Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

Cells use homology‐dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology‐based mechanisms involves nuclease‐dependent DNA end resection, which generates long tracts of single‐stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re‐synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re‐synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single‐stranded gap and terminate further resection.     

Plant metacommunity structure remains unchanged during biodiversity loss in English woodlands

The metacommunity concept provides important insights into large‐scale patterns and dynamics of distributions of interacting species. However, temporal change of metacommunity structure is little studied and has not been previously analysed in the context of biodiversity change. As metacommunity structure is determined by multiple species distributions, it is expected to change as a result of biodiversity loss. To examine this process, we analysed structural change of a southern English woodland metacommunity at two points in time, seven decades apart. During this interval, the metacommunity lost β‐diversity through taxonomic homogenization. We performed an ‘elements of metacommunity structure’ (EMS) analysis to examine metacommunity structure, based upon three structural elements: coherence (i.e. gaps in species range along a structuring gradient), spatial turnover (replacements), and species range boundary clumping. We predicted that metacommunity structure would decrease in spatial turnover and thus become more nested over time. We tested for change in individual structural elements with z‐scores and examined the role of spatial and environmental variables as potential structuring mechanisms through correlation with EMS ordination axes. Our results demonstrated that the metacommunity had a Clementsian structure that was maintained over time. Despite no change in broad structure, coherence and species range boundary clumping increased. Spatial turnover increased along the first structuring gradient but decreased on the second gradient. We hypothesise that this difference between gradients may reflect the presence of competing processes affecting spatial turnover. The mechanisms of biological structuring involved both environmental and spatial factors at the scale of the individual woodland. Therefore, our results suggest that broad metacommunity structure would not be a good landscape‐scale indicator for conservation status. Conversely, knowledge that metacommunity structure does not change over time could assist in long‐term conservation strategy because fundamental metacommunity structural processes are resistant to environmental change.     

High-density lipoprotein binding to bovine adrenal cortex membranes

Binding studies were performed with bovine adrenal cortex membranes, human 125I-labelled high-density lipoprotein (HDL) and modified photoactivable derivatives of 125I-labelled HDL, namely 125I-labelled HDL-amidinophenylazide and 125I-labelled HDL-amidopropionyldithiophenylazide. The purity of the apolipoprotein composition of the 125I-labelled HDL and photoactivable 125I-labelled HDL used in the binding studies was determined by Coomassie blue and silver staining, and by measuring 125I-labelled cpm after SDS-polyacrylamide gel electrophoresis. About 45% of the 125I-labelled HDL binding to the membranes occurred in the presence of excess EDTA and only unlabelled HDL competed for the binding site. The 125I-labelled interaction with this binding site on the membranes did not require calcium. In addition, 40% of the 125I-labelled HDL binding was to an EDTA-sensitive site, and unlabelled HDL and low-density lipoprotein (LDL) competed for the binding site. Consequently, adrenal cortex membranes have binding sites which show cross reactivity for both HDL and LDL. Modification of 58% of the apolipoprotein lysine residues of 125I-labelled HDL with methylazidophenylimidate, a reagent which maintains the positive charge at lysine residues, had little affect on binding to EDTA-sensitive and insensitive sites. In contrast, modification of 35% of apolipoprotein lysine residues of 125I-labelled HDL with N-succinimidyl(4-azidophenyldithio)propionate, a reagent which converts charged amino lysines to amide bonds, showed binding properties which were almost totally inhibited by EDTA.     

Effects of experimentally altered glutathione levels on life span, metabolic rate, superoxide dismutase, catalase and inorganic peroxides in the adult housefly, Musca domestica

Effects of varied levels of glutathione, an intracellular redox buffer, were examined in the adult male housefly in order to study the inter-relationship between enzymic and non-enzymic antioxidant defenses. An increase of over 100% in the concentrations of glutathione was induced by the administration of 3 mM L-2-oxothiazolidine-4-carboxylate (LOC), which increases the intracellular level of cysteine. A decrease in glutathione concentration of up to 85% was achieved by the administration of L-buthionine-SR-sulfoximine (BUS), which irreversibly inhibits glutamylcysteine synthetase. Life spans of houseflies were shortened by a decrease in the glutathione concentration, but were not prolonged by augmentation of glutathione. Metabolic rate and superoxide dismutase activity were independent of glutathione concentration. H2O2 was increased by both experimental regimes, whereas catalase activity was decreased by BUS. Results suggest that catalase activity is influenced by glutathione concentration.     

Changes in strength over a 2-year period in professional rugby union players

The purpose of this study was to assess the magnitude of upper and lower body strength changes in highly trained professional rugby union players after 2 years of training. An additional purpose was to examine if the changes in strength were influenced by the starting strength level, lean mass index (LMI), or chronological age. This longitudinal investigation tracked maximal strength and body composition over 3 consecutive years in 20 professional rugby union athletes. Maximal strength in the bench press and back squat and body composition was assessed during preseason resistance training sessions each year. The athletes completed a very rigorous training program throughout the duration of this study consisting of numerous resistance, conditioning and skills training sessions every week. The primary findings of this study were as follows: (a) Maximal upper and lower body strength was increased by 6.5-11.5% after 2 years of training (p = 0.000-0.002 for bench press; p = 0.277-0.165 for squat); (b) magnitude of the improvement was negatively associated with initial strength level (r = -0.569 to -0.712, p ≤ 0.05); (c) magnitude of improvement in lower body maximal strength was positively related to the change in LMI (an indicator of hypertrophy; r = 0.692-0.880, p ≤ 0.05); and (d) magnitude of improvement was not associated with the age of professional rugby union athletes (r = -0.068 to -0.345). It appears particularly important for training programs to be designed for continued muscle hypertrophy in highly trained athletes. Even in professional rugby union athletes, this must be achieved in the face of high volumes of aerobic and skills training if strength is to be increased.     

Redox modulation of the inotropic response to dobutamine is impaired in patients with heart failure

It has been suggested that oxidative stress contributes to impaired left ventricular (LV) contractility in the setting of heart failure (HF). To test this hypothesis, we studied the effect of an antioxidant on contractility at rest and in response to dobutamine in 10 HF patients. We hypothesized that vitamin C would augment contractility in HF and that this effect would be of a greater magnitude in HF patients compared with patients with normal LV (NLV) function. Data from 10 patients with NLV function who participated in this study are included in this report and have been published elsewhere. A micromanometer-tipped catheter was introduced into the LV. In the experimental protocol, an infusion catheter was positioned in the left main coronary artery. The peak positive rate of change of LV pressure (LV +dP/dt) was measured in response to the intravenous infusion of dobutamine before and during the intracoronary infusion of vitamin C (96 mg/min). Vitamin C had no effect on basal LV +dP/dt in either HF or NLV groups. The infusion of vitamin C augmented the LV +dP/dt response to dobutamine by 22 +/- 4% in the NLV function group. In contrast, vitamin C had no effect on the inotropic response to dobutamine in the HF group. In the control protocol, without vitamin C, no differences were observed between responses to two sequential dobutamine infusions in either group (HF, n = 11; NLV, n = 9). Therefore, a positive effect of vitamin C on contractility was limited to patients with NLV function. The absence of this effect in HF patients may suggest that normal redox responsiveness is lost in this disease state.     

Protein kinase C is regulated in vivo by three functionally distinct phosphorylations

Background: Protein kinase Cs are a family of enzymes that transduce the plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphorylations before it is competent to respond to second messengers.Results We have identified the positions and functions of the in vivo phosphorylation sites of protein kinase C by mass spectrometry and peptide sequencing of native and phosphatase-treated kinase from the detergent-soluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl terminus of protein kinase C βII are phosphorylated in vivo. T500 and S660 are selectively dephosphorylated in vitro by protein phosphatase 2A to yield an enzyme that is still capable of lipid-dependent activation, whereas all three residues are dephosphorylated by protein phosphatase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytosol, and that T500 is not an autophosphorylation site.Conclusion Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.     

Pattern of growth in weight of alate and apterous nymphs of the English grain aphid, Sitobion avenae

Daily increase in fresh weight was recorded for apterous and alate nymphs of S. avenae at 20°C. Comparison with a control group indicated that daily disturbance and weighing of nymphs did not affect significantly their growth, developmental time or survival. The increase in fresh weight of apterous and alate virginoparae at 20°C was best described by logistic equations. Alate virginoparae were significantly heavier than apterous virginoparae at birth and throughout most of their nymphal life, but they experienced a weight loss at the final ecdysis. The relative growth rate did not remain constant, but declined during development. The decline is associated with a decline in honeydew production per unit body weight. The implications of an inconstant relative growth rate and the marked loss in weight at the adult moult in alates are discussed.

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Résumé L'enregistrement de l'augmentation quotidienne du poids frais à 20°C des larves ailées et aptères de S. avenae a montré que des perturbations quotidiennes n'affectent pas significativement la croissance, la durée du développement et la survie. Les équations logistiques décrivent plus exactement l'augmentation de poids frais des aptères et des ailés virginipares à 20°C. Les virginipares ailés étaient significativement plus lourds que les virginipares aptères à la naissance et pendant la plus grande partie de la vie larvaire, mais présentaient une perte de poids à la mue finale. Le taux de croissance relative ne restait pas constant, mais diminuait au cours du développement. La diminution était associée à une diminution de la production de miellat par unité de poids du corps. La discussion porte sur les conséquences de la variation de l'augmentation du poids relatif et de la perte marquée de poids à la mue imaginale.