H2AX: the histone guardian of the genome

At close hand to one's genomic material are the histones that make up the nucleosome. Standing guard, one variant stays hidden doubling as one of the core histones. But, thanks to its prime positioning, a variation in the tail of H2AX enables rapid modification of the histone code in response to DNA damage. A role for H2AX phosphorylation has been demonstrated in DNA repair, cell cycle checkpoints, regulated gene recombination events, and tumor suppression. In this review, we summarize what we have learned about this marker of DNA breaks, and highlight some of the questions that remain to be elucidated about the physiological role of H2AX. We also suggest a model in which chromatin restructuring mediated by H2AX phosphorylation serves to concentrate DNA repair/signaling factors and/or tether DNA ends together, which could explain the pleotropic phenotypes observed in its absence.     

Liquid chromatographic assay for dicloxacillin in plasma

A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans.     

Optimal process synthesis for the production of multiple recombinant proteins

This paper presents a novel solution strategy for the synthesis of multiproduct and multihost protein production processes. There are several possible hosts that may express each of the products, and different downstream processing separation and purification tasks are needed, which in part depend on the host selection. Moreover, alternative unit operations may be available for some of these separation tasks. Finally, these processing units may be arranged in different configurations. A single mixed-integer optimization model represents the different decisions involved in synthesizing a plant for producing multiple proteins. The mathematical model optimizes the profit of the multiproduct plant and allows the decisions to be made simultaneously, namely, the choice of hosts, downstream operations, the configuration and size of units, as well as their scheduling. An example is solved for a plant that must produce four proteins for which there are alternative hosts for their expression (Escherichia coli, Chinese hamster ovary cells, and yeast that, depending on the product, may express it as an extracellular or intracellular protein) that require 15 stages with choices of unit operations as well as in or out of phase operations. Given the very large quantity of novel recombinant proteins for a number of novel therapeutic uses presently being approved or "in the pipeline", multiproduct and multihost recombinant protein production plants have recently been or are being built for the manufacture of these products. The strategy presented in this paper is of crucial value for the optimal utilization of such plants.     

Rate and pattern of mutation at microsatellite loci in maize

Microsatellites are important tools for plant breeding, genetics, and evolution, but few studies have analyzed their mutation pattern in plants. In this study, we estimated the mutation rate for 142 microsatellite loci in maize (Zea mays subsp. mays) in two different experiments of mutation accumulation. The mutation rate per generation was estimated to be 7.7 x 10(-4) for microsatellites with dinucleotide repeat motifs, with a 95% confidence interval from 5.2 x 10(-4) to 1.1 x 10(-3). For microsatellites with repeat motifs of more than 2 bp in length, no mutations were detected; so we could only estimate the upper 95% confidence limit of 5.1 x 10(-5) for the mutation rate. For dinucleotide repeat microsatellites, we also determined that the variance of change in the number of repeats (sigma(m)2) is 3.2. We sequenced 55 of the 73 observed mutations, and all mutations proved to be changes in the number of repeats in the microsatellite or in mononucleotide tracts flanking the microsatellite. There is a higher probability to mutate to an allele of larger size. There is heterogeneity in the mutation rate among dinucleotide microsatellites and a positive correlation between the number of repeats in the progenitor allele and the mutation rate. The microsatellite-based estimate of the effective population size of maize is more than an order of magnitude less than previously reported values based on nucleotide sequence variation.     

Telethonin protein expression in neuromuscular disorders

Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb-girdle muscular dystrophy type 2G (LGMD2G).We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, alpha-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. The possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression.     

Effects of a beta3-adrenergic agonist on glucose uptake and leptin expression and secretion in cultured adipocytes from lean and overweight (cafeteria) rats

The increase in body and white adipose tissue weights induced by a high-fat diet were prevented by treatment with the beta3-adrenergic agonist Trecadrine. Plasma insulin levels were slightly elevated in overweight rats, while a decrease was observed in Trecadrine-treated groups. Insulin-dependent glucose uptake was impaired in adipocytes of the overweight rats in relation to lean animals. The beta3-adrenergic agonist induced an increase in insulin-stimulated glucose uptake by adipocytes as compared to the nontreated animals. In fact, Trecadrine treatment was able to restore to control values the impairment in insulin-mediated glucose uptake induced by the cafeteria diet, suggesting that Trecadrine prevents the development of insulin resistance in overweight animals. Basal leptin secretion was increased in adipocytes of the overweight rats in relation to lean animals. Trecadrine treatment induced a decrease in basal leptin secretion compared to the untreated animals. Insulin-stimulated leptin secretion reached similar levels in adipocytes of the overweight rats as in lean animals. There was a trend for insulin-induced leptin secretion to be lower at 24 h in Trecadrine-treated rats, but it did not reach statistical significance. In conclusion, adipocytes of diet-induced overweight animals have a higher basal leptin secretion, which is reduced by treatment with Trecadrine. However, neither the cafeteria diet nor the Trecadrine treatment significantly alters the ability of adipocytes to increase leptin secretion in response to insulin.     

Molecular simulation of the interaction of κ-conotoxin-PVIIA with the Shaker potassium channel pore

Molecular simulation techniques were appplied to predict the interaction of the voltage-dependent Shaker potassium channel with the channel-blocking toxin kappa-conotoxin-PVIIA (PVIIA). A structural thee-dimensional model of the extracellular vestibule of the potassium channel was constructed based on structural homologies with the bacterial potassium channel Kcsa, whose structure has been solved by X-ray crystallography. The docking of the PVIIA molecule was obtained by a geometric recognition algorithm, yielding 100 possible conformations. A series of residue-residue distance restraints, predicted from mutation-cycle experiments, were used to select a small set of a plausible channel-toxin complex models among the resulting possible conformations. The four final conformations, with similar characteristics, can explain most of the single-point mutation experiments done with this system. The models of the Shaker-PVIIA interaction predict two clusters of amino acids, critical for the binding of the toxin to the channel. The first cluster is the amino acids R2, I3, Q6 and K7 that form the plug of the toxin that interacts with the entrance to the selectivity filter of the channel. The second cluster of residues, R22, F23, N24 and K25, interacts with a channel region near to the external entrance of the pore vestibule. The consistency of the obtained models and the experimental data indicate that the Shaker-PVIIA complex model is reasonable and can be used in further biological studies such as the rational design of blocking agents of potassium channels and the mutagenesis of both toxins and potassium channels.     

Pythium spp. present in irrigation water in the Poniente region of Almería (south-eastern Spain)

Pythium catenulatum, P. diclinum and P. paroecandrum, as well as asexual forms with filamentous sporangia were isolated from irrigation water in the Poniente region of Almería (south-eastern Spain). The taxonomic and morphological details of these fungal isolates are described. P. catenulatum and P. diclinumare new reports to Spain. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Monochromatic ultraviolet light induced damage to Photosystem II efficiency and carbon fixation in the marine diatom Thalassiosira pseudonana (3H)

Low light adapted cultures of the marine diatom Thalassiosira pseudonana (3H) were cultured and incubated for 30 min under different ultraviolet (UV) wavelengths of near monochromatic light with and without background photosynthetically active radiation (PAR, 380–700 nm). Maximum damage to the quantum yield for stable charge separations was found in the UVB (280-320 nm) wavelengths without background PAR light while the damage under PAR was 30% less. UV induced damage to carbon fixation in the cells was described by a function similar to non-linear functions of inhibiting irradiance previously published with the exception that damage was slightly higher in the UVA (320–380). Various measurements of fluorescent transients were measured and the results indicate localised damage most likely on the acceptor side of the Photosystem II reaction center. However, dark adapted measurements of fluorescence transients with and without DCMU do not result in similar functions. This is also true for the relationships between fluorescence transients and carbon fixation for this species of marine diatom. The correlation between the weightings _H from measurements of carbon fixation and the quantum yield for stable charge separation as calculated from induction curves with DCMU and without DCMU is R ² 0.44 and R ² 0.78, respectively. The slopes of the two measurements are 3.8 and 1.4, respectively. The strong correlation between the weightings of the induction curves without DCMU and carbon fixation are due to a loss of electron transport from the reaction center to plastoquinone. Under these experimental conditions of constant photon flux density (PFD) this is manifested as a strong linear relationship between the decrease in the operational quantum yield of Photosystem II and carbon fixation. This revised version was published online in June 2006 with corrections to the Cover Date.