Conservation of RecG activity from pathogens to hyperthermophiles

Maintaining the integrity of the genome is essential for the survival of all organisms. RecG helicase plays an important part in this process in Escherichia coli, promoting recombination and DNA repair, and providing ways to rescue stalled replication forks by way of a Holliday junction intermediate. We purified RecG proteins from three other species: two Gram-positive mesophiles, Bacillus subtilis and Streptococcus pneumoniae, and one extreme thermophile, Aquifex aeolicus. All three proteins bind and unwind replication fork and Holliday junction DNA molecules with efficiencies similar to the E. coli protein. Proteins from the Gram-positive species promote DNA repair in E. coli, indicating either that RecG acts alone or that any necessary protein-protein interactions are conserved. The S. pneumoniae RecG reduces plasmid copy number when expressed in E. coli, indicating that like the E. coli protein it unwinds plasmid R loop structures used to prime replication. This effect is not seen with B. subtilis RecG; the protein either lacks R loop unwinding activity or is compromised by having insufficient ATP. The A. aeolicus protein unwinds DNA well at 60 degrees C but is less efficient at 37 degrees C, explaining its inability to function in E. coli at this temperature. The N-terminal extension present in this protein was investigated and found to be dispensable for activity and thermo-stability. The results presented suggest that the role of RecG in DNA replication and repair is likely to be conserved throughout all bacteria, which underlines the importance of this protein in genome duplication and cell survival.     

An E. coli RuvC mutant defective in cleavage of synthetic Holliday junctions.

Escherichia coli RuvC protein is a specific endonuclease that resolves recombination intermediates into viable products. The structural features needed for RuvC activity were investigated by sequencing three ruvC mutations and relating the base pair changes identified to the activity of the mutant proteins. Each of the three mutations is a single base-pair substitution. ruvC51 converts glycine-15 to an aspartic acid residue. The product of ruvC51 was purified and shown to retain the ability to bind junctions, albeit with a slightly reduced affinity. However, it has lost the ability to resolve these structures by symmetrical cleavage. A multicopy ruvC51 plasmid confers sensitivity to UV light in a ruvC+ strain. The ruvC53 allele causes a glycine-17 to serine substitution while ruvC55 produces a stop codon. Neither of these genes produces a stable product. The results suggest that the N-terminal domain of RuvC may be concerned with cleavage of junctions.     

Presenilin 2 expression in neuronal cells: induction during differentiation of embryonic carcinoma cells

Mutations in the presenilin 1 and 2 (PS1 and PS2) genes cause most cases of early onset Alzheimer's disease. The genes encode two homologous multipass membrane proteins. Since the endogenous expression of PS2 has been poorly analyzed to date, we studied PS2 expression and localization in cultured human neuroblastoma cells and mouse neuronal cells. PS2 was mainly detected as a full-length protein of about 52 kDa in these cells and in brain, in contrast to PS1 that is mainly detected as endoproteolytic N-terminal and C-terminal fragments. Using immunofluorescence we found that like PS1, PS2 colocalized with markers of the endoplasmic reticulum-Golgi intermediate compartment, ERGIC-53 and beta-COP. Double labeling for PS1 and PS2 indicated that both proteins are colocalized in neuroblastoma SH-SY5Y cells. To study PS2 expression during differentiation, mouse embryonic carcinoma P19 cells were treated with retinoic acid. We found minimal PS2 expression in undifferentiated cells, an increase from day 2, and a maximum at day 8 after treatment. PS1 expression remained constant during this period. The differential expression of PS1 and PS2 within the P19 cells following retinoic acid treatment indicates different utilization or temporal requirements for these proteins during neuronal differentiation.     

Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells

Cell wall polysaccharides are synthesized from sugar-nucleotides, e.g. uridine 5'-diphosphoglucose (UDP-Glc), but the metabolic pathways that produce sugar-nucleotides in plants remain controversial. To help distinguish between potentially 'competing' pathways, we have developed a novel dual-radiolabelling strategy that generates a remarkably wide range of 3H:14C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces of D-[1-(3)H]galactose and a 14C-labelled hexose (e.g. D-[U-14C]fructose) in the presence of an approximately 10(4)-fold excess of non-radioactive carbon source. Six interconvertible 'core intermediates', galactose 1-phosphate <--> UDP-galactose <--> UDP-glucose <--> glucose 1-phosphate <--> glucose 6-phosphate <--> fructose 6-phosphate, showed a large decrease in 3H:14C ratio along this pathway from left to right. The isotope ratio of a polysaccharide-bound sugar residue indicates from which of the six core intermediates its sugar-nucleotide donor substrate stemmed. Polymer-bound galacturonate, xylose, arabinose and apiose residues (all produced via UDP-glucuronate) stemmed from UDP-glucose, not glucose 6-phosphate; therefore, UDP-glucuronate arose predominantly by the action of UDP-glucose dehydrogenase rather than through the postulated competing pathway leading from glucose 6-phosphate via myo-inositol. The data also indicate that UDP-galacturonate was not formed by a hypothetical UDP-galactose dehydrogenase. Polymer-bound mannose and fucose residues stemmed from fructose 6-phosphate, not glucose 1-phosphate; therefore GDP-mannose (guanosine 5'-diphosphomannose) arose predominantly by a pathway involving phosphomannose isomerase (via mannose phosphates) rather than through a postulated competing pathway involving GDP-glucose epimerization. Curiously, the ribose residues of RNA did not stem directly from hexose 6-phosphates, but predominantly from UDP-glucose; an alternative to the textbook pentose-phosphate pathway therefore predominates in plants.     

The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction

Considering the well-known antioxidant properties of statins, it seems important to assess their impact on major markers of oxidative stress (superoxide anion radical, nitric oxide, and index of lipid peroxidation) to compare the antioxidative potentials of atorvastatin and simvastatin during the different degrees of hyperhomocysteinemia (HHcy) in rats. This study was conducted on adult male Wistar albino rats (n = 90; 4 weeks old; 100 ± 15 g body mass) in which HHcy was achieved by dietary manipulation. For 4 weeks, the animals were fed with one of the following diets: standard rodent chow, diet enriched in methionine with no deficiency in B vitamins (folic acid, B6, and B12), or diet enriched in methionine and deficient in B vitamins (folic acid, B6, and B12). At the same time, animals were treated with atorvastatin at doses of 3 mg/kg/day i.p. or simvastatin at doses of 5 mg/kg/day i.p. Levels of superoxide anion radical and TBARS were significantly decreased by administration of simvastatin in normal and high-homocysteine (Hcy) groups (p < 0.05). At 4 weeks after feeding with purified diets, the concentrations of the GSH, CAT, and SOD antioxidants were significantly affected among all groups (p < 0.05). Our results indicated that statin therapy had variable effects on the redox status in hyperhomocysteinemic rats, and simvastatin demonstrated stronger antioxidant effects than did atorvastatin.     

Longevity and skeletal muscle mass: the role of IGF signalling,the sirtuins,dietary restriction and protein intake

Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating socio‐economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin‐like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS‐1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging.     

Contrasting principal stratum and hypothetical strategy estimands in multi-period crossover trials with incomplete data

Complete case analyses of complete crossover designs provide an opportunity to make comparisons based on patients who can tolerate all treatments. It is argued that this provides a means of estimating a principal stratum strategy estimand, something which is difficult to do in parallel group trials. While some trial users will consider this a relevant aim, others may be interested in hypothetical strategy estimands, that is, the effect that would be found if all patients completed the trial. Whether these estimands differ importantly is a question of interest to the different users of the trial results. This paper derives the difference between principal stratum strategy and hypothetical strategy estimands, where the former is estimated by a complete-case analysis of the crossover design, and a model for the dropout process is assumed. Complete crossover designs, that is, those where all treatments appear in all sequences, and which compare t treatments over p periods with respect to a continuous outcome are considered. Numerical results are presented for Williams designs with four and six periods. Results from a trial of obstructive sleep apnoea-hypopnoea (TOMADO) are also used for illustration. The results demonstrate that the percentage difference between the estimands is modest, exceeding 5% only when the trial has been severely affected by dropouts or if the within-subject correlation is low.