“
“The bed nucleus of the stria terminalis (BST) is a structure of the limbic system that is involved in behavioral, neuroendocrine and autonomic responses (Alves et al., 2007, Crestani et al., 2007, Crestani et al., 2008a and Dunn and Williams, 1995). Moreover, it has been proposed that the BST is an important center for the regulation of cardiovascular activity (Crestani et al., 2009a, Gelsema et al., 1987 and Ulrich-Lai and Herman, 2009). The electric stimulation of the BST has been reported to evoke pressor as well as depressor responses in anesthetized rats (Dunn and Williams, 1995).

Cardiovascular responses was also observed after chemical stimulation of the BST using either glutamate, d,l-homocysteic acid or noradrenaline (Ciriello and Janssen, 1993, Crestani et al., 2007, Gelsema et al., 1987, Gelsema et al., 1993 and Hatam and Nasimi, 2007). In addition, there is evidence that Epacadostat the BST tonically modulates cardiac baroreflex activity (Alves et al., 2009, Crestani et al., 2006, Crestani et al., 2008b, Li and Dampney, 1994 and McKitrick et al., 1992). Cholinergic synaptic terminals as well as muscarinic and nicotinic cholinergic receptors have been identified in the BST (Clarke et al., 1985, Ruggiero et al., 1990 and Wamsley et al., 1984), thus providing evidence of a cholinergic neurotransmission in the BST. We have previously reported that microinjection of carbachol, a cholinergic

agonist, into the BST of unanesthetized Hydroxychloroquine purchase rats caused an increase in arterial pressure that was followed by a baroreflex-mediated reduction of the heart rate (HR) (Alves et al., 2007). These responses were inhibited by systemic pretreatment with a V1-vasopressinergic receptor antagonist (Alves et al., 2007),

thus suggesting a mediation by acute vasopressin release into the systemic circulation. Moreover, cardiovascular responses to carbachol microinjection into the BST were mediated by activation of local M2-cholinergic receptors (Alves et al., 2007). These results Demeclocycline suggested the existence of a cholinergic mechanism in the BST that integrates cardiovascular and neuroendocrine control and could take part in fluid balance adjustments. However, the neural pathway involved in cardiovascular responses to carbachol microinjection into the BST is yet unknown. Vasopressin, also known as antidiuretic hormone, is a nonapeptide with a potent vasoconstrictor action (Altura and Altura, 1984 and Barer, 1961). This peptide is synthesized by magnocellular neurons located in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus and stored in the posterior hypophysis to further release into the systemic circulation (Swaab et al., 1975). Because cardiovascular responses following carbachol microinjection into the BST were shown to be mediated by an acute release of vasopressin into the systemic circulation (Alves et al.

2%. Whole Target Selective Inhibitor Library blood was centrifuged at room temperature (95 g, for 12 min) to obtain the platelet-rich plasma (PRP). Five hundred microliters of platelet-rich plasma (PRP) were added to 700 μl of washing buffer (140 mM NaCl, 0.5 mM KCl, 12 mM trisodium citrate, 10 mM glucose, 12.5 mM saccharose, pH 6) and again centrifuged (800 g, 12 min). Platelets were gently suspended in Krebs solution containing (mM) 118 NaCl, 25 NaHCO3, 1.2 KH2PO4, 1.7 MgSO4, 5.6 glucose (pH 7.4). Platelet number was adjusted to 1.2 × 108 platelets/ml in the presence of 1 mM CaCl2. Platelet aggregation was performed using an optical aggregometer (Chrono-log, Kordia Life Sciences, Leiden) at 37 °C with 400 μl of washed platelets placed

in glass cuvettes containing a disposable stir bar for constant stirring. Platelet aggregation was carried out in ADP (20 μM) and thrombin (0.05 U/mL)-stimulated platelets. Results were reported as mean ± SEM. The significance of differences among means was assessed by analysis of variance followed by ANOVA test, when several experimental groups were compared with the control click here group. Differences were considered statistically significant if p < 0.05. Four peaks were obtained after crude venom fractionation on the Sephadex G75 gel filtration column (Fig. 1A). All fractions were tested for the presence of PLA2 activity. Peak 3 (FIII) displayed high PLA2 activity. The

proteins contained in this chromatographic peak were further purified using reverse phase-HPLC

performed on a C5 column (Fig. 1B). All eluted peaks were manually collected, Decitabine ic50 lyophilized and screened for PLA2 activity. The main fraction, labeled as LmrTX, had PLA2 activity. Analysis by ESI-MS of the intact protein indicated a molecular mass of the purified protein of 14277.50 Da (Fig. 2). Mass spectrometric analysis was performed in order to obtain a molecular identification and homology study. Digestion of the protein (LmrTX) with trypsin, followed by LC/MS/MS, identified ten peptides. The deduced sequence and measured masses of alkylated peptides of LmrTX are summarized in Table 1. The sequence of each peptide was then submitted separately to the SNAKE database using the protein search program BLAST-p. Using the position matches of the ‘de novo’ sequenced peptides with homologous proteins present in the database, it was possible to deduce their original position on the unknown protein LmrTX. Fig. 3 shows the result of BLAST alignment between LmrTX with the phospholipase A2 from Crotalus durissus terrificus, L. muta muta and L. stenophrys. Amino acid analysis revealed the following composition of LmrTX PLA2: Asx/9, Glx/7, Ser/6, Gly/11, His/2, Arg/9, Thr/8, Ala/5, Pro/5, Tyr/11, Val/2, Met/2, Cys/14, Ile/5, Leu/6, Phe/6 and Lys/11. LmrTX showed a high content of Lys and Arg residues typical of a basic PLA2 protein (data not show).

Whether OFC is able to select the appropriate task structure or just applies this information computed by other frontal cortical regions

see more is not yet known; as is shown in Figure 1B, encoding of decision type predominated across multiple regions of frontal cortex and was not unique to OFC. What is evident is that OFC can utilise information about task structure to promote rapid contingent learning. Unlike research into OFC function, evidence for the role of VMPFC in value-guided decision making has to date been largely driven by human studies. The BOLD signal in this region has often been shown to correlate with the current subjective value of various different types of options

33, 34 and 35]. This holds true even in the case where the particular item has never previously been directly experienced [36]. However, as with the OFC, the functional role of VMPFC value signals remains disputed. Representations of decision value are evident in many brain regions [37], thus an important question is to identify a neural signature of a decision. A version of p38 MAPK inhibitor a biophysically plausible attractor network model of a binary probabilistic choice process [38] suggests decision inputs (values) are initially summed, and then compete via mutual inhibition, producing a later, second signal reflecting the difference in value between the chosen and unchosen options [39••]. Critically, VMPFC activity contained both such signatures in the correct timeframe [39••]. In fact, in many situations when two choice options are presented, the BOLD signal in this region not only correlates positively with the subjective value of a chosen, attended

option, but also negatively with the value Benzatropine of the next best, but rejected option 40, 41 and 42]. Recently, Strait and colleagues have reported comparable antagonistic effects between the values of two sequentially presented options in area 14 in macaques [43•]. Together, this evidence points towards an important role for VMPFC in a competitive value comparison necessary for decision making 3 and 39••]. Nonetheless, while VMPFC activation is common to a range of studies (outside the domain of decision making as well as within), it is not a signature of all decisions and is instead critically dependent on the local context. For instance, VMPFC value comparison signals are not observed when selecting whether to take an available option or to forego this to search for something better in the environment; only when a decision is made to engage with the current option does the VMPFC BOLD signal represent the value of this chosen item [44].

4A and B). To further test the biological activity of the recombinant PnTx3-4 we investigated its effect on blocking Ca2+ channels involved in glutamate release from cortical synaptosomes. To do that, we measured changes in cytosolic Ca2+ in fura-2-loaded synaptosomes (Prado et al., 1996). Synaptosomes depolarized with 33 mM KCl in the presence of 1 mM CaCl2 showed a fast increase in internal calcium concentration

(Fig. 4C). Addition of 16 nM of native PnTx3-4 6 min before KCl depolarization inhibited internal Ca2+ increase by approximately 30%. Addition of similar concentration of the recombinant PnTx3-4 peptide to the preparation GSK3235025 mw also blocked Ca2+ channels, however, the inhibition of internal Ca2+ increase observed was smaller (approximately 20% inhibition). Because the 6xHis-SUMO-PnTx3-4 fusion protein showed to be highly expressed as inclusion

bodies (Fig. 3, lane 2), we chose to improve our purification yield by purifying it from the pellet. To do that, recombinant 6xHis-SUMO-PnTx3-4 present in the pellet was first solubilised in 6 M of Guanidine-HCl (Fig. 5A) and then purified by affinity chromatography Trametinib using a Ni-NTA agarose resin. After removal of the imidazole by dialysis, the N-terminal tag was cleaved off by digestion with SUMO protease I (Fig. 5B, lane 2). The recombinant toxin was purified by RP-HPLC and two peaks with retention times of about 32 and 41 min respectively were observed (Fig. 5D and E). The peak with 32 min retention time Methocarbamol presented one band of 8 kDa that could be recognized by a polyclonal antibody raised against the spider venom (Fig. 5C, lane 1 and 2). This peptide presented no biological activity when tested in the glutamate release assay (Fig. 4D and E) indicating that the peptide was not properly folded. Our next step was to determine the optimized condition necessary to obtain reliably refolded, biologically active PnTx3-4. To do that, we incubated the recombinant PnTx3-4 in a strong denaturing buffer (6 M Gnd-HCl,

50 mM Tris, 10 mM DTT, pH 8.0) to completely unfold the protein. After 4 h of incubation at RT, DTT was removed by filtration (VIVASPIN 6 column; 3 kDa MWCO). The toxin was then diluted into a refolding buffer to a final concentration of 0.1–0.2 mg/mL. Nine different refolding buffers were tested (Table 3), ranging from strong to weak denaturing conditions. Refolding was allowed to proceed for 24 h at 4 °C, samples were submitted to RP-HPLC and tested. We estimated refolding yields by measuring biological activity using the glutamate release assay as described for experiments in Fig. 4; that is, 16 nM of each refolded peptide was added to mouse cortical synaptosomes prior to depolarization with 33 mM KCl in the presence of 1 mM CaCl2 and total glutamate release was measured (Fig. 5F). As our experiments consistently showed that 16 nM of native PnTx3-4 or Ca2+ removal from the medium (by adding 2.

Supplemental XRT was delivered at two dose levels (20 and 44–50.4 Gy)

using a three-dimensional conformal technique. BMS-354825 molecular weight The planning target volume was inclusive of the prostate and proximal seminal vesicles plus margin. In patients with pelvic lymph node risk >10%, this volume was also inclusive of the pelvic nodal basins extending superiorly to the L5–S1 interspace (5). Among patients receiving XRT, 238 received 20 Gy and 427 received doses in the range of 44–50.4 Gy. In this same group, 452 patients were treated to the prostate only and 213 to the whole pelvis. For patients receiving 44–50.4 Gy of XRT, the mPD was 90 Gy (National Institute of Standards and Technologies 99) for 103Pd and 110 Gy (TG-43) for 125I. In those receiving 20 Gy of XRT, the boost was always delivered using 103Pd with an mPD of 115 Gy. Androgen deprivation therapy (ADT) was administered for potential pubic arch interference or adverse disease features. Two hundred seventy-five patients (29.5%) received ADT. This included 167 patients (17.9%) receiving 6 months or less of a leutinizing hormone–releasing

hormone agonist for prostate gland cytoreduction and 108 patients (11.6%) receiving >6 months of a leutinizing hormone–releasing hormone agonist and an oral antiandrogen for adverse pathologic features. In patients receiving ADT, 25 received implant alone and 250 received implant in conjunction with XRT. After brachytherapy, patients were monitored by digital Olaparib ic50 rectal examination and serial PSA measurement at 6-month intervals. The primary end points of this analysis were bPFS, CSS, and OS. Biochemical control was defined as a PSA ≤0.40 ng/mL after nadir (13). Patients dying with either metastatic prostate cancer or castrate-resistant disease in the absence of metastases were classified as experiencing a prostate cancer–related death. Continuous and categorical variables of 6-phosphogluconolactonase interest were

compared using an independent t test and chi-squared analysis, respectively. Comparisons in bPFS, CSS, and OS between the two study cohorts were done using the Kaplan–Meier method. Univariate Cox regression analysis was used to identify predictors of treatment outcome. Those variables with p-value <0.10 were then entered into a multivariate forward conditional Cox regression. Statistical analysis was performed with SPSS v. 13.0 software (SPSS Inc., Chicago, IL). With a median followup of 7.4 years, the 10- and 14-year bPFS, CSS, and OS for the entire Gleason 7 study group were 95.7/95.7%, 98.6/98.6%, and 77.2/64.3%, respectively. Compared with primary Gleason pattern 3, the Gleason pattern 4 patients had a statistically higher pretreatment PSA and percentage of positive biopsy cores (PPCs) (Table 1). The Gleason pattern 4 patients also received XRT more frequently and had a higher incidence and average duration of ADT use.

In selleck screening library secondary endosymbiosis, a red alga or a green

alga was engulfed by a non-photosynthetic protist (Green, 2011 and Reyes-Prieto et al., 2007). Chloroplasts of algae belonging to the heterokonts, which include diatoms, brown algae, raphidophytes and heterotrophic oomycetes, arose from a secondary endosymbiosis event including a red alga. Recent results indicate that the red algal endosymbiont succeeded a green algal endosymbiont related to prasinophytes, as a large number of nuclear genes in diatom genomes have a green algal origin (Jiroutová et al., 2010 and Moustafa et al., 2009). However, this finding is controversial, and has been the subject of criticism for taxonomic sampling bias (Burki et al., 2012 and Deschamps and Moreira, 2012). In addition to the large contribution of genetic material to algal genomes through endosymbiosis (endosymbiotic gene transfer, EGT), several genes have been introduced to nuclear and organelle

genomes independently through horizontal gene transfer (HGT) events. The nuclear genomes selleck chemicals llc of the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum contain several hundred genes that appear to have been acquired from a wide range of bacteria through HGT ( Armbrust et al., 2004 and Bowler et al., 2008). Diatoms (Bacillariophyta) constitute one of the most abundant groups of marine phytoplankton, with an estimated diversity of around 100 000 species (Round et al., 1990 and Van den Hoek et al., 1995). The evolutionary success of diatoms is also reflected in their ecological importance; this group contributes approximately 40% to primary net production in the oceans (Field et al., 1998). This success is suggested to be caused at least in part by the ability of diatoms to respond and adapt to large fluctuations in light irradiance, thereby maintaining high photosynthetic efficiency over a wide range

of environmental conditions (Depauw et al., 2012). Thus far, the chloroplast genome has been sequenced in five diatoms: the centrics Adenylyl cyclase Odontella sinensis and T. pseudonana, and the pennates P. tricornutum, Fistulifera sp. JPCC DA0580 and Synedra acus ( Galachyants et al., 2012, Kowallik et al., 1995, Oudot-Le Secq et al., 2007 and Tanaka et al., 2011). In addition, the chloroplast genomes of the diatom endosymbiont of two dinoflagellates, Durinskia baltica and Kryptoperidinium foliaceum, have also been characterised ( Imanian et al., 2010). These genomes share a highly similar gene set, of which a core set of 86 genes is found in all chromalveolates ( Green, 2011). Two plasmids identified in the pennate diatom Cylindrotheca fusiformis may be associated with chloroplasts, as they hybridise with chloroplast DNA ( Hildebrand et al., 1992 and Jacobs et al., 1992). In support of this view, genes encoding putative proteins with similarity to ORFs found in the C.

Egg yolk (20%; v/v) was added to the each extender and mixed by placing the tube to an orbital shaker for 10 min and centrifuged at 15,000g for 60 min, and the supernatant was filtered through a 0.45 μm membrane filter. Egg yolk phospholipids were then solubilized by adding 0.75% (v/v) Equex-Paste (Minitüb, Tiefenbach, Germany) to the extender. Sperm samples (100 μl) from SD or F344 were transferred into 1.5 ml centrifuge tubes containing 400 μl of each freezing extender and gently mixed by inverting the tube. After dilution, motility analysis was performed

using a phase Obeticholic Acid order contrast microscopy equipped with 20× objective. The sperm samples were then equilibrated at 4 °C for 45 min. After equilibration in the extenders, 150 μl sperm sample from each extender was placed onto a shallow quartz dish (14 mm inner diameter and 2.56 mm deep) and covered with a round coverslip and then inserted into Linkam cryostage (TMS-94) that was mounted on a Nikon microscope.

The ATR cancer samples were then cooled by using various cooling rates (10, 40, 70 and 100 °C/min) to final temperature of −150 °C. For thawing, the quartz dish containing the sperm samples was rapidly removed from the Linkam cryostage and placed on a 37 °C slide warmer in order to have direct contact with the warm surface to achieve about 1000 °C/min warming rate. After warming, motility analysis was performed and the samples were transferred into 1.5 mL Eppendorf tubes containing 150 μL TL-HEPES base solution. All samples were underwent mitochondrial, acrosome and membrane integrity assessment. SYBR-14/Propidium iodide (Live/Dead sperm viability kit, catalog no: L-7011, Molecular Probes, Eugene, OR, USA)

and Alexa Fluor-488-PNA (catalog no: L-21,409, Molecular Probes, Eugene, OR, USA) conjugate were used to determine rat sperm plasma membrane and acrosome integrity, respectively. For plasma membrane integrity, 200 μl TL-HEPES Dipeptidyl peptidase solution was gently added to the tube containing 100 μl thawed sperm (1–2 × 106 spermatozoa/ml). Diluted sperm samples were incubated with 5 μL PI (0.5 μM final concentration) and 10 μL (0.4 μM final concentration) SYBR-14 at 37 °C for 10 min. After staining, 10 μl of sperm sample was placed on a microscope slide, covered with a coverslip and observed under the epifluorescence microscope (Nikon Eclipse 600 using a dual fluorescence filter). The images of stained sperm samples were classified into two groups: sperm head displaying green fluorescence was considered to be membrane intact, whereas sperm displaying red fluorescence in the head was considered to be damaged membrane. 100 sperm per sample were counted as described previously [52]. To evaluate sperm acrosomal integrity, thawed sperm samples were washed to remove freezing extender.

The straws were plunged into liquid N2 for storage. After 1 month, samples were transported to the Integrated Center for Biotechnology (NIB/UECE – Fortaleza, CE, Brazil) for thawing and further analysis. The straws were removed from the liquid nitrogen and randomly thawed on a water bath at 37 °C/1 min 7 days after freezing. Finally, straws were removed, dried, the plug cut off and the contents pushed out into a glass vial that stood in a water bath at 37 °C. Semen samples (two straws per treatment) were immediately evaluated for sperm progressive motility,

morphology and membrane integrity. Thawed semen Metformin nmr was also evaluated by CASA in accordance with previous recommendations. Briefly, a 10 μL aliquot of semen sample was placed on a pre-warmed Makler counting chamber (Sefi Medical Instruments Ltd., Haifa, Israel), allowed to settle for 1 min, maintained at 37 °C and examined in a phase-contrast microcopy system (Olympus BH-2, Tokyo, Japan), with stroboscopic illumination

coupled to a video camera adapted to the www.selleckchem.com/products/Dasatinib.html Sperm Class Analyzer (SCA version 3.2.0; Microptic S.L., Barcelona, Spain). The settings of the instrument were temperature, 37 °C; frame rate, 25 frames/s; minimum contrast, 75; straightness threshold, 80%; low velocity average pathway (VAP) cutoff, 10; and medium VAP cutoff, 45. Three nonconsecutive randomly selected microscopic fields were scanned. The parameters analyzed were number of counted HSP90 cells, total motility (%), progressive motility (%), velocity average pathway (VAP; μm/s), velocity straight line (VSL; μm/s), curvilinear velocity (VCL; μm/s), amplitude of lateral head (ALH; μm), beat cross frequency (BCF; Hz), straightness (STR; %), and linearity (LIN; %) [12]. Twenty-one replicates were performed for each treatment. The results were expressed as mean ± SEM. Data were checked for normality by Shapiro–Wilk test, and for homoscedasticity by Levene’s test using the univariate procedure of the Statistical Analysis System (SAS 6.10,

SAS Institute Inc., Cary, NC, USA). Data were analyzed by General Liner Model (GLM). Comparisons among different cryoprotectants on seminal parameters were analyzed by Tukey test. To evaluate the individual effect of the animals and its interactions with cryoprotectants effect on studied variables, data were evaluated by Fisher’s PSLD test. For all statistical analysis, a significant difference of 5% was considered. Fresh goat semen was yellowish in color and milky in aspect. Total volume of ejaculates was 1.1 ± 0.1 mL, with a sperm concentration of 2.4 ± 0.2 × 109 spermatozoa/mL. Sperm progressive motility of fresh semen was 95.0 ± 2.0%, and mass activity was 3.9 ± 0.2. Percentage of sperm presenting intact membrane was 90.7 ± 3.5% and sperm with normal morphology was 76.1 ± 1.7%, being 33.0 ± 1.8% with functional membrane integrity. Total morphological defects were found in 23.9 ± 1.7%, being 0.6 ± 0.2% classified as primary and 23.

A sépsis é uma síndrome clínica que decorre da ativação de uma resposta inflamatória sistémica desencadeada pela infeção, com consequente lesão tecidular generalizada. Doenças não infeciosas, como a pancreatite aguda, também podem associar-se a um quadro deste tipo, denominado síndrome de resposta inflamatória sistémica (SIRS). A coexistência de SIRS e de infeção é definida como sépsis. A gravidade da

sépsis é estabelecida mediante a existência de disfunção de órgãos e de compromisso hemodinâmico. Daqui surgiram os conceitos de sépsis grave e C59 wnt solubility dmso de choque séptico para designar as situações de sépsis que cursem com sinais de disfunção orgânica e hipoperfusão tecidular persistente, respetivamente 5. Na realidade sépsis, sépsis grave e choque séptico representam um contínuo de gravidade que culmina na falência múltipla de órgãos, tratando-se de um processo dinâmico e que pode evoluir

rapidamente para as formas mais graves 2 and 6. Os princípios de abordagem do doente séptico assentam no reconhecimento de que a adequada ressuscitação nas primeiras horas permite reduzir a mortalidade de forma significativa. Os principais pilares desta abordagem são a precocidade do diagnóstico e a rapidez e eficácia das Enzalutamide mouse intervenções terapêuticas instituídas, consistindo fundamentalmente no suporte das funções vitais e no controlo do foco infecioso. Esta estratégia foi subscrita por várias organizações

médicas e mereceu consenso internacional, dando origem a uma campanha à escala global denominada Surviving Sepsis Campaign (SSC) 3, 4, 7 and 8. Esta campanha serviu de mote à instituição de protocolos de atuação a nível local em diversas instituições hospitalares e à organização dos serviços e treino dos profissionais de saúde para atuação neste contexto. A implementação destas medidas demonstrou um impacto positivo nas taxas de mortalidade observadas 9 and 10. Apesar de a sépsis ser um problema transversal em medicina e do interesse crescente da comunidade médica nesta área, a sua real prevalência Tau-protein kinase e o seu impacto na prática clínica diária permanecem muitas vezes subestimados. Este estudo teve como objetivos avaliar o impacto da sépsis num serviço de gastrenterologia e, simultaneamente, determinar se a abordagem inicial a estes doentes foi a mais adequada, à luz das recomendações vigentes. Foi efetuado um estudo retrospetivo, abrangendo todos os internamentos urgentes ocorridos num serviço de gastrenterologia, durante o período de um ano (de setembro de 2009 a agosto de 2010). O estudo decorreu num hospital terciário, universitário, que integra um serviço de urgência (SU) polivalente. Os doentes admitidos no SU são encaminhados para a urgência geral ou para as diversas especialidades de acordo com a triagem inicial efetuada por enfermeiro, segundo o sistema de Manchester.

These data indicate that epigenetic inheritance of modified Selleck Ganetespib histones may proceed via more than one pathway. Another example of templating comes from Drosophila, in which the centromeric histone variant CID

derived from the sperm is used to template CID deposition at the centromere during embryogenesis [ 34•]. While fertilization can occur with sperm that lack CID, the embryos do not develop normally, and paternal chromosomes lose the ability to recruit maternal CID and re-establish functional centromeres. Thus CID deposition during embryogenesis also appears to depend on a templating mechanism, although it is unclear whether it proceeds via direct or indirect recruitment. Interestingly, several epigenetic marks on the H3 histones appear to be important for proper recycling of old histones to the newly replicated DNA, and these marks have been shown to change under conditions of replication stress [ 35]. However, the mechanism by which nucleosome inheritance is regulated still remains unexplored. Investigations CSF-1R inhibitor into the influence of transcription rate, histone availability, and timing of replication may all provide important insights into how histones provide the genome with a molecular memory. The ability of chromatin to protect DNA from ionizing radiation was established in a seminal study over 20 years ago. When DNA was completely

stripped of its nucleosomes Pyruvate dehydrogenase and exposed to 20 Gy of gamma-radiation, the occurrence of double strand breaks (DSBs) was 10 times greater than that of intact cells [36]. However the discovery that histone variants are intimately tied to proper DNA damage response (DDR) progression is relatively recent. In particular, work has focused on the role played by variants of the H2A family: (γ)H2A.X, H2A.Z and macroH2A. While the localized phosphorylation of H2A.X has been

implicated in the response to DSBs for some time, it is only recently that the behavior of H2A.X in response to clustered DNA lesions has been elucidated. Interestingly, when clustered DSBs were induced by ionizing radiation in skin fibroblasts, H2A.X phosphorylation, monitored by immunostaining, was not limited to the region directly surrounding the break, but occurred throughout the genome in a dose dependent manner [37]. This response, catalyzed by two kinases, ATM and DNA-PK, was transient and not linked to apoptosis. Recently, using ChIP at a defined DSB, a second H2A variant usually involved in transcriptional regulation, H2A.Z, was found at the break site [38]. H2A.Z is deposited at the DSB by the ATP-dependent chromatin remodeler p400, and is thought to re-organize the chromatin surrounding the DSB into a more fluid conformation by promoting H4 acetylation (Figure 3).