Dictyostelium, chemoattractant gradients generate directed cell migration by eliciting signaling events that bias intrinsic motility and favor the production and retention of upgradient pseudopods [3 gradients within the plasma membrane [3 signaling is nonessential for gradient sensing [2 is little understood, despite its clear importance in many cell biological processes [2 and is essential for efficient chemotaxis of Dictyostelium cells. Without PikI, PIP2 levels are reduced by 90%, and while pikI− cells move at normal speeds, they are highly disorientated in cAMP gradients. Following chemotactic stimulation, Ras is efficiently activated in pikI− cells, yet Ras-dependent responses (including activation of PKB) are severely impaired. PikI is phosphorylated by PKB [2 levels are required to couple activated Ras to its downstream effectors and that these levels are regulated by PikI, making it a crucial player in gradient sensing.In neutrophils and eudopods . Phosphomembrane . Althoug sensing , the rolrocesses . We descd by PKB , and in   •pikI− cells, but cell speed is unaffectedGradient sensing is severely impaired in •Dictyostelium and produces 90% of cellular PIP2PikI is the major PIP5K in •PikI is required for active Ras to couple to downstream effectors such as TorC2•Phosphorylation by PKB (downstream of Ras) may increase PikI activity 2, is vital for chemotaxis in Dictyostelium cells. Without PikI, cells are able to move at normal speeds, but gradient sensing is dramatically attenuated as a result of uncoupling of activated Ras from its downstream effectors.Fets et al. show that PikI, a PIP5 kinase that generates PIP To investigate this, we created a strain in which this gene is deleted from the wild-type strain, Ax2 . The pikI− strain has a severe growth defect in liquid medium and impaired phagocytosis  that is strongly phosphorylated by PKBA and PKBR1 within seconds of stimulation with the chemoattractant cAMP  suggesteocytosis D–S1F. Deacterial E and nonpikI− cells toward cAMP was analyzed using a Dunn chamber [pikI− cells is indistinguishable from wild-type  and phospholipase C (PLC), both of which are active in chemotactic signaling. To address whether the pikI− phenotype results from reduced signaling via these enzymes, we knocked out plcA in the pi3k1−5− strain [pi3k1−5− and the pi3k1−5−,plcA− strains was found to be as efficient as wild-type . This suggests that disruption of these pathways cannot explain the severity of the pikI− phenotype and that PIP2 is playing another, more important role in gradient sensing.PikI is predicted to produce PI4,5)PP2, the sild-type A and 1B.-type . We found that expression of the developmentally regulated gene contact sites A (csaA) was normal in pikI− cells , we found that phosphorylation of Erk2 is normal in pikI− cells P2 production. During short-term labeling, the rate of PIP2 labeling was dramatically reduced in pikI− cells, whereas bulk phospholipid labeling was unaffected (2 in pikI− cells are 10-fold lower than in wild-type cells (2 synthesis. This supports RNA sequencing expression data showing that PikI is highly expressed throughout development and is the most highly expressed PIP5K during vegetative growth (data from http://dictyexpress.biolab.si/). None of the other labeled phospholipids were significantly altered Pactivity . cAMP-innaltered F. Attenu defects . We therpikI− strain, cAMP-induced substrate phosphorylation is almost entirely lost, with basal substrate phosphorylation also being very low , mediated by the TorC2 complex , and theI− cells C and 3D.d by Ras , we nextThe two main Ras GTPases involved in chemotaxis are RasC and RasG . RasC li2 levels are low in PikI null cells, this implies that Ras coupling to its downstream effectors is dependent on PIP2. Small G proteins containing polybasic clusters of amino acids bind specifically to the plasma membrane using both lipid anchors and electrostatic interactions with anionic lipids [2 levels might weaken electrostatic interactions, resulting in mislocalization of Ras and, consequently, uncoupling of activated Ras from its effectors. Cell fractionation was used to examine levels of membrane-bound Ras in resting cells and showed that whereas cytoplasmic levels of Ras are increased in pikI− cells, levels of membrane-bound Ras are comparable with wild-type Pc lipids . Low PIP in the membrane [2 may play a more specific role in regulating Ras activity, independent of any role in localizing Ras to the plasma membrane, perhaps by directly affecting the ability of Ras to modulate the activity of its effectors through conformational changes.These results are consistent with recent evidence that many proteins that bind to PIPmembrane . It therT262A) and phosphomimetic (PikIT262E) point mutants and examined their enzymatic properties. It is apparent that whereas PikIT262A is indistinguishable from PikIWT, PikIT262E is significantly more active, with an almost 3-fold increase in Vmax P2 levels during cAMP signaling by in vivo labeling with 32Pi (unpublished data), localized changes in PIP2 levels could occur, as has been suggested in other systems [3 production P2 consumption) can be sharply localized to the leading edge during chemotaxis, and PLC activity may be similarly localized [2. Conversely, PKBR1 is selectively activated at the leading edge [2 levels in regions of high PI3K and PLC activity and providing a feedback loop governing the strength of chemotactic signaling.We have shown that extreme depletion of PIP systems . PIP3 signaling may be dispensable for chemotactic gradient sensing, maintenance of PIP2 is vital.Although PIPeviously , our wor


Drosophila and mouse. Drosophila express a single Homer gene product that is upregulated during sleep. By contrast, vertebrates express Homer as both constitutive and immediate early gene (H1a) forms, and H1a is up-regulated during wakefulness. Genetic deletion of Homer in Drosophila results in fragmented sleep and in failure to sustain long bouts of sleep, even under increased sleep drive. However, deletion of Homer1a in mouse results in failure to sustain long bouts of wakefulness. Further evidence for the role of Homer1a in the maintenance of wake comes from the CREB alpha delta mutant mouse, which displays a reduced wake phenotype similar to the Homer1a knockout and fails to up-regulate Homer1a upon sleep loss. Homer1a is a gene whose expression is induced by CREB. Sustained behaviors of the sleep/wake cycle are created by molecular pathways that are distinct from those for arousal or short bouts, and implicate an evolutionarily-conserved role for Homer in sustaining these behaviors.Sleep is an evolutionarily conserved process that is linked to diurnal cycles and normal daytime wakefulness. Healthy sleep and wakefulness are integral to a healthy lifestyle; this occurs when an organism is able to maintain long bouts of both sleep and wake. Homer proteins, which function as adaptors for group 1 metabotropic glutamate receptors, have been implicated in genetic studies of sleep in both  Sleep is essential for all organisms that exhibit diurnal attention. A current model of sleep/wake control proposes that sleep and wake are created by a “flip-flop” involving reciprocal inhibition between sleep-active versus wake active neural pathways that project broadly to the neocortex and subcortical areas While this model has been successful, it is not sufficient to explain sleep/wake control. First, targeted lesions of wake/active neuronal groups that would be expected to change the amounts of sleep and wake do not do so 2+ signaling. The vertebrate genome includes three Homer genes . Conventional Homer proteins are bipartite, consisting of an N-terminal Enabled/Vasp homology 1 (EVH1) target-binding domain and a C-terminal coiled-coil (CC) domain that mediates self-association. Homer1a and Ania 3 are immediate early gene (IEG) forms that lack the CC domain and function as dominant negatives to disrupt Homer cross-linking homer gene that encodes a cross-linking Homer protein but no Homer-1a homologue Homer proteins function as molecular adaptors that bind to a specific proline-rich sequence in the C-terminus of Group I metabotropic glutamate receptors and other proteins that play a role in Cahomer transcript in adult female Drosophila brains is down regulated with sleep deprivation and increased with sleep The R102homer mutant flies (n = 120) by electronic beam breaks  under 12∶12 light-dark conditions and compared the activity pattern to that of the CS (n = 102) background strain. As DAMS analysis can overestimate the amount of daytime sleep and bout durations of state, both during the day and at night, we also used video recording under the same conditions R102 flies have less sleep compared to controls both by DAMS and video (p<0.0001). The average bout duration of sleep is much reduced in the HomerR102 compared to wildtype flies during the lights on (p<0.0001) and lights off (p<0.001) periods (R102flies (p<0.001) (R102 compared to wildtype both during the day and at night (We assessed sleep-wake activity in  periods . By videp<0.001) . This dip<0.001) . Using 5p<0.001) . This efat night . The difR102 mutant flies is due to deletion of the homer gene, we crossed the HomerR102 flies with a deficiency line Df(2L)ED7007 which lacks a small region of the left arm of chromosome 2 (bands 27A1–27C7) that includes homer (located on 27A1). The homer/Df (2L) ED7007 hemizygotes (n = 10) displayed sleep behavior similar to the homer homozygotes (n = 16). In all sleep wake parameters R102/Df(2L)ED7007homer is not significantly different from R102/homerR102homer but is significantly different from CS/Df(2L)ED7007  and R102homer/CS   . ExpressR102 flies (n = 16) indicate that they are rhythmic under conditions of constant darkness. No significant difference was found in clock period between the Homer null  and the wildtype CS (23.8±0.1 hr).Examination of the circadian clock of HomerR102homer flies, there was continued rebound sleep over the entire 24 hours after the end of deprivation, while in wildtype CS flies the recovery was relatively short . The immediate response in the first 4 hours after the end of deprivation was similar between genotypes. However, in ly short  There waR102 flies showed no increase in average sleep bout duration following 4 or 6 hours of sleep deprivation, but rather, increase their sleep by increasing the number of sleep bouts. Thus, HomerR102 flies are not able to maintain long sleep bouts even under increased sleep drive.Another behavioral response to sleep deprivation is an increase in sleep bout duration We used Homer1a KO, Homer1a heterozygous and wildtype littermate controls as the best available mouse model to study the role of Homer1a in sleep/wake behavior. H1a KO mice show a selective loss of IEG forms of Homer without changes in Homer 1c expression or expression of other synaptic proteins, and are healthy, fertile and robust Analysis of the wake data over 24 hours indicated an overall main effect of genotype . Consistent with the reduced wakefulness during the lights off period, the average wake bout during the dark active period in the Homer1a KO mice is significantly shorter than in either the wildtype or the heterozygote . There aA more formal test of the hypothesis that wake bouts for H1a differ from those of wildtype and heterozygote is given by QQ probability plots as shown for WT and H1a in In mice the distribution of sleep and wake bouts is not normal; there is a mixture of short and long bouts of each state There is no significant difference in the homeostatic response to sleep deprivation as measured by the increase in delta power following 6 hours of sleep deprivation between Homer1a KO, Homer1a heterozygote and wildtype controls . There wExamination of the circadian phenotype by wheel running indicates that the Homer1a KO mice, like the wildtype littermate controls, displayed a circadian rhythm during constant dark conditions . A 1-wayMice with very low levels of CREB due to deletion of the alpha and delta isoforms of CREB also have reduced wakefulness during the early part of their nighttime active period This study indicates that Homer proteins play a role in sustaining sleep/wake behavioral states in Drosophila and mice that are distinct from the predictions of the “flip-flop” model. In Drosophila, lack of Homer leads to alteration in the ability to sustain both sleep and wakefulness but the effect on sleep consolidation is greater. The Homer null flies have no alteration in clock period but do have an altered response to sleep deprivation. Their initial response to sleep deprivation is similar to wildtype but they show recovery sleep over much longer time periods, i.e., their recovery sleep does not efficiently dissipate the drive for sleep. This may, in part, be related to their sleep being fragmented with short bouts, even with increased drive for sleep. The requirement for Homer in Drosophila to sustain sleep is so marked that even during recovery sleep there is no increase in sleep bout duration after 4 and 6 hours of sleep deprivation as normally occurs, and sleep is solely increased by an increased number of sleep bouts.In mice the major difference in the sleep/wake phenotype of the Homer1a knockout is in their ability to sustain long bouts of wakefulness. In mice, wakefulness exists in two different states, i.e., short transient bouts of wakefulness and long bouts of sustained wakefulness that occur in the early part of the lights off period The Homer1a sleep phenotype is unique and different than phenotypes that are commonly associated with sleep disruption. Most of the other studies have focused on sleep homeostasis after deprivation or transition between sleep and wake. The orexin KO mice display reduced waking and increased NREM and REM across the lights on period Our data imply that the traditional wake-active neuronal systems such as noradrenaline cells in the locus coeruleus Differences in the Homer1a KO compared to wildtype or heterozygotes during the lights on and not the lights off period are not because of absence of a functioning circadian clock. Studies involving changing animals from light/dark to dark/dark conditions reveal a robust circadian rhythm in the Homer1a knockouts. Thus, they have a functioning clock. However, the period of the circadian rhythm is significantly less in the knockout compared to the wildtype or heterozygote. It has been demonstrated that light induces Homer-1a expression in rat SCN both in the early and late night E. coli and predicted gene EG667132 bdnf gene is known to parallel delta power; it increases with wakefulness and decreases with recovery sleep We did not find an effect of loss of Homer1a in the response to sleep deprivation as measured by increase in delta power following 6 hours of sleep deprivation or in decline of delta power during recovery sleep. While Homer1a has been proposed as a strong candidate to explain the quantitative trait locus for sleep homeostasis in mice The signaling pathways involved in Homer1a induction include the MAP kinase cascade In conclusion, our data support the notion that Homer scaffolding proteins are required for maintenance of behavioral state and that consolidation of sleep and wake is governed by molecules other than traditionally known neurotransmitters. We note that in a model of competitive actions of H1a versus cross-linking forms of Homer, that up-regulation of H1a is functionally equivalent to down regulation of cross-linking Homer. Thus, despite evolutionary changes in Homer gene structure and copy number, Drosophila and mice share the functional consequence of reduced Homer cross-linking during wakefulness and increased cross-linking during sleep. The cellular substrate of this effect, and whether it is mediated by ascending pathways or targets of these pathways in higher brain areas, will be critical to evaluate in the future.Drosophila melanogaster adult females of homerR102 and the background wild type strain Canton-S [w CS(10)] were collected within 24 hours after eclosion, placed in locomotor activity tubes and were maintained at 25°C in a 12∶12 light: dark cycle on 5% sucrose 1% agar.R102 to create homerR102/Df(2L)ED7007 and homerR102/Df(2L)ED6569 hemizygotes. CS flies were also crossed with the same deficiency lines.To map the altered phenotype to Homer, the deficiency lines Df(2L)ED7007 and Df(2L)ED6569 that are in the genome region of Homer were crossed with HomerAll experiments were performed on male mice, 10 wk of age±1 wk, maintained on 12 light/dark cycle  in a sound attenuated recording room, temperature 22–24°C. Food and water were available ad libitum. Animals were acclimated to these conditions for 10–14 days before beginning any studies. All animal experiments were performed in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Pennsylvania Animal Care and Use Committee.S2F in Hu et al, 2010 The Homer1a null mice were generated as described by CREB mutant αΔ hypomorph mice were generated as described by Graves et al Mice were genotyped using the REDExtract-N-Amp Tissue PCR kit (Sigma) and standard PCR techniques. The primers for genotyping the Homer1a knockout, heterozygote and wildtype mice were as follows:5′-AGTCAAAGAGGTCCCTCTGTTCTTG-3′ (reverse)H1aB2: 5′-TCATGTTTACAGTCCAGTAATGCC-3′ (reverse)H1aB3: 5′-TGTGACACAGAACTCAGCCAAG-3′ (forward)H1aA3: The primers used to genotype the CREB mutant mice were as follows:CAGGGACCATTCCTCATTTCCTCREB alpha/delta 5′ primer: GCTGGGCTTGAACTGTCATTTGCREB alpha/delta 3′ primer: GGACAGGTCGGTCTTGACAAAACREB alpha/delta NEO primer: Rest/activity was recorded using beam breaks in the Drosophila Activity Monitoring System (DAMS)  as described in Naidoo et al, 2007 The DAMS and video data were analyzed using custom software R102 and CS flies for 4 or 6 hours by tapping during the dark phase when the fly was seen to be quiescent. We chose to end all deprivations at ZT 22 because the background CS strain displays the most consolidated rest between ZT16–22. Time matched controls were maintained in the monitors without intervention. After SD, the flies were allowed to recover for 24 hr during which their behavior was recorded by video. Recovery sleep was calculated by subtracting the total sleep per hour on baseline day from total sleep per hour post sleep deprivation.We used video analysis to record two days of baseline behavior and then sleep deprived HomerFor analysis of circadian behavior, activity counts were collected in 1-minute bins in DD over a 6-day period and analyzed using ClockLab (Actimetrics).Sleep-wake behavior was recorded via EEG/EMG as described in Mice were sleep deprived for 6 hours. Deprivation was initiated at lights-on (7:00 AM), and performed through gentle handling To assess the exponential decline in delta power during recovery sleep following sleep deprivation, we calculated the average delta power for consecutive 225×4-second (15 minutes) epochs of NREM sleep between 1 pm and 7 pm. Since there could be other stages  as time progressed, it took more than 15 minutes to accrue the 225×4-second epochs of NREM. We assessed the time for each such measure as the temporal mid-point of the 225 epochs. We examined for each mouse the relationship between the log of delta power and time and specifically computed the linear slope of this relationship as a measure of the time constant of the decline in delta power.Circadian period was assessed as described R102 fly heads probed with this antibody did not produce a signal indicating that the mutant flies do not make full length Homer  and further cleaned using the RNAeasy purification kit (Qiagen).Homer1a gene were designed from a region that includes intron 5, which contributes a nucleotide sequence found in Homer1a but not Homer1c.The relative transcript level was established by the “ΔΔ method” as described previously Homer1a:The following primers and probe were used for 5′-end primer-5′TGTCCTGCTGTAAATAAAGCCATCT-3′;Homer1a contained the 6-FAM at the 5′-end and the Black Hole Quencher (3BHQ_1) at the 3′-end, and had the following nucleotide sequence: 5′-6-FAM-AGCAGCGCCTTGAGTTCTTGTGGCTA-3BHQ_1–3′. The final PCR was performed with Homer1a-specific primers: upper strand primer: 5′-AACACTGTTTATGGACTGGG-3′, lower strand primer: 5′-TGATTGCTGAATTGAATGTGTACCTATGTG-3′. The custom made primers and probes were used at a concentration of 50 μM and 20 μM respectively.3′-end primer-5′-AGTATAATCATCTTAACTATCTAGCACCTTCTG-3′. The probe for A mixed model analysis of variance (ANOVA) was used to estimate least squares mean contrasts of interest between genotypes, overall, and at specific times-of-day.The analysis for comparisons of data from the 24-hour recordings used a mixed model ANOVA, with a factor for genotype  and a repeated measures factor of time period. Separate models operationalized time into: (1) an overall 24 hour time period; (2) 2 twelve-hour time periods (light/dark); and (3) 12 two-hour time periods. A logarithm variance stabilizing transformation was used where indicated by the Shapiro-Wilk statistic and inspection of normal probability plots.y = x line  against one another. When the distributions are the same, the Q-Q line should lie approximately on the = x line . We perfIn addition a novel statistical method that takes into account both the “spike and slab” nature of the distribution of bouts in each state and the fact of that the duration of each bout is dependent on the previous state was used as previously described For the sleep deprivation response, we assessed differences between genotypes in our two measures, i.e., magnitude of sleep homeostatic response and time constant of decline of delta power during recovery sleep using a one-way ANOVA with the main factor being the 3 genotypes. The analysis of delta power changes was identical to that described by Franken et al For comparison of circadian period, we used a one-way ANOVA with the main factor being genotype . Pair-wise comparisons between genotypes were done.Figure S1R102 flies do not make Homer protein. Representative immunoblot showing the absence of D-Homer in the homerR102 flies as detected by an antibody made to full length D-Homer and present in the Canton-S (CS) background strain. Each lane represents a single fly head. B) Representative western blot showing homer expression in individual fly heads following 6 hr sleep deprivation (SD) comparing the three lanes from control flies to 3 from sleep deprived (SD) flies. C) Densitometric quantification of homer expression in female heads (n = 9) following 6 hr SD (▪). The density of each band was normalized to that of the control. Data shown are mean and standard deviation. The mean ratio of Homer protein from sleep deprived flies to controls was 0.62 (95% CI was 0.44 to 0.99). The non-parametric analog to the t-test was also significant at p = 0.039 (Wilcoxon signed rank test). D) The HomerR102/Df and CS/Df hemizygotes display very different sleep phenotypes. There are significant differences in sleep bout number (p≤0.001) and sleep bout length (p≤0.001) between HomerR102/Df and CS, CS/Df and HomerR102/CS. There are no significant differences between Homer/Df and Homer/Homer. Shown are the average percent time sleeping, number of sleep bouts and mean sleep bout length in minutes with standard deviations for HomerR102/ HomerR102 (n = 20); HomerR102/Df (n = 30); CS (n = 16); CS/Df (n = 13) and HomerR102/CS (n = 13).A) Homer(PDF)Click here for additional data file.Figure S2Representative Q-Q plot illustrating the comparison of two probability distributions. The black line falling within the grey lines indicates that the null hypothesis is retained (left panel). The black line outside the grey region rejects the null hypothesis of equality of distribution (right panel).(PDF)Click here for additional data file.Figure S3x-axis represents a double plotted 48-h period of activity. Black bar on top indicates dark phase of the LD cycle. The Homer 1a knockout has a reduced circadian period. C) Expression levels of Homer 1a transcripts in the cerebral cortex of CREB αΔ mutant mice (Mut) and wildtype littermates (WT) after 1 hour of total sleep deprivation (SD) (▪) and spontaneous sleep (SS) (□); mean and standard deviation shown. 1 hour SS (n = 4/group); 1 hour SD (n = 3/group). Wildtype mice display a significant increase in Homer 1a transcript after 1 hour of sleep deprivation compared to the mutant mice. The mean expression level of Homer1a mRNA in the CREB αΔ hypomorph is 50.04±26.66 compared to the wildtype which has 301.98±45.72 . The wildtype mice also express significantly more Homer 1a transcript than mice sleeping spontaneously over 1 h .A) Homeostatic response to 6 hours of sleep deprivation measured as a percent of baseline from one mouse of each genotype . The representative line graphs illustrate the decline in delta power in the 3 strains over 15 consecutive epochs. There was no significant difference in the decline of delta power during recovery sleep. B) Representative activity records of Homer 1a knockout, heterozygote and wildtype mice entrained to an LD 12∶12 cycle and subsequently placed in constant darkness (DD) on day 10 as indicated by arrows. Successive days are plotted from top to bottom, and (PDF)Click here for additional data file.Table S1Average amount of NREM and NREM bout duration for Homer 1a wildtype, heterozygote and knockout mice measured in 2 hour time periods. Values are given for the average plus/minus standard deviation. Values significantly different from Homer1a knockout denoted by a = p<0.05 p>0.01; b = p<0.01 p>0.001; c = p<0.001 p>0.0001; d = p≤0.0001.(PDF)Click here for additional data file.Table S2Average amount of REM and REM bout duration for Homer 1a wildtype, heterozygote and knockout mice measured in 2 hour time periods. Values are given for the average plus/minus standard deviation. Values significantly different from Homer1a knockout denoted by a = p<0.05 p>0.01.(PDF)Click here for additional data file.Table S3Minutes of waking and wake bout duration in the 3 genotypes during the lights on and lights off periods in 2 hour intervals. Values are given for the average plus/minus standard deviation. Values significantly different from Homer1a knockout denoted by a = p<0.05 p>0.01; b = p<0.01 p>0.001; c = p<0.001 p>0.0001; d = p≤0.0001(PDF)Click here for additional data file.Table S4Changes in NREM delta determined in 15 minute intervals following 6 hours of sleep deprivation. Delta is measured as % of baseline (100%). Midpoint is the chronological midpoint time of each interval measured from the end of the sleep deprivation period. There are no significant differences between genotypes.(PDF)Click here for additional data file.


Dinophysis and plankton concentrates. Successive blooms of Dinophysis acuminata, D. acuta and D. caudata in 2006 caused a long mussel harvesting closure (4.5 months) in the Galician Rías (NW Spain) and a record (up to 9246 ng·g resin-week−1) accumulation of toxins in SPATT discs. Best fit of a toxin accumulation model was between toxin accumulation in SPATT and the product of cell densities by a constant value, for each species of Dinophysis, of toxin content (average) in picked cells. Detection of Dinophysis populations provided earlier warning of oncoming diarrhetic shellfish poisoning (DSP) outbreaks than the SPATT, which at times overestimated the expected toxin levels in shellfish because: (i) SPATT accumulated toxins did not include biotransformation and depuration loss terms and (ii) accumulation of toxins not available to mussels continued for weeks after Dinophysis cells were undetectable and mussels were toxin-free. SPATT may be a valuable environmental monitoring and research tool for toxin dynamics, in particular in areas with no aquaculture, but does not provide a practical gain for early warning of DSP outbreaks.From June 2006 to January 2007 passive samplers  were tested as a monitoring tool with weekly monitoring of phytoplankton and toxin content  in picked cells of  Dinophysis have two kinds of lipophilic shellfish toxins (LST): diarrhetic shellfish poisoning (DSP) toxins—okadaic acid (OA) and dinophysistoxins (DTXs)—and pectenotoxins (PTXs) + [m/z 805 [M + H]+, 822 [M + NH4]+, 827 [M + Na]+ and 803 [M − H]− for OA and DTX2; 876 [M + NH4]+, 881 [M + Na]+, 857 [M − H]− and 918 [M + CH3COO]− for PTX2; 894 [M + NH4]+, 899 [M + Na]+ and 875 [M − H]− for PTX2SA. Predominant ions were detected in positive mode for OA, DTX2, PTX2 and PTX2SA. Negative mode was performed only to confirm the toxin spectrum.LC-MS analyses were performed on a Thermo Finnigan Surveyor coupled to an ion-trap mass spectrometer (Thermo Finnigan LCQ-Advantage) equipped with a micro-electrospray ionization interface (μESI). Separations were achieved on a Waters XBridge C18 5-μm (2.1 × 150 mm) column at 35 °C. The mobile phase consisted of 2 mM ammonium acetate at pH 5.8 (A) and methanol (B). A linear gradient elution of 60%–100% B for 20 min followed by 100% B for 2 min and re-equilibration with 60% B for 8 min was used. The flow rate was 0.2 mL min + NH4]+ . Once th4]+ and [M + Na]+ ions for OA, DTX2 and PTX2. The presence of PTX2SA—considered an enzymatic transformation of PTX2 due to poor handling of plankton samples—was not explored as it has never been detected in phytoplankton samples in previous studies in the region [In the case of picked cells, to obtain a discernible signal according to the possibilities of the mass spectrometer, a single ion monitoring (SIM) analysis and positive ionization mode alone were used to record signals of the [M + NHe region ,14.−1 approximately, detection limit (DL) 6 ng µL−1—and PTX2SA—0.3 ng µL−1 approximately, DL 3 ng µL−1—were used to generate a four-point calibration curve with variable injection volumes. Concentration ranges for the calibration lines obtained for each toxin were from 1.5 to 6.0 ng for OA and DTX2, from 1.8 to 7.0 ng for PTX2 and from 0.5 to 3.0 ng for PTX2SA. For picked cells, a working solution of OA and DTX2—4 pg µL−1 approximately, DL 40 pg µL−1—was used from 8 to 40 pg. A four-point calibration curve was generated with variable injection volumes. The linearity of each point was tested following van Trijp and Roos [et al. [For the analyses of plankton concentrates and SPATT extracts, working solutions of OA, DTX2 and PTX2—0.6 ng µLand Roos . Full de [et al. .−1—which accumulated all the particulate toxins available in the filtered seawater following the equation:Ta is the amount of toxin adsorbed by the SPATT; Ccell is the Dinophysis cell concentration per liter of seawater; Ctox is the pg of toxin per cell of Dinophysis and Fads is the rate of toxin adsorption by the SPATT that was given a constant value of 3 L h−1.The adsorption of each type of toxin  by the SPATT resins was mathematically simulated considering that SPATT at each depth behaved as an artificial mussel—with an adsorption rate of 3 L h−1) per cell, for each species of Dinophysis used in the model, from the analyses of picked cells; (ii) another simulation that used the estimate of toxin content (pg cell−1) per cell of Dinophysis  obtained from the analyses of the size-fractioned (77–20 µm) plankton concentrates.Two kinds of simulations were made for each type of toxin  and depth  of SPATT deployment: (i) one simulation that used the average estimate of toxin content  Dinophysis acuminata + Dinophysis acuta for OA; (ii) Dinophysis acuta for DTX2 and (iii) Dinophysis acuta + Dinophysis caudata for PTX2.Data of Dinophysis and in plankton concentrates led to confirmation of D. acuminata and D. acuta as the main sources of OA in Galician shellfish, D. acuta of DTX2 and both D. acuta and D. caudata of PTX2. Discrepancies between toxin per cell estimates and those from plankton concentrates suggest increased amounts of released toxins in the water during late stages of the bloom, which are attached to organic aggregates retained in the filters and not taken up by mussels. Likewise, discrepancies between toxins in plankton concentrates and those accumulated by the SPATT suggest zooplankton and shellfish faecal pellets as the source of PTX2SA in the resins. Model simulations showed best-fit between weekly toxin accumulation by the SPATT discs and the toxin content of Dinophysis cells per volume of water, estimated from a constant value of toxin content for each species, averaged from LC-MS analyses of picked cells. OA and PTX2 can persist “dissolved” in the water for weeks but DTX2 is less stable. Monitoring of Dinophysis cells with appropriate spatial (integrated samples) and temporal frequency provides earlier warning of DSP outbreaks than SPATT resins, which allow detection of toxins with a similar timing than that of detection in shellfish tissues by LC-MS analyses in regulatory centers. In addition, SPATT accumulation of toxins overestimates that taking place in shellfish and subject to loss terms ; further, SPATT continues accumulating toxins for weeks after Dinophysis cells become undetectable and mussels are depurated. SPATT provides a valuable tool for the study of Dinophysis population and their toxin dynamics, in particular if deployed in areas and depths not subject to regular surveillance. Further, it has the potential to be a relatively simple method which may provide a more efficient regulatory action, cost-savings, enhanced environmental toxin detection and a monitoring perspective that is less susceptible to the variability of discrete phytoplankton and shellfish sample collections. However, SPATT does not represent a practical gain for early warning of DSP outbreaks in aquaculture sites subject to routine monitoring of HABs and phycotoxins and for the time being should remain as a complementary tool to traditional sampling.A systematic monitoring of HABs and lipophilic toxins (LC-MS) in picked cells of


The orbitofrontal cortex (OFC) and piriform cortex are involved in encoding the predictive value of olfactory stimuli in rats, and neural responses to olfactory stimuli in these areas change as associations are learned. This experience-dependent plasticity mirrors task-related changes previously observed in mesocortical dopamine neurons, which have been implicated in learning the predictive value of cues. Although forms of associative learning can be found at all ages, cortical dopamine projections do not mature until after postnatal day 35 in the rat. We hypothesized that these changes in dopamine circuitry during the juvenile and adolescent periods would result in age-dependent differences in learning the predictive value of environmental cues. Using an odor-guided associative learning task, we found that adolescent rats learn the association between an odor and a palatable reward significantly more slowly than either juvenile or adult rats. Further, adolescent rats displayed greater distractibility during the task than either juvenile or adult rats. Using real-time quantitative PCR and immunohistochemical methods, we observed that the behavioral deficit in adolescence coincides with a significant increase in D1 dopamine receptor expression compared to juvenile rats in both the OFC and piriform cortex. Further, we found that both the slower learning and increased distractibility exhibited in adolescence could be alleviated by experience with the association task as a juvenile, or by an acute administration of a low dose of either the dopamine D1 receptor agonist SKF-38393 or the D2 receptor antagonist eticlopride. These results suggest that dopaminergic modulation of cortical function may be important for learning the predictive value of environmental stimuli, and that developmental changes in cortical dopaminergic circuitry may underlie age-related differences in associative learning. Learning to associate environmental cues with behaviorally significant outcomes is necessary for organizing and driving behavior. In rodents, this associative learning is thought to be mediated in large part by a highly interconnected neuronal network that includes the orbitofrontal cortex (OFC), amygdala, and piriform cortex In the rat, adult-like dopaminergic projections to the OFC are established between postnatal days 20–35 All procedures in this study were conducted in accordance with guidelines for animal use published by the Society for Neuroscience and the National Institutes of Health, under protocol approved by the University of St. Thomas institutional animal care committee (Protocol #48).We used 97 Male Long-Evans rats  from four age groups: juvenile , adolescent , adult  and aged adult . Results from the two adult groups are presented together because there were no significant differences on any measures between these two groups.To compare the ability of juvenile, adolescent and adult animals to acquire an odor-based association, rats were trained on a digging task similar to that described previously e.g., . First, After training with unscented sand, animals were tested on their ability to perform an odor-association task, in which two sand-filled cups were presented to the rat, with each cup containing sand combined with a different odor . Only one of the two odorized cups contained a Froot Loop. Digging in the Froot Loop-containing cup was considered a “correct” trial, while digging in the empty cup was considered “incorrect” and would result in a short timeout from the task. Sand was odorized using commercially-obtained fragrances, spices and extracts, diluted to a 10% concentration with mineral oil prior to combining with sand. Cups were randomly positioned within the arena for each trial, and no location was used more than three consecutive times. The task continued for a 20 minute session or until rats reached performance criterion , whichever came first. The number of trials to achieve criterion was recorded for each rat; for rats that required more than one session, trials were counted across sessions. Those trials during which animals were inactive or displayed behavior that was not directed toward the sand-filled cups  for 30 consecutive seconds were counted as “distracted” trials, which were analyzed separately. Results from behavioral experiments were analyzed using one-way  analyses of variance (ANOVA), and a significant F test was followed by Tukey’s HSD post-hoc test of group means as noted.Rats from all age groups were trained and tested. Naïve rats began training at different ages, but were tested at all subsequent ages to examine effects of previous experience . This arrangement yielded the following groups of animals: Naïve juveniles (n = 10), naïve adolescents (n = 13), naïve adults (n = 8), experienced adolescents (n = 7), and experienced adults (n = 12).Separately, 18 non-trained rats were euthanized by decapitation, the brain was quickly removed from the head, and samples of brain regions of interest from both right and left hemispheres were quickly dissected, flash-frozen in liquid nitrogen, and stored at −80°C until RNA extraction. Total RNA was extracted from brain samples from juvenile (n = 6), adolescent (n = 6) and adult (n = 6) rats using TRI Reagent with glass fiber filter purification . After extraction, total RNA pellets were resuspended in RNAsecure and DNAse treated using DNAse-free (both from Applied Biosystems/Ambion). The quality and concentrations of RNA were evaluated using a Take Three application and BioTek Synergy 2 multimodal plate reader  to measure the ratio of optical density at 260/280 nm wavelengths. Samples were then diluted to 0.1 µg/µL as recommended by Applied Biosystems, Inc. The RNA samples were stored at −80°C until subsequent use. To obtain cDNA from RNA samples a High Capacity RNA-to-cDNA Kit  was used, the quality and concentrations of cDNA were evaluated using the Take Three application and samples were diluted to identical concentrations.AGGAGTCCCCATCCCAACTCAGCCC; D1 primer sequence TCTAGAAAAGATCCAACCTGTCACA), and TaqMan MGB probe for the genes of interest, labeled with FAM . The thermocycle conditions were: 50°C for 2 min, hold for 10 min at 95°C, followed immediately by 40 cycles of PCR amplification  using a StepOne Real-Time PCR system (Applied Biosystems). Controls for primer efficiency were run in the same experiments as our experimental samples, as were controls for the reporter dye and blank (water-only) controls.Gene expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Assays for rat GAPDH  and rat D1 dopamine receptor (NCBI Reference NM012546.2 and M35077.1) were obtained commercially (Applied Biosystems). Each assay was conducted as a real-time PCR assay in a 48-well format using TaqMan EZ RT-PCR kits . Each 20-µl reaction was run in triplicate, and contained 9 µl cDNA diluted in nuclease-free water, 10 µl of TaqMan Universal PCR Master Mix, and 1 µl of 20X TaqMan Gene Expression Assay Mix  to protect against the possibility of falsely reporting a mean difference between groups because of the arbitrary assignment of cycle value.All mRNA levels were normalized to mRNA levels for a ubiquitous reference gene (GAPDH). Prior to selecting GAPDH as the reference gene, we confirmed that the gene was stably expressed during development, and that its abundance showed a strong correlation with the total amount of mRNA present in the samples. Quantification of normalized samples was performed using the Comparative CT method of Livak and Schmittgen 2O2 for 5 minutes at 25°C. Sections were dried, dehydrated and coverslipped.To assess the distribution of dopamine D1 receptor-expressing cells within OFC, 19 rats were deeply anaesthetized with barbiturate  and transcardially perfused with phosphate buffered saline  followed by 4% formaldehyde freshly depolymerized from paraformaldehyde. Brains were then removed from the skull and cryoprotected in 30% sucrose at 4°C. Sections were cut at 40 µm in the coronal plane using a cryostat and were mounted on electrostatically-charged slides. Immunohistochemical localization of dopamine receptors was performed on slides, using a modification of previously described protocols Each region of interest (ROI) was examined on sections randomly chosen for quantification. The ROIs included lateral (LO) and ventrolateral orbital (VLO) regions of the OFC, and dorsal (APCd) and ventral (APCv) regions of the anterior piriform cortex. A microscope (Nikon Instruments) connected to a computer running Neurolucida software  was used to visualize sections and create boxes which were randomly placed overlying ROIs , and the number of immunopositive cells within the sampling box was determined. At all times, experimenters were blind to the age-group identity of the tissue being quantified. Counts were made of tissue from juvenile (n = 7), adolescent (n = 6) and adult (n = 6) animals. Statistical analyses were performed on raw cell counts using ANOVA, and a significant F test was followed by post-hoc tests of group means using Tukey’s HSD.Experiment 1 above). One day after rats had successfully learned to dig in unscented sand, they proceeded to the odor-association task. Thirty-minutes before starting the odor-association task, these animals were administered a subcutaneous injection of either 0.33 mg/kg dopamine D1 receptor agonist SKF-38393 , 0.125 mg/kg of the dopamine D2 antagonist eticlopride  or saline (n = 6). As before, animals were tested on the odor-association task and the number of trials to criterion  was recorded. Animals that required more than one session were injected thirty minutes before each session, but no more frequently than once in a 12 hour period. Data were analyzed using ANOVA and followed up with Dunnet’s test to compare injected group means to the naïve adolescent group mean.Three additional groups of naïve adolescent rats were trained on the digging task  = 8.27, p<0.002), which was investigated further using Tukey’s HSD post-hoc tests. After learning to dig for palatable reward, juvenile animals naïve to the task were able to learn the predictive value of an odor  in an average of 11.7 trials (±1.8 SE), which was about the same number of trials as naïve adult rats . In contrast, naïve adolescent animals required a significantly greater number of trials to reach criterion , than either the naïve juvenile  or the naïve adult animals  = 3.399; p<0.01). Post-hoc tests with Tukey’s HSD revealed that adolescent animals displayed significantly more distracted behavior than either juveniles or adults (p<0.02). Adult animals displayed an intermediate level of distracted behavior  that was significantly lower than that displayed by adolescent animals (p<0.05), but not significantly different than juvenile animals.Only “active” trials where the rats were engaged in the digging task were used to determine criterion for learning, so the slower learning exhibited by adolescents for the initial odor pairing could not be attributed to an increase in incompatible behavior (such as grooming or investigation). However, a separate analysis of these “distracted” trials  revealed a significant effect of age on the number of distracted trials exhibited in the odor association task  = 5.56, p<0.02). The experienced adolescent animals (which had learned the association task with different odors as juveniles) required significantly fewer trials to meet criteria than naïve adolescents . Prior to selecting GAPDH as the reference gene, we confirmed that it was stably expressed during postnatal development, and that its abundance showed a strong correlation with the total amount of mRNA present in the samples. Indeed, no differences in mean threshold cycle (Ct) values for GAPDH expression were observed among age groups for either the OFC or piriform cortex .U = 78, p<0.01; juvenile vs. adult, U = 52, p<0.01). Similarly, adolescent rats displayed significantly higher levels of D1 receptor mRNA in piriform cortex compared to juvenile and adult levels U = 91, p<0.01).The expression of D1 receptor mRNA in OFC and piriform cortex showed a clear developmental regulation in rats. As illustrated in  = 14.766, p<0.0001; p<0.001 for each comparison). Despite these large changes in the overall number of cells in each cortical area during adolescence, there were no apparent changes in the overall distribution of cells expressing the D1 receptor; cells appeared distributed throughout the regions examined, with no evidence for clustering  = 14.59; p<0.001). Post-hoc analyses revealed that naïve saline-injected adolescents took as long to learn  as uninjected naïve adolescents  or saline-injected controls , or the D2 antagonist eticlopride (0.0125 mg/kg). Control animals were injected with an equivalent volume of saline. The animals were then tested on the odor association task, and the number of trials to criterion was recorded. An ANOVA revealed a significant effect of the injection (Fp>0.20), injection of SKF significantly decreased the number of distracted trials (mean = 2.25±1.4 SE) compared with naïve rats . Similarly, eticlopride reduced the number of distracted trials to a mean of 2.2 trials (±1.4 SE), significantly lower than naïve adolescent rats (p<0.05).Subcutaneous injection of drugs also reduced the number of distracted trials displayed by adolescent rats . While sThe OFC and piriform cortex, along with associated subcortical structures, play a role in encoding the predictive value of cues during odor-guided associative tasks per se. However, releasing suppression of the olfactory nerve input would be expected to degrade associative-cue odor signals relative to background odors The deficit in learning we observed in adolescence could be ameliorated by a single, low-dose, systemic injection of a drug that increased postsynaptic dopamine receptor activation (either the D1 receptor agonist SKF-38393 or the D2 receptor antagonist eticlopride), but the systemic injections employed in this study preclude localizing the site of action of these drugs. In particular, the olfactory bulb is rich with dopaminergic cells, and dopamine has been shown to suppress the input to the olfactory bulb from the olfactory nerve The changes in D1 receptor expression and distribution we observed in the OFC and piriform cortex are some of many ongoing modifications in cortical circuitry during adolescence. Cortical structures important for associative learning in the adult rat are immature at birth, and significant changes in the morphology and physiology of structures involved with olfactory-guided behavior occur during the first four weeks in the rat During adolescence, rats begin to leave the nest What could be the survival advantage of increased distractibility and slower learning during adolescence? Perhaps learning only those associations which are repeatedly confirmed or which are highly rewarding leads to greater behavioral variety, supporting more exploration and risk-taking and driving adolescent animals to leave the nest. In this way, behaviors that are important for survival such as foraging, colonizing new territory, and engaging in social behaviors are promoted. In situations where D1 receptors are more highly activated, either by a highly rewarding situation or through external manipulation (i.e. drugs), behavior becomes more focused and associations learned more rapidly.


There were errors in the Funding section. The correct funding information is as follows: Financial support to the Instituto de Biología Funcional y Genómica (IBFG) from the Fundación Ramón Areces and to our group from the Comisión Interministerial de Ciencia y Tecnología (CICYT) (Spain)/European Union FEDER  program (grant BFU2007-61866 and BFU2010-15085) made this work possible. Dr. Curto was supported by a contract for training in research and technological innovation by Junta de Castilla y León and by Formacion de Personal Investigador (FPU) fellowship. Dr. de Leon and Dr. Hoya were supported by FPU and Junta de Ampliacion de Estudios (JAE)-PREDOC fellowships from the Ministry of Science and Consejo Superior de Investigaciones Científicas (CSIC), Spain, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Mas receptor as shown by diverse studies involving site-specific binding techniques, selective antagonists, and targeted gene deletion. This paper provides an overview of the functional role and the molecular pathways involved in the biosynthesis and activity of Ang-(1–7) in diverse systems.Ang-(1–7) is produced via degradation of Ang II by the human angiotensin converting enzyme, also known as ACE2. In the cardiovascular system, Ang-(1–7) has been shown to produce effects that are opposite to those of Ang II. These include smooth muscle relaxation and cardioprotection. While the roles of Ang-(1–7) in other systems are currently topic of intense research, functional data suggest a relaxation action in gastrointestinal smooth muscles in a way that corroborates the results obtained from vascular tissues. However, more studies are necessary to determine a relevant role for Ang-(1–7) in the gastrointestinal system. The Ang-(1–7) actions are mediated by a distinct, functional, Ang-(1–7) receptor: the  ACE2 is insensitive to classic ACE inhibitors such as lisinopril , 11, ena in ACE2 . 1 receptors in the media layer of thoracic aortas isolated from SHR resulted in significant increases in the expression of ACE2 at the mRNA and protein levels. This effect was consistently associated with increased levels of Ang-(1–7), suggesting that ACE2 generates Ang-(1–7) locally. Corroborating these results, studies by Lavrentyev and Malik [As mentioned above, ACE2 catalyzes the conversion of both Ang I and Ang II into smaller fragments. Donoghue et al.  first shnd Malik  have found Malik  and rat nd Malik . In addition to ACE2, there are other enzymes that have been reported to release Ang-(1–7). Endopeptidases are the first example and have been implicated in the biosynthesis of Ang-(1–7) mainly from Ang I. As an example, prolyl endopeptidase converts Ang I into Ang-(1–7) as shown in NG108-15 neuroblastoma versus glioma hybrid cells  and in eACE has also been reported to be able to release Ang-(1–7). Even though it does not catalyze the generation of Ang-(1–7) from Ang I or Ang II, ACE is able to cleave Ang-(1–9) leading to the generation of Ang-(1–7) . FurtherThe first evidence that pointed a potential role for ACE in the degradation of Ang-(1–7) came from the findings obtained by Kohara et al. . In thisin vitro experiments obtained by Chappell et al. [5-His6 bond of Ang-(1–7) with a high specificity constant. The hydrolysis of Ang-(1–7) catalyzed by ACE involves the N-domain of the enzyme [Indeed, l et al.  showed te enzyme  and leade enzyme . Studies show that Ang-(1–7) has a cardioprotective role mainly attributable to counteraction of the Ang II effects, which contribute to maintaining the vascular homeostasis and attenuating the progression of atherogenesis , 32 amonα1-adrenergic agonists. Later, the authors confirmed their hypothesis [α1-adrenergic agonist phenylephrine, and to Ang II, and improved the sensitivity of the reflex control of the heart rate in SHR. These results were suggestive that Ang-(1–7) activated antihypertensive mechanisms.Campagnole-Santos et al.  were thepothesis  by showi2 − generation [2 −, which is involved in the reduction of endothelial NO availability and in the consequent endothelial dysfunction [Additional studies showed that Ang-(1–7) was responsible for the hypotensive effects of ACE inhibitors like lisinopril and losartan. Experiments using monoclonal antibodies selective for Ang-(1–7) showed that the antihypertensive effects of lisinopril and losartan were reversed by scavenging the circulating Ang-(1–7) in SHR , 37, 38.neration , NO inacneration , and hasfunction , 48. The2-mediated increase in cAMP production. In addition, Strawn et al. [Ang-(1–7) also has antiproliferative effects in the cardiovascular system. Freeman et al.  have shon et al.  have shon et al.  observedn et al. –54. eNOS. These findings have opened new perspectives on the promising vasculo- and atheroprotective effects of Ang-(1–7) suggesting therapeutic potentials for the atherosclerosis, thrombosis, and atherothrombosis. Studies also provide evidence for a role for Ang-(1–7) in reducing thrombus formations. Experiments in rat vena cava have shown that Ang-(1–7) reduces the thrombus weight and platelet adhesion to fibrilar collagen by a mechanism involving NOS and COX metabolites . In addiMas receptor was first cloned and sequenced by Young et al. [The g et al.  in cotraMas receptor and Ang-(1–7) was first established by Santos et al. [Mas-deficient mice, the authors demonstrated that Ang-(1–7) binding was absent in kidneys from Mas-deficient mice, but preserved in wild-type membranes. In addition, the authors also showed that Ang-(1–7) binding was preserved in membranes isolated from AT1- or AT2-deficient animals, suggesting a twofold conclusion: (1) that Ang-(1–7) has limited interactions with AT1 and AT2 receptors and (2) that it mainly binds to Mas receptors [Mas receptors. The results also showed that the Mas receptor has very low affinity for AT1 or AT2 ligands, excluding the hypothesis that Ang-(1–7) would directly interact with AT1 or AT2 receptors [A connection between the s et al. . Using beceptors . These receptors .Mas receptor as the mediator of the Ang-(1–7) effects in the vascular system was also provided by Santos et al. [Mas-deficient mice. The authors were also the first group to develop a selective antagonist for the Mas receptor called D-Ala7-Ang-(1–7) also known as A-779 [Mas receptors in the effects induced by Ang-(1–7). These include indirect  antihypertensive effects [eNOS induced by Ang-(1–7) in human aortic endothelial cells, suggesting the involvement of Mas receptors in this effect. A-779 also blocked the inhibitory effect of Ang-(1–7) on VSMC growth, suggesting the participation of Mas receptors in the antiproliferative action of Ang-(1–7) [The first evidence for a functional role for the s et al. . The autas A-779 , 59. The effects  and loca effects  and mole effects  levels.  effects  have sho effects  demonstrng-(1–7) . Mas receptors in the antithrombotic effect of Ang-(1–7). A-779 dose-dependently inhibits the Ang-(1–7)-induced reduction in venous thrombus weight [2-antagonists, suggesting an important interaction between Mas and AT2 receptors in mediating the atheroprotective effect of Ang-(1–7) [There is also functional evidence directly supporting a role for s weight . In adding-(1–7) . Mas receptors and Ang-(1–7) in the regulation of gastrointestinal smooth muscle motility. Studies by De Godoy et al. in Dr. Rattan's laboratory [Multipronged studies also show a potential role forboratory  have shoboratory . WhetherMas receptor that elicit well-known molecular mechanisms of relaxation such as the release of NO. Limited studies suggest a potential role for Ang-(1–7) in regulating motility of gastrointestinal smooth muscles, but additional studies are necessary to further determine Ang-(1–7) actions and therapeutic potentials in the gastrointestinal system. On the other hand, the effects of Ang-(1–7) in the cardiovascular system are better understood and provide strong evidence for the cardiovascular protective action that can be selectively modulated via Mas ligands and have important therapeutic implications for human therapy.In closing, Ang-(1–7) relaxes smooth muscles via interactions with the


Background and Objectives. To explore the role of cis-regulatory sequences within the β globin gene cluster at chromosome 11 on human γ globin gene expression related to Hb E allele, we analyze baseline hematological data and Hb F values together with β globin haplotypes in homozygous Hb E. Patients and Methods. 80 individuals with molecularly confirmed homozygous Hb E were analyzed for the β globin haplotypes and Xmn I polymorphism using PCR-RFLPs. 74 individuals with complete laboratory data were further studied for association analyses. Results. Eight different β globin haplotypes were found linked to Hb E alleles; three major haplotypes were (a) (III), (b) (V), and (c) (IV) accounting for 94% of Hb E chromosomes. A new haplotype (Th-1) was identified and most likely converted from the major ones. The majority of individuals had Hb F < 5%; only 10.8% of homozygous Hb E had high Hb F . No association was found on a specific haplotype or Xmn I in these individuals with high Hb F, measured by alkaline denaturation. Conclusion. The cis-regulation of γ globin gene expression might not be apparent under a milder condition with lesser globin imbalance such as homozygous Hb E. Beside producing abnormal variant, hemoglobin E (HbE), the G→A substitution in codon 26 (Glu→Lys) of the echanism . However disease . Althougγ globin expression and HbF production in these patients was thought to be one of the main genetic factors responsible for clinical heterogeneity found in Hb E/β thalassemia by reducing globin imbalance and ameliorating ineffective erythropoiesis. Through erythroid development, the γ globin expression was regulated by interactions between cis-acting sequences within the β globin cluster and trans-acting factors such as BCL-11A, cMYB, and TOX [ cis associated with high HbF is Xmn I polymorphism located at −158 upstream to the Gγ globin genes [β globin gene cluster in HbE/β thalassemia has shown that there was no other variant elsewhere which has a comparable level of association with that of Xmn I site and the T allele  was almost always in cis with the HbE alleles [cis-acting sequences on Hb F production under the less hematopoietic stress and globin chain imbalance in Hb E disorders, we analyzed 7 known single nucleotide polymorphisms within the 50 kb of the β globin gene cluster to construct Hb E-linked β globin haplotypes together with Xmn I polymorphism. The β globin haplotypes of Hb E have been characterized previously in Thailand and within the region of Southeast Asia but they were principally utilized to identify origin, spread, and anthropology of different ethnic groups found with Hb E [β globin haplotypes and Xmn I (representing cis-regulatory sequences) on propensity of the γ globin expression. Moreover the association analysis between Hb E alleles and persisting Hb F production in our Thai population might be useful to confirm the findings and apply in a more clinical significant syndrome such as Hb E/β thalassemia.Variation of postnatal  and TOX –8. The min genes . In a re alleles . To furtith Hb E . Howeverith Hb E  and idenEthylene diamine tetraacetic acid (EDTA) blood was obtained from 80 Thai individuals with homozygous HbE after informed consent. They were parents, siblings, and unrelated individuals who were tested due to microcytic anemia at Department of Pediatrics, Faculty of Medicine Siriraj Hospital as a part of an ongoing project studying natural history and genotype-phenotype correlation in Hb E disorders in Thailand. This study was approved by a local ethical committee at Mahidol University. Complete hematological and hemoglobin studies have been performed using standard techniques as described previously . QuantifMnl I restriction sites, one at the 5′end used as an internal control for digestion (135 bp) and another one linked to CD26 (171 and 62 bp). Subsequently we digested the PCR products using Mnl I using the conditions recommended by the manufacturer ; Hb E mutation (G→A) abolishes this Mnl I site therefore digestion demonstrated the 233 bp and 135 bp   less than 0.05 were considered to be statistically significant.SPSS statistical software package Version 10.0  was used for data analysis. Paired and unpaired β globin haplotypes associated with HbE alleles have been constructed with two novel ones, (− − − − − + −) and (+ + − + + + −). Three major haplotypes based on Orkin et al. [Xmn I +, −, and +, respectively. Therefore only 71.43% of HbE allele is linked with the T allele (or + genotype) of Xmn I. Only the frameworks 2 (+ −) and 3 (− +) based on Antonarakis et al. [β globin haplotypes is shown in From 80 individuals with homozygous Hb E, we could successfully genotypes all 8 polymorphic sites in 77 cases  had high level of Hb F , while the rest had Hb F less than 5%. Distribution of steady stage Hb F percentages in all 74 homozygous Hb E is shown in P < 0.05, γ globin gene [Seventy-four individuals who had all complete hematological and hemoglobin data with molecular results including Hb E genotypes and their linked eviously . In summeviously . At the hildhood . While ibin gene –23. Howeβ globin haplotypes and Xmn I polymorphism in these eight individuals with high Hb F compared to the rest. Moreover, we failed to identify any association between eight β globin haplotypes and the baseline Hb F levels determined by alkaline denaturation in all 74 cases (data not shown). Even though the average alkali F levels were albeit increased in homozygous Hb E with Xmn I (+ +) compared to (+ −) it was statistically insignificant within the same sex , Hb E alleles were linked with the three major haplotypes; (a) (III), (b) (V), and (c) (IV), consisting with previous studies in our population and supporting the hypothesis of multiple origins of Hb E in this part of the world [βS haplotype reported from previous studies [ε-Hind II site is different suggesting that these two novel haplotypes may arise from interallelic gene conversion than an independent origin [β globin haplotypes in other studies which had a limited number of homozygous condition and used the haplotype analyses mainly from Hb E heterozygotes [To the best of our knowledge, this study, herein, provides the most extensive analyses of the he world –13. In B studies , 24. Howt origin . Moreoveozygotes , 12.β globin haplotype (implying cis-regulatory sequences) and the predisposition of Hb F production. We found several individuals with homozygous Hb E and high levels of Hb F in which other extrinsic factors such as intercurrent infection, hematopoietic stress, and erythropoiesis could not be accounted for. These individuals may provide a novel, rather less complicated natural model for further study on molecular mechanism controlling γ globin expression as they all have identical β globin gene defects (Hb E) and their linked β globin haplotypes could be easily constructed. However, we did not succeed to identify any positive correlation, even with the Xmn I polymorphism suggesting that Hb F production found in these homozygous Hb E individuals might be mainly controlled by trans-acting mechanism(s) including HBS1L and BCL11A [β globin haplotypes and clinical severity of Hb E/β thalassemia patients were found in Thai patients and this was accounted by a link between the probable functional SNP, Xmn I, and the propensity to increase Hb F production [β thalassemia and this might not be enough to trigger the switching of postnatal γ globin expression as the Hb E/β thalassemia syndrome does. Interestingly, our study showed that Hb E alleles in our study population are linked to both T (+) and C (−) alleles of the Xmn I site suggesting a high heterogeneity of Hb E alleles within Thai population and highlighting a great consideration for future determination of haplotype and SNPs, which links to the phenotypes related to Hb F and clinical severity.In this study, we further addressed the correlation between the d BCL11A , 26. On oduction . It is pXmn I and the β globin haplotypes as a marker could not be confirmed using homozygous Hb E condition. However, this might suggest a limitation of the model used in this study as the influence of cis-regulatory elements might not be strong enough to be detected under a condition with slighter hematopoietic stress such as homozygous Hb E. In conclusion, the association of Hb F production with the


Here we demonstrate that the relative contribution of Ih and Ikx to the rods' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells.This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1 In rod and cone photoreceptors Ih has been characterized with electrophysiological recording techniques h has been recorded in these neurons h is to generate, along with an ionic conductance named Ikx, a band-pass filter effect in rod responses to light h in rods and bipolar cells have been described in some detail but the actual role of the individual HCN isoforms in retinal processing remains unclear.Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are widely expressed in both central and peripheral nervous system where, upon activation by hyperpolarization of an inwardly rectifying current . The protocol was approved by the Animal Care Committee of the University Of Pisa, Italy .Animals were kept in a local facility with water and food ad libitum, under a 12∶12 h light∶ dark cycle with illumination levels below 60 lux. Special care was exercised to limit any suffering and discomfort associated with the experimental procedures that were all conducted under deep anesthesia.−/−, HCN2−/− and littermate controls (HCN+/+) were used for immunolabeling, RT-PCR, western blotting analysis, whole cell recordings and ERG experiments. HCN1−/+ animals were obtained from The Jackson Laboratory −/+ animals were crossed with C57Bl/6J wild-type mice in order to obtained hybrid HCN1−/+. These animals were intercrossed to produce HCN1−/− and HCN1+/+ littermates. Genotyping was done by PCR using primer 1F1 (5′-TAATGTTCTCGCAGCCTATG-3′), 2F1 (5′-CCTCAATGAAAACTGCAAGGAGC-3′) and 1R4 (5′-AAGATTGGGCACTACACGCT-3′). HCN2−/− mice have been described previously −/+ animals on a hybrid 129Sv/C57Bl/6J background were intercrossed to generate HCN2-deficient and control animals. Genotyping was done by PCR using primers 14 F (5′-GGTCCCAGGCACTTCCATCCTTT-3′), 156 R (5′-GGAAAAATGGCTGCTGAGCTGTCTC-3′) and 16 F (5′-CAGCTCCCATTTGCCCTTGTGC-3′).Adult HCN1All the experimental procedures involving animals were carried according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research . Animals were kept in a local facility with water and food ad libitum, under a 12∶12 h light∶ dark cycle with illumination levels below 60 lux.Adult mice were deeply anaesthetized with urethane 20% W/V in 0.9% saline before eye-enucleating. The retinas in the eyecup were immersion-fixed for 20 min in 4% paraformaldehyde in 0.1 M phosphate buffer saline  at room temperature and then washed 3 times for 10 min in PBS. Tissue was cryoprotected in scalar dilution  of sucrose in PBS. Eyecups were then included in Tissue Tek Optimal Cutting Temperature (OCT) compound  and sectioned at −20°C into a cryostat. Serial sections of 18 µm in thickness were collected on super-frost plus slides (Fluka Biochemika).Sections were washed 3 times for 10 min in PBS and then incubated in 1% bovine serum albumin (BSA) and 0.3% Triton-X 100 in PBS 0.1 M for 45 min in order to block unspecific binding and induce membrane permeability. Sections were incubated for 48 h at 4°C with primary antibodies  diluted in 1% BSA and 0.03% Triton-X 100 in PBS. Sections were washed in PBS and incubated in secondary antibodies  diluted in 1% BSA in PBS for 2–3 h at room temperature, washed in PBS and cover slipped with Vectashield (Vector Laboratories). Retinal sections were visualized with a confocal microscope equipped with a krypton-argon laser ; files were processed with image manipulation software .AGGTTAATCAGATACATACACC, reverse: GAGTGCGTAGGAATATTGTTTT, 231-bp amplicon; HCN2: CGGCTCATCCGATATATCCA, reverse: AGCGCGAACGAGTAGAGCTC, 230-bp amplicon; PCR conditions: 15 min 95°C; 40 cycles: 10 s 95°C, 40 s 60°C, 40 s 72°C. The identity of PCR products was verified by agarose gel electrophoresis GGCTCTTGAAATGGACCCTTC, reverse: CAGCCAATGCTTGATCATATTCTT, 91-bp amplicon Total RNA was extracted from mouse retina using RNeasy Fibrous Tissue kit (Qiagen). For RT- PCR, 1 µg of total RNA was retro transcribed with both random hexamer and oligo (dT) primers using the Quant Tect Reverse Transcription Kit (Qiagen). Conventional RT-PCR was used to examine the expression of HCN1-2. We used the following primer sets: HCN1: forward: 2/CO2 bubbled AMES medium integrated with sodium bicarbonate (Sigma-Aldrich), and the vitreous delicately removed with forceps. A retina was laid vitreal side down on filter paper, made to adhere to it by weak transmural suction, and slices of 250 µm thickness were cut with a manual tissue chopper . Once secured within the recording chamber slices were visualized in the near infrared (LED peak emission at 780 nm) with a CCD camera attached to an upright microscope  while being continuously perfused with the same AMES medium at a temperature of 24°C. HCN inhibition was obtained by adding 3 µM ivabradine  to the perfusing medium 2SO4, 15 KCl, 10 NaCl, 5 Pipes, corrected to a pH of 7.20 with KOH/HCl. The back-filling solution also contained 0.4 mg/ml Amphotericin-B (Sigma-Aldrich) pre-dissolved in DMSO at 60 mg/ml. Recordings were made with an Axopatch 1D amplifier, low-pass filtered at 500 Hz and digitized at 5 kHz (200 Hz/1 kHz during input impedance measurement), and acquired by pClamp 9 software . Membrane potentials were not corrected for the liquid junction and Donnan potentials Isolation of the dark adapted retinas (>3 hrs) and slicing were performed with a naked eye under dim illumination in the far red . Following anesthesia by i.p. injection of 2,2,2-tribromethanol , each retina was rapidly extracted through a corneal incision into cold OThe general procedure for animal preparation, anesthesia, ERG recording, light stimulation and data analysis has been previously described in detail in Della Santina et al. The full field illumination of the eyes was achieved via a Ganzfeld sphere 30 cm in diameter, whose interior surface was coated with a highly reflective white paint. Two stimulus patterns were adopted: brief flashes that generated the typical ERG response (a- and b-waves) and sinusoidal time varying luminance stimuli eliciting periodic responses.5 Φ (Photoisomerisation Rod−1) per flash. Calibrated neutral density filters were used to attenuate the intensity of the flashes.An electronic flash unit (SUNPAK B3600 DX) generated a stimulus whose energy decayed in time with a τ = 1.7 ms. A short-wavelength band-pass filter, 7.5 nm half bandwidth , was used, which gave a scotopic effective λ of 492 nm. Because the maximal energy of the band-pass filtered flashes was not sufficient to elicit saturating a-wave responses, these were obtained by delivering flashes of white light whose scotopic efficacy was evaluated according to Lyubarsky and Pugh Sinusoidal changes in luminance at various temporal frequencies and modulation depth were generated by a light-emitting diode (LED) source (peak wavelength: λ = 520). The luminance of sinusoidal stimuli is expressed as:A light stimulus unit developed in our laboratory generated sinusoidal temporal patterns The recorded signals were averaged in synchrony with the stimulus luminance periodicity and a discrete Fourier analysis was performed to estimate amplitude and phase of the first harmonic. Corrections were made to allow for the amplifier's filter properties. The frequency response curves (FRCs) reported in the results were obtained by plotting the amplitude of the first harmonic as a function of the temporal frequency.+/+ and HCN1−/− or HCN 2−/− mice are shown in +/+ littermates. As expected, the signal for the HCN1 or 2 transcripts is missing in the respective HCN knockout mice. Confocal images of immunofluorescence-stained trans retinal sections are shown in −/− and HCN2−/− do not show any specific staining for HCN1 or HCN2 proteins, respectively. Collectively, these data are evidence that the two knockout mouse lines used in our study are valid animal models. We thus shifted to electrophysiology to investigate the functional role of the HCN1 and HCN2 isoforms in the outer retina.Retinal transcripts of HCN1-2 isoforms detected by RT-PCR from HCNh in generating the initial nose was first proposed for lower vertebrate rods +/+, HCN1−/− and HCN2−/− mice.We investigated the role of the HCN channels in rod bright flash responses with patch clamp recordings obtained in dark-adapted mouse retinal slices. It has long been assumed that the initial sharp transient of voltage responses of rods to bright flashes, commonly referred to as nose, reflects the activation by membrane hyperpolarization of a current flowing through the HCN channels. A role of Ih in the rods of HCN+/+ (n = 19) and HCN2−/− (n = 3) mice, but not in those of HCN1−/− mice (n = 5). In HCN1−/− rods, instead, an inward-rectifying current with instantaneous kinetics was present, activating negative of −74/−81 mV (n = 5). HCN1−/− rods, in contrast to HCN+/+ and HCN2−/−, also did not display tail currents following the hyperpolarizing steps suggesting that tail currents are entirely due to the deactivation of Ih. Based on tail currents, Ih in HCN+/+ rods activates negative of −60/−67 mV. These results are consistent with the notion that HCN1 is the sole isoform expressed by rods.We measured in voltage-clamp the membrane current changes evoked by step hyperpolarization or depolarization. The results are illustrated in dark.) of rods. Note that this value is likely to be more depolarized than the unperturbed Vdark, due to shunting introduced by the finite seal resistance of the patch pipette on the cell's membrane +/+ (n = 22) and HCN2−/− (n = 4) animals expressed a typical nose in response to bright flashes , was absent in the photocurrent at all potentials (lower traces). It must then arise from the action of voltage-gated currents downstream of phototransduction. In HCN+/+ (n = 4) and HCN2−/− (n = 2) mice the nose became sharper and more pronounced with hyperpolarization into the range of activation of Ih (−/− rods (n = 2), hyperpolarization had the effect of suppressing the nose  and in agreement with a recent study dark but not upon hyperpolarization . Positive values indicate the presence of a nose as a rapid depolarization immediately after the peak response, whereas values near zero correspond to a plateau without the nose. All rods expressed a nose at Vdark values more depolarized than −35/−40 mV, independently of the presence of the HCN1, HCN2 or Ih. On the other hand, when Ih was absent in rods  the nose was not present for Vdark more hyperpolarized than −40/45 mV. These observations indicate that more than one current can contribute to the nose of the rod response to bright flashes: Ih plays a greater role at hyperpolarized potentials, with other currents acting in a more depolarized range. By examining the outward currents expressed by rods in both normal and HCN deficient mice, a candidate was identified having slow kinetics, activating upon depolarization positive of −57/−50 mV, and not showing inactivation  causes a generalized reduction of the FRC band-pass profile in all mice models.The temporal response properties of the outer retina can be better appreciated by examining its FRC profile obtained with the ERG, complemented by a single cell analysis in photoreceptors. The results of the ERG experiments are illustrated in −/− rods (n = 2), but not in normal rods (n = 2). It is thus clear that Ih is not the only current able to shape the frequency response of rods and that, depending on their actual membrane potential, the relative contribution of Ih and of other currents such as Ikx will vary. Based on these and other experiments shown in h will contribute more at more hyperpolarized membrane potentials. This may explain why the FRCs measured with the ERG maintained a degree of band-pass behavior even during phamacological blockade of Ih  in the hyperpolarizing response, which has been described in retinas of all animal species. For decades there has been a general consensus on the idea that this fast nose was due to the activation of Ih, the voltage dependent current that flows through HCN channels. I−/− mice the b-wave of the ERG in response to bright flashes is slower than in normal controls, while in HCN2−/−, appreciable kinetics changes also occur in the temporal course of the response to dim flashes (see with little influence from the inner segment currentsThe present study also contributes to clarify the functional significance of the different HCN isoforms expressed in the outer retina. Convergent evidence from immunolabelling and electrophysiological studies strongly indicate that HCN1 isoforms are mainly expressed at the inner segments of the photoreceptor  and 2A wshes see . This is−/− or HCN2−/− the FRCs behave as though partial pharmacological blockade of HCN was induced, thus causing attenuation of the normal band-pass profile. An almost full suppression of the band-pass profile may then be obtained by pharmacological inhibition of the residual HCN still expressed in HCN2−/− or HCN1−/− respectively (In a previous study on rats we have shown that the most evident effect of the HCN pharmacological inhibition can be observed on the profile of the FRC of the ERG. Here we confirm this observation also in mice and show that in either HCN1ectively .The membrane impedance of normal, HCN-deficient rods and rod bipolar cells are in substantial agreement with the FRCs of the ERG response. An important implication of this finding is that the gating properties of the HCN channels in photoreceptors and bipolar cells may also be inferred from non invasive ERG recordings. It is important to note, however, that the data from single cells reflect the filtering properties of their membrane suggesting that the impact of the HCN on the visual signals at the retinal output is bound to be determined also by the cascade of stages where the channels operate. Accordingly, the ERG's b-wave, whose main determinants are the rod bipolar cells, must reflect the operation of the second stage. It seems therefore reasonable to assume that the impact of HCN on processing of visual information would be further enhanced in the subsequent stages of the visual system including those in the retina and in the central pathways.