Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.     

Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Structuring detergents for extracting and stabilizing functional membrane proteins

Background

Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.

Methodology/Principal Findings

Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by ¹H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.

Conclusion/Significance

These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.     

Cultivar‐specific effects of pathogen testing on storage root yield of sweetpotato,Ipomoea batatas

The accumulation and perpetuation of viral pathogens over generations of clonal propagation in crop species such as sweetpotato, Ipomoea batatas, inevitably result in a reduction in crop yield and quality. This study was conducted at Bundaberg, Australia to compare the productivity of field‐derived and pathogen‐tested (PT) clones of 14 sweetpotato cultivars and the yield benefits of using healthy planting materials. The field‐derived clonal materials were exposed to the endemic viruses, while the PT clones were subjected to thermotherapy and meristem‐tip culture to eliminate viral pathogens. The plants were indexed for viruses using nitrocellulose membrane‐enzyme‐linked immunosorbent assay and graft‐inoculations onto Ipomoea setosa. A net benefit of 38% in storage root yield was realised from using PT materials in this study. Conversely, in a similar study previously conducted at Kerevat, Papua New Guinea (PNG), a net deficit of 36% was realised. This reinforced our finding that the response to pathogen testing was cultivar dependent and that the PNG cultivars in these studies generally exhibited increased tolerance to the endemic viruses present at the respective trial sites as manifested in their lack of response from the use of PT clones. They may be useful sources for future resistance breeding efforts. Nonetheless, the potential economic gain from using PT stocks necessitates the use of pathogen testing on virus‐susceptible commercial cultivars.     

Menadione increases hepatic tight-junctional permeability. Its effect can be decreased by butylated hydroxytoluene and verapamil.

Infusion of menadione at two different doses [2.7 mg and 5.5 mg in 100 microliters of dimethyl sulphoxide (DMSO)] into perfused rat livers for 30 min caused no or a 6-fold increase respectively in junctional permeability to horseradish peroxidase as compared with controls receiving 100 microliters of DMSO alone. The total glutathione (GSH) contents in these livers measured at the end of the experiments were 115% and 53%, compared with the controls. The free-radical scavenger butylated hydroxytoluene (BHT) (final concn. 5 microM) protected against the GSH depletion caused by the higher dose of menadione and partially decreased the menadione-induced increase in junctional permeability. Verapamil, a Ca2(+)-channel blocker which was added into the perfusion medium (final concn. 40 microM) 10 min before the infusion of 5.5 mg of menadione, completely abolished the effect of menadione on junctional permeability. Menadione exposure therefore increases tight-junctional permeability in the liver; this may involve a depletion of GSH and a subsequent increase in intracellular Ca2+.     

Characterization of pea chloroplastic carbonic anhydrase. Expression inEscherichia coli and site-directed mutagenesis

A cDNA encoding the mature, chloroplast-localized carbonic anhydrase in pea has been expressed inE. coli. The enzyme is fully active and yields of up to 20% of the total soluble protein can be obtained from the bacteria. This expression system was used to monitor the effects of site-directed mutagenesis of seven residues found within conserved regions in the pea carbonic anhydrase amino acid sequence. The effects of these modifications are discussed with respect to the potential of various amino acids to act as sites for zinc coordination or intramolecular proton shuttles.     

Pure acceleration is the primary determinant of speed to first-base in major-league baseball game situations

The purposes of this research were to (a) quantify interval sprint times between Home-Plate and the Foul-Line and the Foul-Line and First-Base, (b) determine if differences exist in interval velocities and acceleration between left- and right-handed batters or between-position groupings, and (c) to quantify determinants of time to First-Base in Major-League Baseball players during actual games. A total of 1,896 sprint times to the Foul-Line (13.7 m) and First-Base (27.4 m) were recorded in 302 baseball players by a single coach, positioned in the dugout with a hand-held stopwatch. Interval velocities and accelerations were computed between Home-Plate and the Foul-Line and the Foul-Line and First-Base; average velocity and acceleration were also determined over the entire 27.4 m. Velocity and acceleration for left-handed batters were greater than for right-handed batters from Home-Plate to the Foul-Line and from Home-Plate to First-Base; however, there were no differences in velocity or acceleration from the Foul-Line to First-Base. Interval velocity was significantly greater for outfielders and infielders compared with that for catchers from Home-Plate to the Foul-Line and from the Home-Plate to First-Base. Outfielders were faster than catchers from the Foul-Line to First-Base; no other between-group differences were evident. Accelerations from Home-Plate to the Foul-Line and from Home-Plate to First-Base were greater for outfielders compared with infielders and catchers. Infielders accelerated at greater rates than did catchers between these intervals. There were no between-position differences in acceleration from the Foul-Line to First-Base. These data indicate that time to First-Base is most affected by acceleration from Home-Plate to the Foul-Line. Coaches should implement strategies that encourage players to sprint maximally over the first 13.7 m to maximize chances of successfully reaching First-Base.     

Soil Aggregates and Associated Organic Matter under Conventional Tillage,No-Tillage,and Forest Succession after Three Decades

Impacts of land use on soil organic C (SOC) are of interest relative to SOC sequestration and soil sustainability. The role of aggregate stability in SOC storage under contrasting land uses has been of particular interest relative to conventional tillage (CT) and no-till (NT) agriculture. This study compares soil structure and SOC fractions at the 30-yr-old Horseshoe Bend Agroecosystem Experiment (HSB). This research is unique in comparing NT and CT with adjacent land concurrently undergoing forest succession (FS) and in sampling to depths (15–28 cm) previously not studied at HSB. A soil moving experiment (SME) was also undertaken to monitor 1-yr changes in SOC and aggregation. After 30 years, enhanced aggregate stability under NT compared to CT was limited to a depth of 5 cm, while enhanced aggregate stability under FS compared to CT occurred to a depth of 28 cm and FS exceeded NT from 5–28 cm. Increases in SOC concentrations generally followed the increases in stability, except that no differences in SOC concentration were observed from 15–28 cm despite greater aggregate stability. Land use differences in SOC were explained equally by differences in particulate organic carbon (POC) and in silt-clay associated fine C. Enhanced structural stability of the SME soil was observed under FS and was linked to an increase of 1 Mg SOC ha⁻¹ in 0–5 cm, of which 90% could be attributed to a POC increase. The crushing of macroaggregates in the SME soil also induced a 10% reduction in SOC over 1 yr that occurred under all three land uses from 5–15 cm. The majority of this loss was in the fine C fraction. NT and FS ecosystems had greater aggregation and carbon storage at the soil surface but only FS increased aggregation below the surface, although in the absence of increased carbon storage.     

A discoordinate increase in the cellular amount of 3-hydroxy-3-methylglutaryl-CoA reductase results in the loss of rate-limiting control over cholesterogenesis in a tumour cell-free system.

Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant systems prepared from both normal rat liver and Morris hepatoma 3924A. The rate of cholesterol synthesis per cell was 9-fold greater in the tumour system than in that from normal liver, and the tumour systems showed the loss of rate-limiting control at the hydroxymethylglutaryl-CoA reductase (HMGR)-catalysed step. The apparent absence of rate-limiting control over cell-free tumour cholesterogenesis was traced primarily to a discoordinate and dramatic increase in the amount of HMGR in the tumour relative to the liver system. Preliminary evidence for an altered control of the post-lanosterol portion of the pathway was also obtained with the tumour system.