Comparison of two combinations of cyanine dyes for prelabelling and gel electrophoresis

We report the use of IC‐OSu ethyl‐Cy3 and ethyl‐Cy5 N‐hydroxysuccinimide ester (NHS) cyanine dyes, which have similar chemical properties as the CyDye? DIGE fluor minimal dyes for pre‐electrophoresis labelling. Multiple sample analyses in different laboratories indicate that the use of IC‐OSu ethyl‐Cy3 and ethyl‐Cy5 NHS ester cyanine dyes produces equivalent results to those obtained with DIGE CyDyes, and allows sample multiplexing and accurate quantitation for differential proteome analysis.     

Identifying novel genes involved in both deer physiological and human pathological osteoporosis

Osteoporosis attacks 10% of the population worldwide. Humans or even the model animals of the disease cannot recover from porous bone. Regeneration in skeletal elements is the unique feature of our newly investigated osteoporosis model, the red deer (Cervus elaphus) stag. Cyclic physiological osteoporosis is a consequence of the annual antler cycle. This phenomenon raises the possibility to identify genes involved in the regulation of bone mineral density on the basis of comparative genomics between deer and human. We compare gene expression activity of osteoporotic and regenerating rib bone samples versus autumn dwell control in red deer by microarray hybridization. Identified genes were tested on human femoral bone tissue from non-osteoporotic controls and patients affected with age-related osteoporosis. Expression data were evaluated by Principal Components Analysis and Canonical Variates Analysis. Separation of patients into a normal and an affected group based on ten formerly known osteoporosis reference genes was significantly improved by expanding the data with newly identified genes. These genes include IGSF4, FABP3, FABP4, FKBP2, TIMP2, TMSB4X, TRIB, and members of the Wnt signaling. This study supports that extensive comparative genomic analyses, here deer and human, provide a novel approach to identify new targets for human diagnostics and therapy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Superoxide-mediated Formation of Tyrosine Hydroperoxides and Methionine Sulfoxide in Peptides through Radical Addition and Intramolecular Oxygen Transfer

The chemistry underlying superoxide toxicity is not fully understood. A potential mechanism for superoxide-mediated injury involves addition to tyrosyl radicals, to give peptide or protein hydroperoxides. The rate constant for the reaction of tyrosyl radicals with superoxide is higher than for dimerization, but the efficiency of superoxide addition to peptides depends on the position of the Tyr residue. We have examined the requirements for superoxide addition and structurally characterized the products for a range of tyrosyl peptides exposed to a peroxidase/ system. These included enkephalins as examples of the numerous proteins and physiological peptides with N-terminal tyrosines. The importance of amino groups in promoting hydroperoxide formation and effect of methionine residues on the reaction were investigated. When tyrosine was N-terminal, the major products were hydroperoxides that had undergone cyclization through conjugate addition of the terminal amine. With non-N-terminal tyrosine, electron transfer from to the peptide radical prevailed. Peptides containing methionine revealed a novel and efficient intramolecular oxygen transfer mechanism from an initial tyrosine hydroperoxide to give a dioxygenated derivative with one oxygen on the tyrosine and the other forming methionine sulfoxide. Exogenous amines promoted hydroperoxide formation on tyrosyl peptides lacking a terminal amine, without forming an adduct. These findings, plus the high hydroperoxide yields with N-terminal tyrosine, can be explained by a mechanism in which hydrogen bonding of to the amine increases is oxidizing potential and alters its reactivity. If this amine effect occurred more generally, it could increase the biological reactivity of and have major implications.Free radical-mediated oxidative damage occurs in numerous diseases and is thought to contribute to the aging process. The primary radical generated by the reduction of oxygen is superoxide (), a relatively benign radical that nevertheless must be removed by superoxide dismutases (SODs)² for an organism to survive in an aerobic environment (1). A number of potentially damaging reactions of have been identified (1–4). One of these, which has received relatively little attention, is the addition of to other radicals to form hydroperoxides (5, 6). This reaction has been shown to occur readily with tyrosine and Tyr-containing dipeptides, resulting in the formation of tyrosine hydroperoxides (5–7). Hydroperoxides are potentially damaging reactive oxygen species. Formation on proteins can result in detrimental structural and functional changes (8). Protein hydroperoxides are also oxidants that can injure other biomolecules.Tyrosyl radicals are generated in many physiological situations and proteins are major targets for reactive oxidants (9). In proteins exposed to free radicals, regardless of the initial site of attack, the resultant radical commonly localizes to Tyr (10–13). Tyrosyl radicals are also produced from tyrosyl peptides through the action of peroxidases such as myeloperoxidase, and are generated during the catalytic cycle of enzymes such as ribonucleotide reductase and cyclooxygenase (14). Tyrosyl radicals undergo a variety of subsequent reactions. They readily dimerize to form dityrosine, which has been well documented as a product of oxidative injury (15, 16). Another oxidative biomarker, nitrotyrosine, is also formed via tyrosyl radicals (4, 15, 17). However, one of their most favored reactions is with (5, 7, 18, 19). The reaction has a rate constant several times higher than that for dimerization (7, 20) and is favored over dityrosine formation in situations where both tyrosyl and radicals are generated (7, 20).The reaction of with phenoxyl radicals results in either repair of the parent phenol (reaction 2, Fig. 1b) or addition to form a hydroperoxide (reaction 3). With tyrosine, most of the reacts by addition (7, 20). The structure of tyrosine hydroperoxide has not been determined directly but inferred from NMR studies of the corresponding monoxide derivative formed by slow decomposition (7). These were shown to be bicyclic compounds formed by conjugate addition of the amino group to the phenol ring (HOHICA, designated I and named in full in Fig. 1b, proposed to arise from reactions 5 and 6).Open in a separate windowFIGURE 1.a, experimental system used for the generation of superoxide and tyrosyl (TyrO^·) radicals. b, proposed mechanism for the formation and decomposition of tyrosine hydroperoxide derivatives. R and R′ represent OH and H, respectively, for Tyr, or amino acid residue(s) for the peptides. Reaction 1 shows peroxidase-mediated formation of Tyr radicals, which can either dimerize (not shown) or react with by electron transfer (reaction 2) or addition (reaction 3). Addition results in the formation of hydroperoxides (o- and p-isomers, only the o-isomer shown) that may exist transiently and decompose to release ¹O₂ (reaction 4) or form a stable species that can undergo conjugate addition of the terminal amino group are shown (when R′= H, reaction 5). An equivalent reaction is proposed for non-N-terminal Tyr (R′= amino acid residue) in which conjugation involves the amide nitrogen. Hydrolysis of the hydroperoxides that are modified by conjugate addition gives the corresponding hydroxide derivatives (I, 3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indol-2-carboxylic acid or HOHICA) in reaction 6. c, possible alternative hydrolysis products (mono-oxygenated derivatives). II, 3,4-dihydroxyphenylalanine derivatives from the o-isomer; III, 4-alanyl-4-hydroxy-cyclohexadienone (HACHD) derivatives from the p-isomer.Hydroperoxide formation has been observed with peptides but only when tyrosine is N-terminal or the reaction is promoted by amino compounds (5). The amine effect has implications for hydroperoxide formation on proteins, but the mechanism is not understood. It has also been postulated that the repair mechanism involves singlet oxygen release from an intermediate (reaction 4) rather than electron transfer (reaction 2) (18), but this has not been studied experimentally.The objectives of this investigation were to determine the structures of the hydroperoxide and any other superoxide addition products, and to understand the mechanism of formation, using a range of synthetic and physiological tyrosyl peptides. These include the opioids Leu- and Met-Enkephalin (Leu-Enk, YGGFL; and Met-Enk, YGGFM, respectively) and Endomorphin 2 (Endo2, YPFF). The opioids have a free N-terminal Tyr that is essential for activity and are potential physiological targets for inactivation by addition. We also investigated whether the presence of a Met residue (as in Met-Enk) influences Tyr-hydroperoxide formation on the peptide and whether addition results in the formation of methionine sulfoxide. If so, this could be a physiological mechanism for production of methionine sulfoxide, which is one of the most prevalent products of oxidative stress (21, 22).Peptides were exposed to a xanthine oxidase (XO) system to generate and hydrogen peroxide (H₂O₂) plus horseradish peroxidase (HRP) to catalyze the reaction of H₂O₂ with the peptide to give the tyrosyl radical (Fig. 1a). Products were analyzed using a general hydroperoxide assay (Fe²⁺/xylenol orange or FOX assay) and by liquid chromatography/electrospray mass spectrometry (LC/MS). We have obtained structural information on the hydroperoxides, identified a mechanism of rapid intramolecular oxidation of Met residues via a hydroperoxide intermediate, and provide an explanation for why amino groups facilitate the addition of to the tyrosyl radical.     

Germline mutation in ATR in autosomal- dominant oropharyngeal cancer syndrome

ATR (ataxia telangiectasia and Rad3 related) is an essential regulator of genome integrity. It controls and coordinates DNA-replication origin firing, replication-fork stability, cell-cycle checkpoints, and DNA repair. Previously, autosomal-recessive loss-of-function mutations in ATR have been demonstrated in Seckel syndrome, a developmental disorder. Here, however, we report on a different kind of genetic disorder that is due to functionally compromised ATR activity, which translates into an autosomal-dominant inherited disease. The condition affects 24 individuals in a five-generation pedigree and comprises oropharyngeal cancer, skin telangiectases, and mild developmental anomalies of the hair, teeth, and nails. We mapped the disorder to a ～16.8 cM interval in chromosomal region 3q22-24, and by sequencing candidate genes, we found that ATR contained a heterozygous missense mutation (c.6431A>G [p.Gln2144Arg]) that segregated with the disease. The mutation occurs within the FAT (FRAP, ATM, and TRRAP) domain-which can activate p53-of ATR. The mutation did not lead to a reduction in ATR expression, but cultured fibroblasts showed lower p53 levels after activation of ATR with hydroxyurea than did normal control fibroblasts. Moreover, loss of heterozygosity for the ATR locus was noted in oropharyngeal-tumor tissue. Collectively, the clinicopathological and molecular findings point to a cancer syndrome and provide evidence implicating a germline mutation in ATR and susceptibility to malignancy in humans.     

Complete genome sequences of three Propionibacterium acnes isolates from the type IA(2) cluster

Propionibacterium acnes is an anaerobic Gram-positive bacterium that has been linked to a wide range of opportunistic human infections and conditions, most notably acne vulgaris (I. Kurokawa et al., Exp. Dermatol. 18:821-832, 2009). We now present the whole-genome sequences of three P. acnes strains from the type IA(2) cluster which were recovered from ophthalmic infections (A. McDowell et al., Microbiology 157:1990-2003, 2011).     

Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.     

The combined effect of environmental and host factors on the emergence of viral RNA recombinants

Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3'-phosphoadenosine-5'-phosphate pAp, inhibits the activity of the Xrn1p 5'-3' ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.     

Secondary metabolites from Calotropis procera (Aiton)

Three new metabolites, 5-hydroxy-3,7-dimethoxyflavone-4′-O-β-glucopyranoside (1), 2β,19-epoxy-3β,14β-dihydroxy-19-methoxy-5α-card-20(22)-enolide (4) and β-anhydroepidigitoxigenin-3β-O-glucopyranoside (5), along with two known compounds, uzarigenine (2) and β-anhydroepidigitoxigenin (3), were isolated from Calotropis procera (Asclepiadaceae). The structure elucidation was accomplished mainly by nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric methods. To examine putative antimicrobial or cytotoxic activities, various bioassays were performed. Uzarigenine (2) demonstrated moderate cytotoxicity.     

Spatio-temporal visual properties in the ascending tectofugal system

Our study compares the spatio-temporal visual receptive field properties of different subcortical stages of the ascending tectofugal visual system. Extracellular single-cell recordings were performed in the superficial (SCs) and intermediate (SCi) layers of the superior colliculus (SC), the suprageniculate nucleus (Sg) of the posterior thalamus and the caudate nucleus (CN) of halothane-anesthetized cats. Neuronal responses to drifting gratings of various spatial and temporal frequencies were recorded. The neurons of each structure responded optimally to low spatial and high temporal frequencies and displayed narrow spatial and temporal frequency tuning. The detailed statistical analysis revealed that according to its stimulus preferences the SCs has markedly different spatio-temporal properties from the homogeneous group formed by the SCi, Sg and CN. The SCs neurons preferred higher spatial and lower temporal frequencies and had broader spatial tuning than the other structures. In contrast to the SCs the visually active SCi, as well as the Sg and the CN neurons possessed consequently similar spatio-temporal preferences. These data support our hypothesis that the visually active SCi, Sg and CN neurons form a homogeneous neuronal population given a similar spatio-temporal frequency preference and a common function in processing of dynamic visual information.     

Mild Calorie Restriction Induces Fat Accumulation in Female C57BL/6J Mice

This study investigated the effects of mild calorie restriction (CR) (5%) on body weight, body composition, energy expenditure, feeding behavior, and locomotor activity in female C57BL/6J mice. Mice were subjected to a 5% reduction of food intake relative to baseline intake of ad libitum (AL) mice for 3 or 4 weeks. In experiment 1, body weight was monitored weekly and body composition (fat and lean mass) was determined at weeks 0, 2, and 4 by dual energy X‐ray absorptiometry. In experiment 2, body weight was measured every 3 days and body composition was determined by quantitative magnetic resonance weekly, and energy expenditure, feeding behavior, and locomotor activity were determined over 3 weeks in a metabolic chamber. At the end of both experiments, CR mice had greater fat mass (P < 0.01) and less lean mass (P < 0.01) compared with AL mice. Total energy expenditure (P < 0.05) and resting energy expenditure (P < 0.05) were significantly decreased in CR mice compared with AL mice over 3 weeks. CR mice ate significantly more food than AL mice immediately following daily food provisioning at 1600 hours (P < 0.01). These findings showed that mild CR caused increased fat mass, decreased lean mass and energy expenditure, and altered feeding behavior in female C57BL/6J mice. Locomotor activity or brown adipose tissue (BAT) thermogenic capacity did not appear to contribute to the decrease in energy expenditure. The increase in fat mass and decrease in lean mass may be a stress response to the uncertainty of food availability.