-making mechanism

Geographic patterns of koala retrovirus genetic diversity, endogenization, and subtype distributions

Koala retrovirus (KoRV) subtype A (KoRV-A) is currently in transition from exogenous virus to endogenous viral element, providing an ideal system to elucidate retroviral–host coevolution. We characterized KoRV geography using fecal DNA from 192 samples across 20 populations throughout the koala’s range. We reveal an abrupt change in KoRV genetics and incidence at the Victoria/New South Wales state border. In northern koalas, pol gene copies were ubiquitously present at above five per cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers below one, while the env gene was detected in all animals and in a majority at copy numbers above one per cell. These results suggest that southern koalas carry partial endogenous KoRV-like sequences. Deep sequencing of the env hypervariable region revealed three putatively endogenous KoRV-A sequences in northern koalas and a single, distinct sequence present in all southern koalas. Among northern populations, env sequence diversity decreased with distance from the equator, suggesting infectious KoRV-A invaded the koala genome in northern Australia and then spread south. The exogenous KoRV subtypes (B to K), two novel subtypes, and intermediate subtypes were detected in all northern koala populations but were strikingly absent from all southern animals tested. Apart from KoRV subtype D, these exogenous subtypes were generally locally prevalent but geographically restricted, producing KoRV genetic differentiation among northern populations. This suggests that sporadic evolution and local transmission of the exogenous subtypes have occurred within northern Australia, but this has not extended into animals within southern Australia

Multicentury changes in ocean and land contributions to the climate-carbon feedback

Author Posting. © American Geophysical Union, 2015. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Global Biogeochemical Cycles 29 (2015): 744–759, doi:10.1002/2014GB005079.Improved constraints on carbon cycle responses to climate change are needed to inform mitigation policy, yet our understanding of how these responses may evolve after 2100 remains highly uncertain. Using the Community Earth System Model (v1.0), we quantified climate-carbon feedbacks from 1850 to 2300 for the Representative Concentration Pathway 8.5 and its extension. In three simulations, land and ocean biogeochemical processes experienced the same trajectory of increasing atmospheric CO2. Each simulation had a different degree of radiative coupling for CO2 and other greenhouse gases and aerosols, enabling diagnosis of feedbacks. In a fully coupled simulation, global mean surface air temperature increased by 9.3 K from 1850 to 2300, with 4.4 K of this warming occurring after 2100. Excluding CO2, warming from other greenhouse gases and aerosols was 1.6 K by 2300, near a 2 K target needed to avoid dangerous anthropogenic interference with the climate system. Ocean contributions to the climate-carbon feedback increased considerably over time and exceeded contributions from land after 2100. The sensitivity of ocean carbon to climate change was found to be proportional to changes in ocean heat content, as a consequence of this heat modifying transport pathways for anthropogenic CO2 inflow and solubility of dissolved inorganic carbon. By 2300, climate change reduced cumulative ocean uptake by 330 Pg C, from 1410 Pg C to 1080 Pg C. Land fluxes similarly diverged over time, with climate change reducing stocks by 232 Pg C. Regional influence of climate change on carbon stocks was largest in the North Atlantic Ocean and tropical forests of South America. Our analysis suggests that after 2100, oceans may become as important as terrestrial ecosystems in regulating the magnitude of the climate-carbon feedback.We are grateful for support from the U.S. Department of Energy Office of Science and the National Science Foundation (NSF). J.T.R. and F.H. received support from the Regional and Global Climate Modeling Program in the Climate and Environmental Sciences Division of the Biological and Environmental Research (BER) Program in the U.S. Department of Energy Office of Science. J.T.R., K.L., E.M., W.F., J.K.M., S.C.D., and N.N.M. received funding from the NSF project “Collaborative Research: Improved Regional and Decadal Predictions of the Carbon Cycle“ (AGS-1048827, AGS-1021776, and AGS-1048890). The Community Earth System Modeling project receives support from both NSF and BER.2015-12-0

Role of Dimerization of the Membrane-associated Growth Factor Kit Ligand in Juxtacrine Signaling: The Sl17H Mutation Affects Dimerization and Stability—Phenotypes in Hematopoiesis

The Kit ligand (KL)/Kit receptor pair functions in hematopoiesis, gametogenesis, and melanogenesis. KL is encoded at the murine steel (Sl) locus and encodes a membrane growth factor which may be proteolytically processed to produce soluble KL. The membrane-associated form of KL is critical in mediating Kit function in vivo. Evidence for a role of cytoplasmic domain sequences of KL comes from the Sl17H mutation, a splice site mutation that replaces the cytoplasmic domain with extraneous amino acids. Using deletion mutants and the Sl17H allele, we have investigated the role of the cytoplasmic domain sequences of KL in biosynthetic processing and cell surface presentation. The normal KL protein products are processed for cell surface expression, where they form dimers. Both Sl17H and the cytoplasmic deletion mutants of KL were processed to the cell surface; however, the rate of transport and protein stability were affected by the mutations. Deletion of cytoplasmic domain sequences of KL did not affect dimerization of KL. In contrast, dimerization of the Sl17H protein was reduced substantially. In addition, we have characterized the hematopoietic cell compartment in Sl17H mutant mice. The Sl17H mutation has only minor effects on hematopoiesis. Tissue and peritoneal mast cell numbers were reduced in mutant mice as well as in myeloid progenitors. Interestingly, long-term bone marrow cultures from Sl17H mice did not sustain the long-term production of hematopoietic cells. In addition, homing of normal hematopoietic progenitors to the spleen of irradiated Sl17H/Sl17H recipient mice was diminished in transplantation experiments, providing evidence for a role of Kit in homing or lodging. These results demonstrate that the membrane forms of KL exist as homodimers on the cell surface and that dimerization may play an important role in KL/Kit-mediated juxtacrine signaling

Exploring the sensitivity of interannual basin-scale air-sea CO2 fluxes to variability in atmospheric dust deposition using ocean carbon cycle models and atmospheric CO2 inversions

Author Posting. © American Geophysical Union, 2007. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 112 (2007): G02012, doi:10.1029/2006JG000236.Estimates of sources/sinks of carbon dioxide (CO2) at the Earth's surface are commonly made using atmospheric CO2 inverse modeling, terrestrial and oceanic biogeochemical modeling, and inventory-based studies. In this study, we compare sea-air CO2 fluxes from the Time-Dependent Inverse (TDI) atmosphere model and the marine Biogeochemical Elemental Cycling (BEC) model to study the processes involved in ocean carbon cycling at subbasin scales. A dust generation and transport model, based on analyzed meteorology and terrestrial vegetation cover, is also used to estimate the interannual variability in dust and iron deposition to different ocean basins. Overall, a fairly good agreement is established between the TDI and BEC model results for the net annual patterns and seasonal cycle of sea-air CO2 exchange. Sensitivity studies with the ocean biogeochemical model using increased or reduced atmospheric iron inputs indicate the relative sensitivity of air-sea CO2 exchange. The simulated responses to changes in iron inputs are not instantaneous (peak response after ∼2−3 years). The TDI model derived seasonal cycles for the Southern Ocean (South Atlantic) are better matched by the BEC model by increasing (decreasing) iron inputs through atmospheric aerosols. Our results suggest that some of the interannual variability in TDI model air-sea CO2 fluxes during the past decade may be explainable by dust variability that relaxes/increases iron limitation in high-nitrate, low-chlorophyll (HNLC) ocean regions.S. C. Doney and N. Mahowald acknowledge support from NASA grant NNG05GG30G. J. K. Moore was funded by NSF grant OCE-0452972