Spectroscopic and functional characterization of an environmentally sensitive fluorescent actin conjugate

Rabbit skeletal muscle F-actin has been selectively labeled at a cysteine residue with the environmentally sensitive fluorophore 6-acryloyl-2-(dimethylamino)naphthalene. The fluorescent actin conjugate behaves similarly to native actin with respect to the polymerization kinetics, critical monomer concentration, and ability to form F-actin paracrystals. Upon polymerization to F-actin, the absorption of the actin conjugate is red-shifted, whereas the fluorescence emission is blue-shifted 740 wavenumbers and is accompanied by a decrease in the fluorescence bandwidth of 470 wavenumbers. These large shifts in the spectral properties of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) in actin provide a simple method for obtaining a spectral discrimination between the G- and F-actin populations during the polymerization reaction. Steady-state fluorescence techniques were used to study the environment of the fluorophore in the monomeric and polymeric forms of actin. Fluorescence emission spectral analysis and quenching and polarization studies of G-actin-Prodan indicated that the fluorophore lies immobile on the protein surface but with one of its faces in full contact with the solvent. In F-actin, the fluorophore has a limited exposure to the solvent and is located in a dielectric environment similar to those seen for Prodan in polar, aprotic solvents or buried within a protein matrix [Macgregor, R. B., Jr., & Weber, G. (1986) Nature (London) 318, 70-73]. Additionally, our results demonstrate that the Prodan molecule conjugated to F-actin is completely immobile during its fluorescence lifetime, exhibits an increase in the resonance energy transfer (RET) from tryptophan residues compared to that observed in G-actin, and shows evidence of homologous RET within the polymer.     

Specific recognition of calmodulin from Dictyostelium discoideum by the ATP, ubiquitin-dependent degradative pathway

Calmodulin purified from Dictyostelium discoideum is selectively degraded by rabbit reticulocyte extracts in the presence of ubiquitin and ATP. This protein forms a 1:1 covalent conjugate with ubiquitin. Analyses of the cyanogen bromide fragments of the protein conjugate indicate that lysine 115 on calmodulin is the ubiquitin conjugation site. Bovine brain calmodulin which contains a trimethyllysine residue at this position is not a substrate for conjugation with ubiquitin, and its degradation rate is not affected by ATP and ubiquitin. These results suggest that the trimethyllysine residue in mammalian calmodulin may function in protecting the protein from degradation by the ATP, ubiquitin-dependent pathway. Since there are eight lysine residues in Dictyostelium calmodulin, the specific conjugation of ubiquitin to lysine 115 may provide a good model system to delineate the structural features required for the conjugation and to follow the degradative steps in the pathway.     

Structural Dynamics of Troponin I during Ca2+-Activation of Cardiac Thin Filaments: A Multi-Site F?rster Resonance Energy Transfer Study

A multi-site, steady-state Förster resonance energy transfer (FRET) approach was used to quantify Ca²⁺-induced changes in proximity between donor loci on human cardiac troponin I (cTnI), and acceptor loci on human cardiac tropomyosin (cTm) and F-actin within functional thin filaments. A fluorescent donor probe was introduced to unique and key cysteine residues on the C- and N-termini of cTnI. A FRET acceptor probe was introduced to one of three sites located on the inner or outer domain of F-actin, namely Cys-374 and the phalloidin-binding site on F-actin, and Cys-190 of cTm. Unlike earlier FRET analyses of protein dynamics within the thin filament, this study considered the effects of non-random distribution of dipoles for the donor and acceptor probes. The major conclusion drawn from this study is that Ca²⁺ and myosin S1-binding to the thin filament results in movement of the C-terminal domain of cTnI from the outer domain of F-actin towards the inner domain, which is associated with the myosin-binding. A hinge-linkage model is used to best-describe the finding of a Ca²⁺-induced movement of the C-terminus of cTnI with a stationary N-terminus. This dynamic model of the activation of the thin filament is discussed in the context of other structural and biochemical studies on normal and mutant cTnI found in hypertrophic cardiomyopathies.     

Population biology of grey gurnard (Eutrigla gurnardus (L.); Triglidae) in the coastal waters of Northwest Wales

The grey gurnard Eutrigla gurnardus (L.) has been identified by ICES as a potential commercial species in the NE Atlantic with recommendations made to derive information on population biology for stock assessment purposes. However, data on the population biology of this species is limited. In this study, data on the age, growth and maturity of grey gurnard were collected by otter trawling in the coastal waters of northwest Wales and Eastern Anglesey. Total length (TL) of fish sampled ranged between 2.1–33.0 cm (male) and 1.9–36.9 cm (female) with the majority of female (70.8%) fish between 11 and 20 cm TL and male fish (70.5%) between 11 and 18 cm TL. The percentage of fish >20 cm TL was larger for females (30.4%) compared to males (17.6%). Total weight (TW) for female and male grey gurnard in the stratified subsample ranged from 1.9 to 499.9 g for females and 2.1–390.0 g for males, with the majority of female (66.3%) and male (76.1%) fish between 10 and 60 g. TL/TW relations for male and female fish and both sexes combined were: TW = 0.006TL^3.07, TW = 0.007TL^3.03 and TW = 0.007TL^3.05 respectively. Age structure (based on otolith reading) ranged between 0.5 and 7.5 years old for females and 0.5 to 5.5 years old for male with the majority of female (41.7%) and male (46.0%) fish aged as 1.5 years old. The age structure of female and male grey gurnards was significantly different with the majority of older fish (>2.5 years) being female. The von Bertalanffy growth functions were calculated as L_t = 32.4[1 ? e^{?0.24(t + 1.41)}] for males, L_t = 45.9[1 ? e^{?0.16(t + 1.37)}] for females and L_t = 44.0[1 ? e^{?0.18(t + 1.20)}] for both sexes combined. Instantaneous rates of total mortality were similar for males and females and the combined Z value 1.00 year^?1 with the natural mortality rate estimated as 0.33 year^?1. The size at 50% maturity (L₅₀) was estimated to be 25.3 cm TL for males, females and for both sexes combined. Age at 50% maturity (A₅₀) was 3.2 years for both males and females. The results of this study provide the first information on the population biology of E. gurnardus in the Irish Sea, the first detailed study in the NE Atlantic since 1985 and helps to address the data gap identified by ICES in knowledge of the population biology of this species.     

Does the uniform packing of sand in a cylinder provide a uniform penetration resistance?

Sand packed to a constant dry bulk density, is frequently used as an artificial growth medium in which to simulate the effects of a constant mechanical impedance on root growth. This research aimed to determine whether conventional packing resulted in constant mechanical impedance and to test alternative packing regimes. Perspex cylinders 300 mm tall with a 49 mm internal diameter were packed with moist sand to uniform and varying bulk densities to examine which type of packing gave the greatest uniformity of penetration resistance (PR) with depth. The cylinders packed to a constant bulk density (1.48, 1.55, and 1.6 Mg m^-3) all had measured PR profiles which increased markedly with depth by approximately 1, 1.5 and 3 MPa, respectively, within the top 100 mm. Between 100–300 mm depth, these same cylinders showed reductions in PR of up to 1, 2 and 2 MPa respectively. These results show that sand packed to a constant bulk density with depth would not provide a uniform mechanical impedance to plant roots.By packing sand to different bulk densities at different depths, we obtained packed cylinders that had much more uniform PR profiles (with average values of 0.25, 1.40 and 2.30 MPa). Below a depth of 50 mm, the coefficients of variation for replicate cylinders packed in this way were 12%, 5% and 18% for the 0.25, 1.40, and 2.30 MPa treatments respectively. For experiments with single plants, the lower PR values that were unavoidable near to the surface (< 50 mm) can be avoided by sowing seeds at the base of a funnel inserted into the cylinder. Treatments such as these can provide reproducible growth media, with adequate water/nutrient and aeration status for the study of plant response to uniform mechanical impedance.     

Effect of sorghum resistant to Stenodiplosis sorghicola (Coquillett) (Diptera: Cecidomyiidae) on oviposition and development of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Abstract Helicoverpa armigera oviposition preference for, and larval development on sorghum hybrids with differing resistance to sorghum midge, Stenodiplosis sorghicola , were investigated. When H. armigera larvae were fed seed of resistant and susceptible hybrids in the laboratory there were no differences in larval and pupal sizes or the rate of development. The same result was recorded when larvae fed on panicles on plants in a glasshouse. On some sampling occasions, significantly more eggs were laid on panicles of resistant hybrids in the field. This occurred when plants were in plots and also in a mixed planting. Midge-resistance status did not affect levels of egg parasitism. In a field study using recombinant inbred lines between a midge-resistant and a midge-susceptible line, no relationship was found between level of resistance and oviposition of H. armigera . We conclude that, although midge-resistant hybrids are sometimes preferred for oviposition by H. armigera, the resistance per se does not determine this preference. Egg survival, larval survival, development and resultant damage are not significantly affected by the midge-resistance status of the host.     

Using Common Boundaries to Assess Methane Emissions: A Life Cycle Evaluation of Natural Gas and Coal Power Systems

There is consensus on the importance of upstream methane (CH₄) emissions to the life cycle greenhouse gas (GHG) footprint of natural gas systems, but inconsistencies among recent studies explain why some researchers calculate a CH₄ emission rate of less than 1% whereas others calculate a CH₄ emission rate as high as 10%. These inconsistencies arise from differences in data collection methods, data collection time frames, and system boundaries. This analysis focuses on system boundary inconsistencies. Our results show that the calculated CH₄ emission rate can increase nearly fourfold not by changing the magnitude of any particular emission source, but by merely changing the portions of the supply chain that are included within the system boundary. Our calculated CH₄ emission rate for extraction through pipeline transmission is 1.2% for current practices. Our model allows us to identify GHG contributors in the upstream supply chain, but also allows us to tie upstream findings to complete life cycle scenarios. If applied to the life cycles of power systems and assessed in terms of cumulative radiative forcing, the upstream CH₄ emission rate can be as high as 3.2% before the GHG impacts from natural gas power exceed those from coal power at any point during a 100‐year time frame.     

Genotoxic stress and cellular stress alter the subcellular distribution of human T-cell leukemia virus type 1 tax through a CRM1-dependent mechanism

Human T-cell leukemia virus type 1 Tax is a predominantly nuclear viral oncoprotein that colocalizes with cellular proteins in nuclear foci known as Tax speckled structures (TSS). Tax is also diffusely distributed throughout the cytoplasm, where it interacts with and affects the functions of cytoplasmic cellular proteins. Mechanisms that regulate the distribution of Tax between the cytoplasm and nucleus remain to be identified. Since Tax has been shown to promote genome instability by perturbing cell cycle progression and DNA repair mechanisms following DNA damage, we examined the effect of genotoxic stress on the subcellular distribution and interacting partners of Tax. Tax localization was altered in response to various forms of cellular stress, resulting in an increase in cytoplasmic Tax and a decrease in Tax speckled structures. Concomitantly, colocalization of Tax with sc35 (a TSS protein) decreased following stress. Tax translocation required the CRM1 nuclear export pathway, and a transient interaction between Tax and CRM1 was observed following stress. These results suggest that the subcellular distribution of Tax and the interactions between Tax and cellular proteins respond dynamically to cellular stress. Changes in Tax distribution and interacting partners are likely to affect cellular processes that regulate cellular transformation.