Overweight and obesity are highly prevalent in developed countries and are also becoming more frequent in the developing world. Overweight and obese patients have elevated levels of several inflammatory markers and this inflammatory state might contribute to their increased vascular risk. We summarize the effects of lifestyle changes, antiobesity agents, and bariatric surgery on serological inflammatory markers in overweight and obese patients. Most studies showed a decrease in inflammation with all 3 interventions. However, it remains to be established whether the decrease in inflammatory markers induced by lifestyle changes or (where indicated) with antiobesity agents or bariatric surgery will translate into reduced vascular morbidity and mortality in overweight and obese patients. Overweight and obesity are highly prevalent in developed countries . Approxiα (TNFα) [α [Adipose tissue is not metabolically inert but synthesizes and secretes proinflammatory mediators, including interleukin 6 (IL-6) , tumor nα (TNFα) , 10, 11,α (TNFα) , and monα (TNFα) , 13. Sevα (TNFα) –26. Compα (TNFα) , 26–28, TNFα) [α , 26–30, TNFα) [α , as wellTNFα) [α , 32, IL-TNFα) [α , solubleTNFα) [α , 34, 35,TNFα) [α , 37, solTNFα) [α –38, and TNFα) [α . In obesTNFα) [α –41 and wTNFα) [α .The activation of inflammation that is present in obesity might contribute to the increased vascular morbidity and mortality of these patients, since elevated levels of inflammatory markers are associated with increased vascular risk –46. In tLifestyle measures are the cornerstone of the management of obesity . In randSeveral studies evaluated the effects of diet and exercise alone or in combination on inflammatory markers in obese patients.n = 11–100) assessed the effects of low-calorie, very low-calorie, low-fat, or low-carbohydrate diets on inflammatory markers in obese patients , the brief duration of the intervention, the insufficient sample size, or the degree of body weight reduction. Overall, the greatest reductions in serological markers of inflammation were observed in studies where an at least 10% weight loss was achieved.Interestingly, recent studies demonstrated that brief periods of reduced food intake can increase resistance to ischemia reperfusion injury in rodents and modify circulating levels of pro-inflammatory agents in humans , 82. Then = 8–199) evaluated the effects of exercise training, resistance training, or aerobic exercise training for 2–12 months on inflammatory markers . Finally, it has been reported that both strenuous exercise (which can lead to muscle damage) and overtraining (which might induce oxidative stress) might act as an inflammatory stimulus and raise inflammatory markers .n = 12–190, duration 4 weeks to 2 years) reported a significant decrease in BMI in overweight [P = .71) [α levels were less consistent with both significant decreases [More data are available on the effects of combining both diet and exercise on inflammatory markers  26, 29,, 94–114.erweight , 112, oberweight , 112 anderweight , 114. Inerweight , 98–114;P = .71) . In accoP = .71) . The majP = .71) , 110–113P = .71) , 96, 109P = .71) , 111. LiP = .71) , and sVCecreases , 105 andα levels) whereas exercise training had no effect; however, weight loss was observed only in patients on diet [α levels) in obese postmenopausal women despite a similar weight and fat loss in both groups [α levels increased significantly in the 3 intervention groups but the increase was smaller in the combined diet and exercise group and probably represents a chance finding [α levels did not change significantly [Finally, 4 studies assessed the separate and combined effects of exercise and diet intervention on several cytokines. Nicklas et al. reported that an 18-month dietary intervention resulted in significant reductions in hsCRP and IL-6 concentrations  differences in the studied population , in the baseline concentration of cytokines and inflammatory status of each patient, as well as in the laboratory methods used for the measurement of cytokines.Orlistat induces weight loss by reducing intestinal fat absorption (by up to 30%) by binding to pancreatic lipases and partially inhibiting the hydrolysis of dietary fat (triglycerides) into absorbable free fatty acids and monoacylglycerols . CompareA limited number of studies evaluated the effects of orlistat on inflammatory markers in obese patients . In someα levels was also reported after treatment with orlistat [α in some studies was greater in the diet plus orlistat group compared with patients who were only on diet even after correcting for the greater weight loss observed in the orlistat group [A reduction in serum IL-6 , 129 andorlistat , 129. Ororlistat . Interesat group , 129. OvSibutramine lowers body weight by increasing satiety and by reducing appetite through the selective inhibition of the neuronal reuptake of serotonin and noradrenaline within the hypothalamus , 131. Inα and increased serum levels of IL-10 when given for 3–6 months . The Food and Drug Administration also stated that sibutramine is not to be used in patients with a history of coronary heart disease, stroke or transient ischemic attack, heart failure, heart arrhythmias, peripheral arterial disease, or uncontrolled hypertension . These recommendations were based on the results of the Sibutramine Cardiovascular Outcome Trial (SCOUT), which showed an increased risk of nonfatal cardiovascular events with sibutramine compared with placebo in patients with known or high risk for cardiovascular disease .It should emphasized that the Committee for Medicinal Products for Human Use of the European Medicines Agency recently concluded that the risks of sibutramine are greater than its benefits and recommended the suspension of marketing authorization of this agent across the European Union , malabsorptive , and combination procedures (Roux-en-Y gastric bypass) . All forP = .05) and there was a trend for significant decrease also at 12 months (P = .08) [per se is the main factor that causes the fall in hsCRP levels, since bariatric surgery results in substantially greater weight loss than lifestyle changes or antiobesity agents. The lack of correlation between weight loss and hsCRP fall might be attributed to the small number of patients included in these studies, which limits their statistical power.Several studies reported a reduction in serum hsCRP levels after Roux-en-Y gastric bypass  158–166166. ThisP = .08) . In a reP = .08) , laparosP = .08) , and gasP = .08) , also reP = .08) . The redn = 26) did not report significant changes in IL-6, TNFα, sTNFR-2, sICAM-1, and sVCAM-1 levels following gastric bypass [α levels [Other markers of inflammation, including IL-6 , 165, ILc bypass . Howeverc bypass . Serum lc bypass , 172, ILc bypass , sTNFR-2c bypass , 170, MCc bypass , solublec bypass , 182, sIc bypass , 182, anc bypass  also falc bypass , 182. Hoc bypass , sVCAM-1c bypass , and sICc bypass  did not α levels  were notα levels , suggestα levels (α levels after liposuction [α levels [In contrast to bariatric surgery, removal of abdominal subcutaneous fat by liposuction does not appear to reduce hsCRP, IL-6, or TNFα levels  [183, 18osuction . In the α levels . Even thExisting evidence suggests that lifestyle measures, antiobesity agents, and bariatric surgery reduce serum levels of inflammatory markers in obese patients. The lack of significant change in some studies appears to be due to small sample size and/or small weight loss achieved. It appears that the reduction in inflammation is primarily driven by weight loss . In a re


Migratory marine vertebrates move annually across remote oceanic water masses crossing international borders. Many anthropogenic threats such as overfishing, bycatch, pollution or global warming put millions of marine migrants at risk especially during their long-distance movements. Therefore, precise knowledge about these migratory movements to understand where and when these animals are more exposed to human impacts is vital for addressing marine conservation issues. Because electronic tracking devices suffer from several constraints, mainly logistical and financial, there is emerging interest in finding appropriate intrinsic markers, such as the chemical composition of inert tissues, to study long-distance migrations and identify wintering sites. Here, using tracked pelagic seabirds and some of their own feathers which were known to be grown at different places and times within the annual cycle, we proved the value of biogeochemical analyses of inert tissue as tracers of marine movements and habitat use. Analyses of feathers grown in summer showed that both stable isotope signatures and element concentrations can signal the origin of breeding birds feeding in distinct water masses. However, only stable isotopes signalled water masses used during winter because elements mainly accumulated during the long breeding period are incorporated into feathers grown in both summer and winter. Our findings shed new light on the simple and effective assignment of marine organisms to distinct oceanic areas, providing new opportunities to study unknown migration patterns of secretive species, including in relation to human-induced mortality on specific populations in the marine environment. Understanding spatiotemporal dynamics of marine vertebrates is essential to determine when and where animals are exposed to human impacts Although recent advances in tracking technology are helping to fill the current gap in marine migration knowledge, studies are usually restricted to a few individuals often tracked for short periods due to logistical and financial constraints Migrating birds with known moulting patterns provide a singular opportunity to validate the utility of biogeochemical markers in the marine environment. As feathers grow, the elements and their isotopic forms assimilated through the diet are incorporated into the keratin structure. Once formed, feathers become metabolically inert, thus integrating the composition of the local food web where feathers were grown Twice a year, millions of seabirds travel tens of thousands of kilometres across the equator to move between wintering and breeding areas, enhancing their susceptibility to threats posed by human activities Calonectris diomedea, a long-distance migrant that breeds on temperate northeast Atlantic and Mediterranean Islands and winters in major upwelling areas of the Atlantic Ocean 13C), nitrogen (δ15N), sulphur (δ34S), hydrogen (δ2H ) and oxygen (δ18O) and the elemental concentrations of selenium (Se), lead (Pb) and mercury (Hg) in feathers from the tracked birds moulted at the end of the breeding season and at the wintering grounds Here, we explored the value of using biogeochemical analyses as intrinsic markers to understand long distance movements of vertebrates in the marine environment. To do so, we studied Cory's shearwaters 13 C, 2,14F = 66.6, P<0.001; δ15 N, 2,12F = 40.1, P<0.001; δ34 S, 2,15F = 146.8, P<0.001; δ2 H, 2,14F = 74.0, P<0.001; δ18 O, 2,12F = 48.5, P<0.001) as well as in elementary content . These differences allowed us to assign any individual to a relatively restricted breeding area using isotopic signature (100% correct classification) and, to a lesser extent, elemental composition (72.0% correct classification) of primary feathers , Brazil-Falklands (n = 5), Agulhas (n = 4), Canary (n = 2) Currents, with the South Central Atlantic Ocean (n = 2) and with the Gulf of Guinea (n = 1). These oceanic areas were not different from those previously reported for the species 13 C, 2,7F = 28.1, P<0.001; δ15 N, 2,9F = 45.9, P<0.001; δ34 S, 2,7F = 34.0, P<0.001; δ2 H, 2,8F = 21.3, P = 0.001; δ18 O, 2,9F = 2.36, P = 0.079) and could also be used to assign birds to specific wintering oceanic areas . Indeed, elemental analyses of feathers moulted in wintering areas were more successful in assigning birds to their breeding origin than to their wintering areas .st primary and 8th secondary feathers from those birds that returned, obtaining year-round GLS data and feather samples from 9, 7 and 9 birds, respectively. Feathers were analysed for stable isotopes of carbon, nitrogen, sulphur, hydrogen and oxygen and for elemental concentrations of selenium, lead and mercury.Transoceanic migrations can currently be investigated using global location sensing (GLS) devices based on recording light levels, which can be deployed on a bird all year-round and will give 2 positions per day with an accuracy of 186±114 km 13C, δ15N, δ34S, δ2H and δ18O values were determined. Isotope ratios are expressed conventionally as δ values in parts per thousand (‰) according to the following equation: δX = [(Rsample/Rstandard) - 1]×1000, where X (‰) is 13C, 15N, 34S 2H or 18O and R are the corresponding ratio 13C/12C, 15N/14N, 34S/32S, 2H/1H or 18O/16O related to the standard values. Rstandard for 13C is Pee Dee Belemnite (PDB), for 15N is atmospheric nitrogen (AIR), for 34S is troilite of the Canyon Diablo Meteorite (CDT) and for 2H and 18O is Vienna Standard Mean Ocean Water (V-SMOW). The isotopic ratio mass spectrometry facility at the Serveis Científico-Tècnics of Universitat de Barcelona (Spain) applies international standards  while Duke Environmental Stable Isotope Laboratory of Duke University (USA) uses internal keratin standards previously calibrated against NIST and IAEA reference materials , all of them inserted every 12 samples to calibrate the system and compensate for any drift over time. Replicate assays of standard materials indicated measurement errors of±0.1, ±0.2, ±0.3, ±1.5 and ±0.1‰ for carbon, nitrogen, sulphur, hydrogen and oxygen respectively but these are likely underestimates of true measurement error for complex organics like feathers.All feathers were washed in a 0.25 M sodium hydroxide solution, rinsed thoroughly in distilled water to remove any surface contamination, dried in an oven at 60°C to constant mass, and ground to a fine powder in a freezer mill  operating at liquid nitrogen temperature. Subsamples of 0.4 mg of feather powder for carbon and nitrogen, about 3.5 mg for sulphur, and 0.25 mg for hydrogen and oxygen analyses were weighed to the nearest µg, placed into tin and silver capsules and crimped for combustion. Samples were oxidized in a Flash EA1112 and TC/EA coupled to a stable isotope mass spectrometer Delta C and Delta Plus XL, respectively through a Conflo III interface , where the δTo determine trace element concentrations, 50 mg of feather powder was digested in 1 ml of nitric acid (69–70%) and 0.5 ml of hydrogen peroxide (30%) using Teflon® bombs during 12 hours at 60°C. The result of the digestion was diluted into 7 ml of distilled water. Quantitative analysis was performed using the ICP-AES technique  at Serveis Científico-Tècnics of Universitat de Barcelona (Spain). Accuracy of analysis was checked by measuring certified reference material .Element concentrations were log-transformed to achieve normality. Differences among breeding and among wintering populations in stable isotope and element values were tested with one-way ANOVA. Tests among wintering populations do not include the Canary Current, South Central Atlantic and Gulf of Guinea because fewer than four birds were found in each area. To assess whether feather composition could be linked to specific oceanic areas, we used classificatory discriminant analyses (SPSS 2003) on the composition of both type of feathers in relation the breeding and wintering areas. Discriminant analyses were carried out separately for stable isotope signatures of C, N, S, H and O and for combined element concentrations of Se, Pb and Hg. We tested models by jackknife cross-validation. Models were built step by step including independent variables according to the Wilks' Lambda criterion, and breeding and wintering areas were weighted according to the sample size.Table S1Biogeochemical composition of summer and winter feathers. Stable isotope signatures (‰) and log-transformed element concentrations (ng g-1) for primary feathers (P1) according to the breeding areas and for secondary feathers (S8) according to the wintering areas. Values are means±standard deviation and sample size is shown in brackets. Significant differences among breeding and among wintering populations are indicated by *** P<0.0001, ** P<0.05 and * P<0.1. ANOVA-test among wintering populations do not include Canary Current, South Central Atlantic and Gulf of Guinea. Standard coefficients from discriminant functions on original data (explained variance in brackets) are conducted separately for stable isotopes and element concentrations.(0.06 MB DOC)Click here for additional data file.


However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′- or 3′-blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short-patch BER) or several nucleotides (long-patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through  N-glycosylic bond  termini . The ensproteins .As experimental data accumulate for non-mammalian systems, it is becoming clear that there are significant differences in the strategies employed by different species during the post-excision events that take place in BER . Additioin vitro assays that allow the steps of the repair process to be monitored . We also examined the repair kinetics of a U:G mispair, measuring the level of fully repaired product at different times  efficiently and reproducibly converted the uracil-containing DNA to a form susceptible to igestion . Important times . We founTo assess whether the first step of the repair process is dependent on uracil DNA glycosylase (UDG) activity, we examined the effect of uracil-DNA glycosylase inhibitor (Ugi) on the reaction. This small peptide is a specific inhibitor of UDGs from the UNG family, and has been successfully used to detect UDG-dependent uracil excision in cell extracts from different organisms  end and a 3′-α,β-unsaturated aldehyde  and Fpg, respectively  prior to PAGE analysis . Therefore, it probably represents a 5′-dRP blocked terminus.We next analyzed the nature of the 5′ terminus, using DNA substrates in which the damaged strand was labeled at the 3′ end . As the ubstrate , lane 9.appeared , lane 8,Taken together, these results indicate that AP sites generated after uracil excision by Arabidopsis cell extracts may be processed either by AP endonucleases or by AP lyases, yielding either 5′- or 3′-blocked ends, respectively.As shown in Repair reactions were performed incubating Arabidopsis extracts with a duplex oligonucleotide labeled with fluorescein at the 5′ end of the upper strand, and with Alexa Fluor at the 5′ end of the lower strand . We founWe then investigated whether the single-nucleotide insertion observed in the presence of dCTP was template-dependent. We performed repair reactions with DNA substrates containing either a U:G or a U:A pair, in the presence of a single deoxynucleotide  . Only dCThe standard model of LP mammalian BER proposes that, after base excision and AP site incision, the damaged strand undergoes displacement 3′ to the nick, and that the resulting flap structure is finally excised by FEN1 before DNA ligation can occur . As expeTaken together, these results suggest that excision repair of uracil by Arabidopsis whole-cell extracts may arise by single-nucleotide insertion, and by an extended (at least 3-nt-long) DNA synthesis involving strand displacement and flap excision.In mammalian cells, it has been proposed that SP BER is Pol β-mediated, whereas LP BER is dependent of replicative DNA polymerases δ and/or ε . This isHpaII-sensitive product; neither in the presence of dCTP nor in the presence of all four dNTPs  in Arabidopsis whole-cell extracts, which measures DNA repair synthesis in plasmid substrates treated with UV-C, methylene blue or cisplatin, but does not allow the unambiguous distinction of the relative contributions of NER and BER, especially for oxidative lesions induced by methylene blue , but its role in uracil repair is still unknown. Therefore, the assay system described here allows reliable monitoring of uracil-initiated BER in Arabidopsis cells.A major aim of this work was to extend the biochemical analysis of BER to plants by developing an assay for the specific detection of this important repair pathway in Arabidopsis. We used similar cell extracts to those of previous studies  involved catalyze β,δ-elimination. This avoids the need for an AP endonuclease to clear the 3′-blocked end, but implies that 3′-phosphoesterase activity may be required to restore conventional 3′-OH. A possible candidate for this role in Arabidopsis is a previously described plant 3′-phosphoesterase that specifically binds to single-strand DNA breaks , and only the latter has been shown to perform incisions in DNA containing abasic sites  involved in the repair synthesis step during plant BER by performing repair assays in the presence of two inhibitors. Our results indicate that DNA synthesis after uracil excision is aphidicolin resistant and ddCTP sensitive. In contrast, the DNA synthesis observed during NER in Arabidopsis cell extracts is both aphidicolin- and ddTTP-resistant . As this is accompanied by the accumulation of AP incision and nucleotide insertion products, it is likely that the rate-limiting steps are neither uracil excision nor AP processing, but rather some of the factors required during gap filling and/or ligation. In fact, we found that the exogenous addition of heterologous DNA ligase and DNA polymerase β activities, as well as of the Arabidopsis scaffold protein AtXRCC1, to some extent increase the level of fully repaired product. However, it is also conceivable that the repair rate is additionally controlled by some inhibitory factor(s) in the extracts. In mammalian cells, the poly(ADP-ribose) polymerase PARP-1 has a high affinity for the single-strand-break intermediates generated during BER  were plated on 10-cm-diameter Petri dishes containing 25 ml of 0.44% (w/v) MS medium , supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar, pH 5.8. Plates were transferred to the growth chamber under long-day conditions (16-h light/8-h dark) at 23°C. After 15 days, whole plants were harvested, frozen in liquid nitrogen and stored at −80°C.Sterilized seeds of wild-type 2, and the resulting powder was resuspended in 2–3 volumes (w/v) of homogenization buffer containing 25 mm HEPES-KOH, pH 7.8, 100 mm KCl, 5 mm MgCl2, 250 mm sucrose, 10% glycerol, 1 mm DTT and 1 μl ml−1 protease inhibitor cocktail (Sigma-Aldrich). The homogenate was incubated for 30 min at 4°C and centrifuged at 13 000 g for 1 h. The supernatant was filtered through a 20-μm nylon mesh and dialyzed overnight against 25 mm HEPES-KOH, pH 7.8, 100 mm KCl, 17% glycerol and 2 mm DTT. Protein concentration was determined by the Bradford assay with BSA standards, and the extract was stored in small aliquots at −80°C.Seedling extracts were prepared by introducing several modifications to previously published methods . Human DNA polymerase β was from Trevigen (http://www.trevigen.com). E. coli T4 DNA ligase was purchased from Promega (http://www.promega.com) and HpaII was purchased from Roche (http://www.roche-applied-science.com). E. coli Nth and Fpg DNA glycosylases/lyases were prepared as previously described .escribed . Partialhttp://www.operon-biotech.com), and were purified by PAGE before use. Double-stranded DNA substrates were prepared by mixing a 5-μm solution of the upper-strand oligonucleotide  with a 10-μm solution of the lower-strand oligonucleotide , heating to 95°C for 5 min and slowly cooling to room temperature (25°C). DNA containing a natural AP site opposite guanine was prepared by incubating a DNA duplex containing a U:G mispair, prepared as above, with E. coli UDG (0.5 U) at 30°C for 5 min.Oligonucleotides used to prepare DNA substrates  were synm HEPES-KOH, pH 7.8, 70 mm KCl, 5 mm MgCl2, 1 mm DTT, 0.4 mm EDTA, 2 mm ATP, 36 μg BSA, 1 mm NAD, 2% glycerol, 20 μm each deoxynucleotide , substrate DNA (40 nm) and 70 μg of extract. After incubation at 30°C for 3 h, reactions were stopped by adding 20 mm EDTA, 0.6% SDS and 0.5 mg ml−1 proteinase K, and the mixtures were incubated at 37°C for 30 min. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol-precipitated at −20°C in the presence of 0.3 mm NaCl and 16 μg ml−1 glycogen. Samples were resuspended in 10 μl of 90% formamide, and were heated at 95°C for 5 min. Where indicated, DNA was resuspended in 5 μl of SuRE/Cut Buffer L containing 5 U of HpaII (Roche) and incubated at 37°C for 1 h. Reactions were then stopped by adding 5 μl of 90% formamide and then heating at 95°C for 5 min. Reaction products were separated in a 12% denaturing polyacrylamide sequencing gel (40 × 20 cm) containing 7 m urea. Fluorescein- or Alexa-labeled DNA was visualized in an FLA-5100 imager, and data were analyzed using Multigauge .Repair reactions (50 μl) contained 45 mm MgCl2. Control AP lyase reactions were performed by incubating a DNA containing a natural AP site opposite guanine with E. coli Nth or Fpg at 37°C, for 30 or 60 min, respectively. Control AP endonuclease reactions were performed by incubating a DNA containing a natural AP site opposite guanine with human APE1 (10 U) at 37°C for 60 min, supplemented with 2.4 U of human DNA polymerase β when generating a control dRP-lyase product. When analyzing 5′ ends, reaction products were stabilized by the addition of freshly prepared sodium borohydride  to a final concentration of 300 mm, incubation at 0°C for 30 min and desalting in a microspin G-25 column . All reaction products were purified, separated and analyzed as described above.Repair reactions (50 μl) were performed at 30°C for 3 h in the buffer indicated above (with no dNTPs), either in the presence or absence of 5 m


L mediates neural recognition byCa+2, Mg+2-independent homophilic binding. In the human and murine immune systems, L1 binds to the “classical” vitronectin receptor, αVβ3, and fibronectin receptor, α5β1, respectively, and abstains from homophilic binding. Homophilic L1 binding probably involves antiparallel alignment of several interactive domains. Integrin binding is mediated by a short segment of immunoglobulinlike domain 6, which includes two RGD repeats in rodent L1 and one RGD motif in human L1. L1 is modulated in activated leucocytes in vitro in parallel to L-selectin, and diverse cell types release intact L in vivo and in vitro. Released L1 can bind to laminin and adheres to the extracellular matrix of sciatic nerve, M21 melanoma, and possibly spleen and other tissues. It can support integrin-dependent cell migration and preliminary data implicate it in tumor development and transnodal lymphocyte migration.The cell-adhesion molecule L1 was originally described in the nervous system.It has recently been detected in CD4


In this article this hypothesis has been tested theoretically.Recently originalization was proposed to be an effective way of duplicate-gene preservation, in which recombination provokes the high frequency of original (or wild-type) allele on both duplicated loci. Because the high frequency of wild-type allele might drive the arising and accumulating of advantageous mutation, it is hypothesized that recombination might enlarge the probability of neofunctionalization (Pneo.Results show that through originalization recombination might not only shorten mean time to neofunctionalizaiton, but also enlarge Pneo cannot be expected to be small.Therefore, recombination might facilitate neofunctionalization via originalization. Several extensive applications of these results on genomic evolution have been discussed: 1. Time to nonfunctionalization can be much longer than a few million generations expected before; 2. Homogenization on duplicated loci results from not only gene conversion, but also originalization; 3. Although the rate of advantageous mutation is much small compared with that of degenerative mutation, P Xenopus Laevis 0, decreaneo for unlinked duplicate genes in large populations, simulation results in a larger population (N μneo = 0.2) are shown in Figure neo > 0.2) (not shown). However, even when N μneo = 0.2, the results sufficiently indicate that Tneo for unlinked duplicate genes is shortened when positive selection is strong , Tneo for unlinked gene duplication is larger than that for linked under the either DNR or HI selective model; and Tneo for unlinked gene duplication becomes greatly prolonged under the HI selective model , the evolutionary behavior of an advantageous mutation is similar to that of a nearly neutral mutation . Therefonon can be prolonged in a larger population (roughly N μnon > 0.1); and x0 is kept higher in the population. So prolonged Tnon, shortened Tneo and high x0 might jointly result in larger Pneo for unlinked gene duplication. In order to validate this prediction, direct observations of Pneo are also carried out.In our previous studies ,16, we o0, y1, y2, y3, y4, y5, y6, y7, y8, respectively. Fitnesses of individuals with various genotypes are shown in Table non) = 10-4 and that of advantageous (μneo) = 10-6. Initially let y0 = 1, and y1 = y2 = y3 = y4 = y5 = y6 = y7 = y8 = 0.Now consider a model involving neofunctionalziation and nonfunctionalization. In the gene pool, there are nine possible chromosomal haplotyes in the population, "00", "01", "02", "10", "11", "12", "20", "21", "22", whose frequencies are denoted as yneo can be approximately expressed as y2+y5+y8 or y6+y7+y8, and the probability of nonfunctionalization as y1+y4+y7 or y3+y4+y5. Because under the DNR and HI selective model described above, y4, y5, y7 and y8 are quite small and close to 0, Pneo is approximately equal to y2 or y6, and the probability of nonfunctionalization is approximately equal to y1 or y3. So only dynamic changes of y2 and y1 are shown in numerical results as the proxies for the probabilities of neofunctionalization and nonfunctionalization, respectively, and y0 is treated as a proxy of non-resolution  [Numerical results are shown in Figure ization) .-6), for unlinked gene duplication, an equilibrium is quickly reached for y0, y1, and low-level y2, while for linked duplication, y0 continually decrease with increasing y1 and very low (close to 0) y2 ; and y2 increase continually up to ~0.5. However, for unlinked gene duplication, y0 is only kept high for a period of time and then crashes while y2 increases suddenly up to be very high (~1) , for linked duplication, y, for lingure non ,16,20-23neo. First, under strong positive selection, when N is small (roughly N μnon < 0.1), Pneo for unlinked gene duplication under both DNR and HI selective models are all close (see Table neo/μnon [non) > 0.1), both predictions from Equation 1 and 2 are different from our observations under the DNR selective model in simulation , simulation results of Pneo with positive selection coefficient (s) are shown in Table neo for unlinked gene duplication under the DNR selective model are also close to Walsh's prediction - μneo/μnon [neo becomes different from expectations from Equation 1 and 2; and Pneo for unlinked gene duplication is larger than that for linked under both the DNR and HI selective models  enlarges Pneo, which is quite consistent with observations of Pneo in Table Second, in a given larger population (N μneo/μnon . If s beneo were obtained under two extreme conditions: linked (r = 0) and unlinked (r = 0.5). However, in most real cases 0 < r < 0.5, so Pneo with these conditions are also simulated, and results are shown in Figure neo becomes larger under both DNR and HI selective models. This reinforces our conclusion that recombination enlarges Pneo under strong selection.Third, these observations of Ped r = 0.. Howeverneo = 10-6, or μnon = 10-4 and μneo = 10-6. They also can be changed into other more realistic values, for example μnon = 10-6, and μneo = 10-9 [One might argue that these parameters used in above analyses are not realistic enough, for example μo = 10-9 ,14,23, bneo and dynamic changes of genotypes observed directly in simulation are quite consistent with predictions from numerical results. In our previous studies, it has been observed that high x0 at the equilibrium can be broken by genetic drift in finite populations [The sudden crash of the balance of chromosomal haplotype frequencies for unlinked gene duplication in numerical results shown in Figure ulations ,16. In tAccording to our theoretical results presented in this study and previous studies, several views on the evolution of gene duplication should be revised and reconsidered.-6) [non ≤ 0.01), can mean time to nonfunctionaliztion be simply estimated to be on the order of the reciprocal of degenerative mutation rate for gene duplication - ~ 1/(2 μnon) [non > 0.1), especially for unlinked gene duplication [non is prolonged dramatically even in a modest population (0.1 < N μnon ≤ 1) [0) in the population retards nonfunctionalization apparently, because at nonfunctionalization x0 must be 0. In nature populations, population sizes are usually not small  [non is usually larger than expected in previous studies (~106 generations).It was commonly considered that for gene duplication, mean time to nonfunctionalization is a few million generations or less (assume degenerative mutation rate is ~10-6) . In ligh(2 μnon) ,22,23. Hlication ,16,20-23non ≤ 1) ,16. The non ≤ 1) ,16. High104~105) , so Tnonn (the rate of non-synonymous nucleotide substitution) and ds (the rate of synonymous nucleotide substitution) of duplicate genes are both small. However, in originalization, under purifying selection, dn of duplicate genes are small while ds are large. This prediction might be applicable to distinguish the effect of originalization from that of gene conversion on genomic evolution.Homogenization is often argued to originate from gene conversion. However, in this study, it is observed that under recombination originalization can also result in homogenization. This result is obtained from the principles of traditional population genetics, under a theoretical framework completely different from gene conversion. In our previous studies on originalization, the effect of gene conversion was neglected. Moreover, in originalization, the wild-type allele is buffered with high frequency on both duplicated loci, which retards the divergence of duplicate genes, while in gene conversion, it is not certain that the wild-type allele is converted on duplicated loci. And during gene conversion, dneo is equal to ~μneo/μnon, regardless of recombination. However, under strong positive selection, in larger populations (N μnon > 0.1) Pneo becomes larger under recombination than that under linkage , the conclusion of PAt the genic level speciation is a differential process accompanied by differential adaptations . It has Methods of simulation and numerical analyses have been described in detail in our previous studies ,16.CX conceived of the study, carried out the most works, and drafted the manuscript. YXF participated in the design of the study. RH and SQL performed some simulation works. All authors read and approved the final manuscript.lflf27@yahoo.com.cnCheng Xue, GuangDong Institute for Monitoring Laboratory Animals, and Key Laboratory of Laboratory Animals in GuangDong, 105 Road Xingang West, Guangzhou, 510260, China. E-mail: labking@sohu.comRen Huang, GuangDong Institute for Monitoring Laboratory Animals, and Key Laboratory of Laboratory Animals in GuangDong, 105 Road Xingang West, Guangzhou, 510260, China. E-mail: Shu-Qun Liu, Laboratory for Conservation and Utilization of Bio-resources, Yunnan University,shuqunliu@gmail.comYunnan, China. E-mail: Yunxin.fu@uth.tmc.eduYun-Xin Fu, Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Yunnan, China, and Human Genetics Center, School of Public Health, University of Texas at Houston, Houston, Texas USA. E-mail: 0, y1, y2, y3, y4, y5, y6, y7, y8 be the frequencies of chromosomal haplotypes, "00", "01", "02", "10", "11", "12", "20", "21", "22", respectively. The fitness of individual genotypes is shown in Table 1 = 0; Under the HI selective model, s1 = 1. Because y0+y1+y2+y3+y4+y5+y6+y7+y8 = 1, only 7 of them are independent. Here we focus on the first 7 frequencies. Therefore, mean population fitness (w) and differential changes of chromosomal haplotype frequencies are given byConsider a pair of duplicated loci on the same chromosome in a random mating, diploid population without considering genetic drift. Let yneo is advantageous mutation rate and μnon is degenerative mutation rate.where r is recombination rate between duplicated loci, s is positive selective coefficient, μ


The incidence of obesity has risen dramatically over the last few decades. This epidemic may be affected by exposure to xenobiotic chemicals. Bisphenol A (BPA), an endocrine disruptor, is detectable at nanomolar levels in human serum worldwide. Adiponectin is an adipocyte-specific hormone that increases insulin sensitivity and reduces tissue inflammation. Thus, any factor that suppresses adiponectin release could lead to insulin resistance and increased susceptibility to obesity-associated diseases.a) the effects of low doses of BPA and estradiol (E2) on adiponectin secretion from human breast, subcutaneous, and visceral adipose explants and mature adipocytes, and b) expression of putative estrogen and estrogen-related receptors (ERRs) in these tissues.In this study we aimed to compare We determined adiponectin levels in conditioned media from adipose explants or adipocytes by enzyme-linked immunosorbant assay. We determined expression of estrogen receptors (ERs) α and β, G-protein–coupled receptor 30 (GPR30), and ERRs α, β, and γ by quantitative real-time polymerase chain reaction.2. Adipose tissue expresses similar mRNA levels of ERα, ERβ, and ERRγ, and 20- to 30-fold lower levels of GPR30, ERRα, and ERRβ.BPA at 0.1 and 1 nM doses suppressed adiponectin release from all adipose depots examined. Despite substantial variability among patients, BPA was as effective, and often more effective, than equimolar concentrations of EBPA at environmentally relevant doses inhibits the release of a key adipokine that protects humans from metabolic syndrome. The mechanism by which BPA suppresses adiponectin and the receptors involved remains to be determined. Polycarbonates are used in numerous consumer products, including food and water containers, baby bottles, linings of metal food and beverage cans, medical tubing, epoxy resins, and dental fillings. Small amounts of BPA can migrate from polymers to food or water, especially when heated . Dozen oBPA has been reported to alter several metabolic functions . However2), suggesting that it should mimic or compete with endogenous estrogens only at the micromolar range. Yet, BPA at nanomolar doses often displays stronger estrogen-like activities than E2 itself. Several speculations have been proposed to reconcile this discrepancy: a) BPA binds differently within the ligand-binding domain of ERα or ERβ and recruits dissimilar coregulators (b) BPA elicits rapid responses by binding to membrane-anchored ERs (c) BPA binds to estrogen-related receptor γ (ERRγ), an orphan nuclear receptor belonging to the ERR family of receptors that do not directly bind E2  α and β , its bingulators ; b) BPA ored ERs , an as-yored ERs , or G-prored ERs ; and c)  bind E2 . BPA was bind E2 .a) compare the effects of low doses of BPA and E2 on adiponectin secretion from human breast, subcutaneous (SC), and visceral (VIS) adipose explants; b) examine whether they exert direct effects on isolated mature adipocytes; c) determine the effects of an ERα/ERβ antagonist  on adiponectin release; and d) compare the expression of ERα, ERβ, GPR30, ERRα, ERRβ, and ERRγin breast, SC, and VIS adipose tissue.The objectives of the present study were to a) samples from breast reduction, b) abdominal SC samples from abdominoplasty, and c) matched VIS  and SC samples from morbidly obese individuals undergoing gastric bypass surgery.The study was approved by the Institutional Review Board of Christ Hospital . Surgical samples were obtained from patients who gave written informed consent. Three types of adipose specimens were obtained: 2 and BPA  and ICI  in ethanol at 50–100 mM. Solvent controls (≤ 0.001% ethanol) were included in all experiments. At the end of a 6-hr incubation, explant weights were determined and conditioned media (CM) were collected.We cut tissue into small (~ 2 × 2 × 2 mm) explants and placed them into 48-well polystyrene plates  containing glucose- and phenol red-free Dulbecco’s modified Eagle medium supplemented with 10 mM HEPES, 2 mM glutamine, 2 mM pyruvate, and 1% charcoal-stripped fetal bovine serum . We prepared stock solutions of EWe used SC adipose tissue from abdominoplasty to prepare mature adipocytes as described by Adiponectin in CM was quantified by a fluorescent-sandwich ELISA, optimized in our laboratory using a matched monoclonal antibody pair against human adiponectin . These antibodies recognize epitopes in the globular head of adiponectin and detect all isoforms. Black 96-well plates  were coated with the capture antibody and blocked with 0.5% BSA. Plates were then coincubated with the detection antibody and recombinant human adiponectin (R&D) or CM aliquots. After 2 hr, we added streptavidin-conjugated horseradish peroxidase and a fluorimetric substrate . We read fluorescence at 325 nm excitation and 420 nm emission, using a Gemini XPS fluorescent microplate reader . The lowest detectable level was 100 pg/mL. We validated assay parameters against commercial plates from the same vendor.B2M) expression  at all doses except 0.1 nM. On the other hand, BPA generated a clear U-shaped response, being significantly suppressive at both the 0.1 and 1 nM doses but not at higher doses. 2, or ICI in explants from individual patients. Suppression of adiponectin by BPA and E2 was significant in five of eight and five of six samples tested, respectively. We also examined several samples for the effects of 1 nM ICI. In this case, three of five samples showed significant inhibition.2 on adipose tissue other than the breast. For that, we obtained SC abdominal adipose samples from nine women undergoing abdominoplasty. in vitro was highly variable, ranging from 7.1 ng/100 mg/6 hr in one patient to 155.2 ng/100 mg/6 hr in another.We next explored the effects of BPA and E2 on adiponectin release in an SC abdominal sample from one patient, selected as a representative. Both compounds generated U-shaped curves, with BPA significantly inhibiting adiponectin at the 0.1, 1, and 10 nM doses, whereas E2 was effective at the 1 and 10 nM doses. 2 was effective only in four of nine samples. We examined the effect of 1 nM ICI in four samples, only one of which showed significant inhibition.2 significantly inhibited adiponectin release from mature adipocytes at most doses examined, albeit without exhibiting dose-dependent effects. ICI at all doses examined significantly inhibited adiponectin release  and SC adipose samples from several morbidly obese patients undergoing gastric bypass surgery. 2 did not reach statistical significance. We saw a more profound inhibition of 65% and 50% by both BPA and E2 in VIS explants at 6 and 24 hr, respectively.To examine whether adiponectin responsiveness to BPA or E2, and ICI. 2 were effective in suppressing adiponectin release from SC explants at 0.1 and 1 nM. E2 at 1 and 10 nM significantly suppressed adiponectin release from VIS explants, whereas BPA had no effect at all doses examined. Surprisingly, 1 nM ICI suppressed adiponectin release from VIS explants by as much as 70% but had no effect on SC explants.Matched VIS  and SC explants, obtained from a morbidly obese man , were incubated for 6 hr with different doses of BPA, E2. ERα, ERβ, GPR30, ERRα, ERRβ, and ERRγ in breast and SC adipose tissue, compared with VIS adipose tissue, which was used as a reference. All six receptors were more highly expressed in breast adipose tissue (from 1.8- to 7.3-fold) than VIS adipose tissue. The expression of GPR30 and ERRα was approximately the same in VIS and SC adipose tissue (1.4- to 1.5-fold), whereas ERα, ERβ, and ERRβ were moderately higher (from 1.7- to 2.1-fold) in SC tissue. Notably, expression of ERRγ was much lower (0.3-fold) in SC than in VIS adipose tissue.We next examined breast, VIS, and SC adipose tissue, each pooled from four or five women, for expression of putative receptors that may mediate the actions of BPA and/or EERα) presented as 100%. Expression levels of ERβ and ERRγ were 50% and 20%, respectively, relative to ERα. On the other hand, expression of ERRα, ERRβ, and GPR30 was < 1% of ERα, indicating a significantly lower abundance.2. The suppressive effects of BPA were not confined to one adipose tissue type but were present in all depots examined: breast, SC, and VIS. We also report for the first time similar mRNA expression levels of ERα, ERβ, and ERRγ in VIS adipose tissue. The expression of ERα, GPR30, ERRα, and ERRγ was higher in breast than in either VIS or SC fat. The relative expression of these receptors in VIS adipose tissue was ERα > ERβ > ERRγ >>> GPR30 = ERRα = ERRβ. The role of any of these receptors in mediating the suppressive actions of BPA or E2 on adiponectin release remains to be determined.This study provides the first evidence that BPA at environmentally relevant doses inhibits a key adipokine that protects humans from the sequelae of the metabolic syndrome. BPA at low nanomolar concentrations suppressed adiponectin release from human adipose tissue explants as well as from isolated mature adipocytes. Despite a substantial variability among patients, BPA was as effective, and often more effective, than equimolar concentrations of E2 was ineffective and ICI did not antagonize BPA. However, only the highest BPA dose (100 μM) was effective. Previous studies on direct actions of BPA on rodent adipocytes have used very high doses. To support the premise that BPA has adverse metabolic effects in humans, it is essential to study its actions on human tissues. Whereas the value of live rodents and murine adipocyte cell lines as experimental models is undisputed, adipocyte biology is sufficiently different between rodents and humans to warrant prudence . For exain vitro and its responsiveness to BPA or E2 were highly variable among patients. This variability results from the combined effects of genetic, nutritional, and hormonal factors, as well as the state of obesity, clinical conditions, and history of drug use. Because all but one of the patients were women, we did not determine the effect of sex. Serum adiponectin levels are moderately higher in women than in men, but hormone replacement therapy does not alter adiponectin release in either pre- or postmenopausal women . Thus, in terms of the control of adiponectin release, ICI does not behave as a typical ERα/ERβ antagonist. The suppressive effect of ICI also differentiate the putative receptor in human adipose tissue from the ncmER reported by 2, we examined published values of their binding affinity to several putative receptors. Although BPA has a lower median effective concentration (EC50) for ERβ than for ERα (2. On the other hand, the EC50 for BPA for GPR30 is 630 nM (The finding that both BPA and E for ERα , it is ss 630 nM  and is as 630 nM .2 in ER-negative breast cancer cell lines (GPR30 in human adipose tissue, albeit at very low abundance compared with either ERα or ERβ (ERRγ, whose expression level in VIS adipose tissue was 4- to 5-fold lower than that of ERα and ERβ. The ERRs are orphan nuclear receptors that are constitutively active and do not bind estrogens (ERRγ is expressed in a tissue-specific manner (2 or BPA on adiponectin release. It would also be of interest to examine whether BPA at low doses affects adipogenesis, lipogenesis/lipolysis, or the release of other adipokines.GPR30 is a seven-transmembrane receptor that increases the activity of second messengers such as adenylate cyclase and mitogen-activated protein kinase in response to Ell lines . Notablyα or ERβ . Anotherstrogens . ERRγ isc manner , but litThe growing interest by scientists and the public alike in BPA has placed this compound at the center of the debate over potential adverse effects of man-made chemicals found in the environment on fetal/neonatal development, reproductive fecundity, metabolic homeostasis, and carcinogenesis. Yet, attribution of such actions to BPA has been controversial. Differences of opinion and disagreements over data interpretation underlie the inability of several expert panels, convened periodically since 1999, to convince regulatory agencies that BPA poses hazards to human health. There is a growing recognition that the roles of genetic predisposition and environmental factors in the epidemic of obesity and related diseases should not be ignored. Given the endurance of BPA in the environment, its presence in serum from humans worldwide, and the suppression of adiponectin release at nanomolar concentrations, BPA may indeed be the bona fide endocrine disruptor that adversely affects metabolic homeostasis and its manifestations.


Human islet amyloid polypeptide (hIAPP), a pancreatic islet protein of 37 amino acids, is the main component of islet amyloid, seen at autopsy in patients with type 2 diabetes mellitus (DM2). To investigate the roles of hIAPP and islet amyloid in DM2, we generated transgenic mice expressing hIAPP in their islet beta cells. In this study, we found that after a long-term, high-fat diet challenge islet amyloid was observed in only 4 of 19 hIAPP transgenic mice. hIAPP transgenic females exhibited severe glucose intolerance, which was associated with a downregulation of GLUT-2 mRNA expression. In isolated islets from hIAPP males cultured for 3 weeks on high-glucose medium, the percentage of amyloid containing islets increased from 5.5% to 70%. This ex vivo system will allow a more rapid, convenient, and specific study of factors influencing islet amyloidosis as well as of therapeutic strategies to interfere with this pathological process. Islet amyloid polypeptide (IAPP), also referred to as amylin, is a 37 amino acid protein producedin the pancreatic islet beta cells. Human IAPP(hIAPP) is implicated in the pathophysiology of type 2 diabetes mellitus (DM2) sinceit forms proteinaceous tissue deposits in the pancreatic islets (“islet amyloid”) –3.IsletThe generation of C57Bl/6J hIAPP transgenic mice with a rat insulin-2 gene promoter fragment(position −695 to +8 relative to the transcription start site) linked to thehIAPP gene has previously been described . The hIAAt 14 months afterthe start of the high-fat diet, nonfasted mice were anaesthetized with an i.p.injection of midazolam (0.4 mg/mouse) , and a combination of fluanison (0.9 mg/mouse), and fentanyl (0.02 mg/mouse) .D-glucose  wasinjected i.p. (1 g/kg) and blood was sampled from the retrobulbar, intraorbital,capillary plexus before glucose administration and after 10, 30, 60, and120 minutes. The samples were taken in heparinized tubes and stored on ice.Following centrifugation, plasma was separated and stored at −20°C untilanalysis. After the 120-minute blood sample, tissue was sampled (see below),and trunk blood was obtained for measurement of IAPP levels. The blood wascollected in EDTA-tubes and kept on ice until centrifugation at 1500 g for 5minutes at 4°C. Plasma was stored at −80°C.All animal experiments were approved by the Animal Welfare Committee of Utrecht University/University Medical Center Utrecht, The Netherlands.125I-labelled human insulin as tracerand rat insulin as standard . Free and bound radioactivitywas separated by use of an anti-IgG antibody (Linco). The sensitivity of theassay is 12 pmol/l and the coefficiency of variation <3% at both low andhigh levels. Glucose was determined in 10 ul plasma by the glucose oxidasemethod.IAPP levels weremeasured in 25–100 ul plasma by RIA as described , using aμm were stained with Congo red for detection ofislet amyloid by polarized light microscopy (“apple-green” birefringence) and fluorescencelight microscopy (red-coloured autofluorescence). At least 10 islets per mousewere examined. The percentage of individual islet areas occupied by amyloid, asindicated by Congo red positive staining, was visually estimated and scored asfollows: 0% = score 0, between 0 and 26% = score 1, 26–50% = score 2, 51–75% =score 3, and 76–100% = score 4. The Amyloid Index (range: 0–100) of anindividual mouse was calculated as (1 × N1 + 2 × N2 + 3 × N3 + 4 × N4) × 25/n, where N1is the number of islets with score 1, N2 the number with score 2, and so on,and n is the total number of islets investigated. The degree of islet amyloidformation was determined with the investigator being unaware of the geneticstatus of the animals .Pancreatic tissue samples were fixed in 3.7% phosphate-buffered formalin(pH 7.4) for 24–48 hours and paraffin embedded. Sections of 5 35S-labelledoligonucleotide probe covering the nucleotide sequence 247–276 of mouse GLUT-2 cDNA [35S-labelled oligonucleotide probe specific for hIAPP mRNA [To examine the cellular expression of GLUT-2 mRNA, paraffin sections weresubjected to in situ hybridization using a previously described protocol  and a 35APP mRNA .n = 5–8 per animal)were randomly selected from different parts of thesections from 4 mice, 2 males and 2 females in each group. The transgenic mice analyzedwere rated as negative for amyloid. The labelled area, that is, graindensity within an islet, and total islet area were measured, using NIH-imagesoftware, and the density of labelling was expressed as percentage of the totalislet area [In situ hybridization radiolabelling was examined in a bright field microscope, and images were captured with a digital camera . To quantify the density of labelling forGLUT-2 mRNA within islets, areas of insitu hybridization radiolabelling were calculated. Islets , and fixed in 0.5 mL isletsfixative  for2 hours at room temperature. Fixative was removed and islets were washed with0.5 mL 0.1 M Sörensen buffer. Sörensen buffer was removed and islets were resuspendedin 30 μm frozen sectionswere cut onto Superfrost Plus microscope slides (Menzel-Gläser) and stored at−20°C until further use. Sections were fixed in acetone for 1′, rehydrated in PBS for 15′, stained with heamatoxylin for 1′, washed in running tap water for 5′, and stained with Congo red  for 30′. After dehydration in anaugmenting ethanol series  and xylene (twice), sections wereenclosed with Depex. Amyloid-containing paraffin sections of hIAPP transgenicmouse pancreatic tissue were used as positive control for the Congo red staining.From the gelatin-embedded islet blocks, 5 For the detection of amyloid, Congo red-stained islet sections were examined usinga fluorescence microscope. Amyloid deposits were visible as bright redautofluorescent areas without cells, which showed a green birefringence uponvisualization with polarized light. An islet was scored as amyloid positive if at least 2 successive sections of thatislet contained Congo red-positive amyloid deposits. The scoring was performedin a “blind” fashion, that is, with the investigator unaware of the source ofthe islets.P-valuesindicate the probability level of random difference between groups, or ofrandom correlation, respectively. P-values <.05 were considered to represent statistical significance. NonparametricT-tests were used to compare 2 independent samples . Data of the mRNA in situ hybridizations were analyzed by Student'sunpaired t-test. Differences in percentage of amyloid-positive islets between 2days and 3 weeks of culture were analyzed byuse of one-way analysis of variance (ANOVA). Probability values of less than.01 were considered significant.Values are means ±SEM, unless stated otherwise. n = 8) and NT (n = 5) mice, and 59 ± 1.1 g versus 64 ± 3 g in female hIAPP (n = 11) and NT (n = 6) mice. Plasma IAPP levels were 462 ± 78 pmol/L in male hIAPP mice versus 195 ± 32 pmol/L in male NT mice, and 346 ± 81 pmol/L in female hIAPP mice versus 130 ± 21 pmol/L in female NT mice, being significantly higher in hIAPP mice ofboth genders (P < .01) without anygender difference.Body weight after 14 months on the high-fat diet did not differ between the groups, being 57 ± 0.8 g versus 58 ± 0.9 g in malehIAPP (P < .05 or P < .01) in association with increasedinsulin levels 30 minutes after glucose administration (P < .01). Hence, hIAPP overproduction was associated with severeimpairment of glucose elimination in female but not in male mice after high-fat diet.After 14 months on the high-fat diet, nonfasted plasma glucose and insulin levels were notdifferent between hIAPP and NT mice of the same gender. However, both for thehIAPP and NT mice, plasma insulin levels were higher in males as compared tofemales . When glIslet amyloid was detected in 4/19 hIAPP mice on high-fat diet but in none of the 11 NT mice. TheAmyloid Index for these 4 mice was 11.0 ± 6.2 (average and SD). There was no gender difference in isletamyloid formation in hIAPP transgenic mice .P = .02, As expected, a strong hIAPP mRNA labeling was observed in the islets of alltransgenic mice, while it was lacking in all NT mice Figures .GLUT-2 n = 8). Of all islet cultures, 22–30 islets were scored for the presence of amyloid. The percentage of amyloid-positive islets significantly increased (P < .001) from 5.5 ± 3.4% (n = 4) after 2 daysof culture to 70 ± 3.1% (n = 8) at the end of the culture period. Thus, the percentageof amyloid-positive islets increased more than 10 times in three weeks ofculture at high glucose conditions in this ex vivo islet amyloidosis system. An example of a cultured hIAPPtransgenic islet containing amyloid is shown in The percentage of 3-week survival of hIAPP transgenic islets was 83.8 ± 1,0% (P = .024) [In this study, transgenic mice overproducing the amyloidogenic hIAPP intheir pancreatic islet beta cells, as well as NT control mice, were fed ahigh-fat diet for 14 months, in order to evaluate the impact on islet amyloidformation and glucose homeostasis when combining these two potentiallydiabetogenic factors. We anticipated a marked islet amyloid formation in thehIAPP transgenic mice on the high-fat diet because we previously observed thatcrossbreeding the hIAPP transgenic mice with the Obese mouse (being severlyinsulin resistant) resulted in extensive islet amyloid formation . Also,w = .024) . This in = .024)  ascompa = .024) . Other mIn this study, we also observed severe glucose intolerance in female but not in male hIAPP transgenicmice. The finding that in all groups of mice plasma insulin levels failed toreturn to basal within 2 hours after the glucose load is in accordance withhigh-fat diet inducing insulin resistance . Also,tIslet GLUT-2 mRNA expression was reduced in hIAPP transgenic versus NT mice, and thisreduction appeared more severe in female than in male mice. Beta cell GLUT-2expression is known to correlate with glucose responsiveness of the cells .HoweverIn conclusion, this in vivo study shows that promoting insulinresistance over a long period of time by giving a high-fat diet for 14 monthspromotes islet amyloid formation in hIAPP transgenic mice, although lessextensively than in the severe insulin-resistant Obese, leptin-deficient hIAPP mice. This suggests that the degree ofinsulin resistance is important for extensive development of islet amyloid. Inaddition, we observed a remarkable gender difference in that severe glucoseintolerance was observed only in female hIAPP transgenic mice given high-fat diet and notin males. We suggest that this gender difference is due to the high level of circulatingIAPP rather than to islet amyloid formation. If and how this glucoseintolerance might be mediated by downregulation of beta cell GLUT-2 geneexpression, as observed in the hIAPP mice, is presently unknown.The rationale for the ex vivo study was toexamine if amyloid would be formed in isolated and cultured pancreatic isletsfrom hIAPP mice, to such a degree that it would be detectable with lightmicroscopy. Since both the present and previous  invivo


To investigate the neuroprotective effect of α2-adrenergic agonist brimonidine in the presence of glutamate-induced neurotoxicity, oxidative stress, and hypoxia on in vitro cultures of purified rat retinal ganglion cells (RGCs).Purified RGC cultures were obtained from retinas of 6–8-day old Wistar rats, following a two-step immunopanning procedure. After 72 h of cultivation, the neuroprotective effect of brimonidine  was investigated by culturing the RGCs under glutamate, oxidative, and hypoxic stress for a further 72 h, 24 h, and 12 h, respectively. Glutamate neurotoxicity was induced by adding glutamate (25 μM), while oxidative stress was induced by substituting the culture medium with B27 supplement without antioxidants, and hypoxia was induced by cultivation in a controlled-atmosphere incubator with oxygen levels 5% of the normal partial pressure. The RGC viability under each stress condition normalized to that under normal condition was evaluated as live cell percentage based on a total of 7–8 full repeated experiments.The cell survival percentages of cultures exposed to glutamate, oxidative, and hypoxic stress were 58.2%, 59.3%, and 53.2%, respectively. Brimonidine dose dependently increased RGC survival in the presence of glutamate (80.6% at 1 µM), oxidative (79.8% at 1 µM), and hypoxic  stress. In the presence of α2-adrenergic antagonist yohimbine (10 μM), brimonidine (1 μM) showed no protective effects on RGC viability.At a concentration of 0.1 µM or higher, brimonidine increased survival of purified rat RGCs in the presence of glutamate neurotoxicity, oxidative stress, and hypoxia. The neuroprotective effect of brimonidine is mediated via α2-adrenergic receptors at the RGC level. Glaucoma is the second leading cause of blindness in the world, and various mechanisms of glaucomatous optic neuropathy (GON) have been thought to cause retinal ganglion cell (RGC) death leading to visual loss . ElevateBrimonidine is a highly selective α2-adrenergic receptor agonist . Brimoni2+ [Because of the wide use and importance of brimonidine as an antiglaucoma drug and its potential in retarding the progression of glaucomatous visual field damage of open angle glaucoma patients through action beyond IOP reduction , further2+ -30.The aim of our study is to examine the neuroprotective effect of brimonidine against glutamate-induced neurotoxicity, oxidative stress, and hypoxia, using purified rat RGC cultures.All animal studies were in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Research. Poly-L-lysine, BSA (BSA), L-glutamine, human recombinant brain-derived neurotrophic factor (BDNF), rat recombinant ciliary neurotrophic factor (CNTF), and yohimbine hydrochloride (Y-3125) were obtained from Sigma . The papain dissociation system was from Worthington Biochemical ; mouse antirat SIRP (CD172a) monoclonal antibody (MAB 1407P), and mouse antirat and mouse Thy1.1 monoclonal antibody (MAB 1406) were obtained from Chemicon International . The live/dead viability cytotoxicity kit (L-3224) was obtained from Molecular Probes . Brimonidine tartrate was obtained from Allergan, Inc. . B27 supplement minus antioxidants (AO-) was from Gibco . Unless named, B27 supplement was with antioxidants.2, following the two-step immunopanning procedure as follows [2, 5% CO2, 75% N2 at 37 °C) before each experiment in serum-free B27 complete medium containing neurobasal medium (Gibco) with 1 mM L-glutamine (Sigma), B27 supplement (Gibco), 40 ng/ml BDNF, 40 ng/ml rat CNTF, and 10 μM forskolin. After completing 72 h of cultivation, RGCs were subjected to the following:RGC cultures were obtained from the retinas dissected from enucleated eyes of 6–8 day-old Wistar rats , euthanized by inhalation with CO follows ,32. TissControl coverslips were moved to freshly prepared neurobasal medium containing B27 supplement and placed in normoxic conditions without glutamate. Test coverslips for glutamate neurotoxicity were then transferred to freshly prepared neurobasal medium containing both B27 supplement and glutamate (25 μM). These were then cultivated for a further 72 h.Control coverslips were moved to freshly prepared neurobasal medium with B27 supplement normally containing potent antioxidants , while coverslips for oxidative treatment were transferred to neurobasal medium containing B27 without these five antioxidants (AO-), which induced oxidative stress ,34. The 2, 5% CO2, 90% N2 at 37 °C) for 12 h.Control coverslips were moved to freshly prepared neurobasal medium with B27 supplement and placed in normoxic conditions, while hypoxic stress was induced by placing the cultures in a hypoxic environment  of brimonidine; these were added separately to each of the test cultures.We studied the effect of yohimbine (10 μM), a specific α2-adrenergic receptor antagonist on the neuroprotective effect of brimonidine (1 μM) by adding brimonidine alone, brimonidine with yohimbine, and yohimbine alone to RGCs cultured under glutamate neurotoxicity, oxidative stress, and hypoxia. Eight separate, repeated, full experiments were performed with yohimbine.At the end of cultivation, the surviving RGCs were processed for viability by labeling with calcein-AM (2 μM), a component of the live/dead viability/cytotoxicity kit . Live RGDunnett’s test was used to determine if test groups were significantly different from controls. A p value of <0.05 was considered significant.After completing 72 h of cultivation in the presence of glutamate alone, 58.2±12.5% of the RGCs survived compared to controls . In the In the yohinbine experiments, the neuroprotective effect of brimonidine at 1.0 μM against glutamate neurotoxicity was replicated (p<0.001). RGC survival in the presence of brimonidine and yohimbine and of yohimbine alone was 51.9±7.4% and 58.2±7.7%, respectively ; these wIn the presence of oxidative stress (AO-), RGC survival was reduced to 59.3±4.1% compared to the AO+ control group in normal cultivating conditions . In the In the yohinbine experiments, the neuroprotective effect of 1.0 μM brimonidine against oxidative stress was replicated p<0.001, . In the Under hypoxic conditions , RGC surIn the yohinbine experiments, the neuroprotective effect of brimonidine against hypoxia was replicated p<0.001, . In the We have demonstrated using an in vitro model of purified rat RGC culture that brimonidine is neuroprotective at the level of the RGC. The neuroprotective effect of brimonidine was present in three different stress situations—glutamate induced neurotoxicity, oxidative stress, and hypoxic stress. Brimonidine, at a 1 μM concentration, significantly increased RGC viability under all three stresses; these stresses have been implicated in the development of GON ,17,35-37Baptiste et al.  used mix2+ increases in retinal neurons in culture, the effect of which was reversed by yohimbine [The neuroprotective pathways triggered by brimonidine were effectively blocked by the selective α2-adrenergic antagonist yohimbine. Previously, α2-adrenergic antagonists, like rauwolscine and yohimbine, were shown to reverse the neuroprotective effects of α2-adrenergic agonists in models of optic nerve injury and photoreceptor light-induced damage ,38,39. Mohimbine . Our in 2+ through modulation of the L-type Ca2+ channel or glutamate receptor activity [2+ through the L-type Ca2+ channel, whereas over 3 µM of brimonidine was required for the modulation of glutamate-induced Ca2+ increase. Thus, the mechanism of neuroprotection observed in our study may be partly attributed to L-type Ca2+ channel modification because the effect was observed at a briminodine concentration of 1 µM.Our results showed a similar percentage protection effect over three different concentrations of brimonidine and under three different stresses. It is possible that a final common pathway of the three neurotoxic insults may be countered by the effect of brimonidine on α2-adrenergic receptors. The α2-adrenergic receptors are expressed in the inner plexiform and RGC layers of the retina in various mammalian species, such as rats and humans -45. Actiactivity ,40,46. Ibcl-2 and bcl-xl [Under our culture conditions, hypoxia mainly induced glutamate-independent apoptosis . Anti-apd bcl-xl -49. ThusIn contrast to glutamate- or hypoxia-induced neurotoxicity, oxidative stress induced by using B27 without antioxidative agents in the current model mainly induced necrosis by activation of the calpain/catepsin pathway . The mecPharmacologically, brimonidine can activate the α2-adrenergic receptor at a concentration of 2 nM or higher. Studies with monkeys showed that the vitreous humor brimonidine concentration was 82 nM after topical application of 0.2% brimonidine . In huma−7 M or higher. In an attempt to search for effective treatment of GON beyond IOP-lowering therapy, the potential of brimonidine or other α-2-selective adrenergic agonists to be able to affect not only the glutamate-induced apoptosis pathway but also the glutamate-independent apoptotic or calpain/catepsin-dependent necrotic pathway in RGCs may merit further study.In summary, we first found that brimonidine acting via the α2-adrenergic receptor was neuroprotective on purified rat RGCs exposed to glutamate-induced neurotoxicity, oxidative stress, or hypoxic stress at concentrations of 10


Baseline glucagon levels were higher in transgenic mice  than in wildtype animals . In contrast, the glucagon response to protein was impaired in transgenic animals . Baseline insulin levels did not differ between the groups, while the insulin response, as the glucagon response, was impaired after protein challenge (P = .018). Glucose levels were not different between the groups and did not change significantly after protein gavage. Acetaminophen was given through gavage to the animals (2 mg/mouse) to estimate gastric emptying. The plasma acetaminophen profile was similar in the two groups of mice. We conclude that disturbances in glucagon secretion exist in mice with β-cell specific overexpression of human IAPP, which are not secondary to changes in gastric emptying. The reduced glucagon response to protein challenge may reflect a direct inhibitory influence of hIAPP on glucagon secretion.Exogenous administration of islet amyloid polypeptide (IAPP) has been shown to inhibit both insulin and glucagon secretion. This study examined  It isc islets , and exoc islets , 3, 5–7.c islets –11.IAPPdiabetes –14.We h α-cells , 16. Whe α-cells , to contβ-cell expression of hIAPP on a C57BL/6J/6xDBA/2 background weregenerated as previously described [Hemizygous transgenic micewith islet escribed . Transgeescribed . ThetraμL) were administered through a gastric tube (outer diameter 1.2 mm). After 15, 30, 60, and 120 minutes, blood samples, 75 μL each, werecollected. Blood was kept in heparinized tubes containing 5 μL Trasylol,immediately centrifuged whereupon plasma was separated and stored at −20°C untilanalysis for glucose, glucagon, insulin, and acetaminophen.Following a four-hour period after removal of food from the cage, femaletransgenic and wildtype animalswere anesthetized with an intraperitoneal injection of midazolam  as well as a combination offluanison (0.4 mg/mouse) and fentanyl . Thirty minutes later, a blood sample was taken from the retrobulbar,intraorbital, capillary plexus in heparinized tubes. Then, whey protein  and acetaminophen dissolved in saline  with a guinea pig antiglucagon antibody, radioiodine labelled glucagon as tracer and glucagon standard. CV ofthe assay is 8% and the sensitivity of the assay is 10 pg/mL. The antibodies donot cross-react with GLP-1. Plasma insulin was determined with radioimmunoassay(Linco) with a guinea pig antirat insulin antibody, radioiodine labelled humaninsulin as tracer and rat insulin as standard. Plasma acetaminophen wasdetermined with a colorimetric assay . Plasma glucose was determinedwith the glucose oxidase method.t-test. For estimation ofglucagon secretion, the increase in plasma glucagon levels during the first 15minutes after protein gavage was estimated by subtracting baseline glucagonvalues from the 15-minute glucagon values. The area under the glucagon andinsulin curves (AUCs) were also calculated using the trapezoid rule.Means ± SEM are shown. Statisticalcomparisons were performed with the Student's n = 6) than in thewildtype animals . Glucagon levels at 15, 30,and 60 minutes after protein administration did not differ significantlybetween the groups, whereas the levels after 120 minutes were, again,significantly, higher in the transgenic animals (P = .008). Glucagonsecretion was estimated as the change in glucagon levels during the first 15minutes after protein gavage. This 15-minute glucagon response to proteinadministration was impaired in the transgenic animals, being 21 ± 2.7 pg/mL intransgenic mice versus 38 ± 5.7 pg/mL in wildtype mice (P = .027). The suprapasal AUC forglucagon for the entire 120-minute study period did not differ significantlybetween the groups, being 4.3 ± 1.1 μg/mL × 120 minutes in wildtype mice versus3.9 ± 0.9 μg/mL × 120 minutes in transgenic mice.P = .018) . Theinsulin response to protein ingestion was impaired in transgenic mice; thesuprabasal AUC for insulin for the 120-minute study period was 27.2 ± 3.1 nmol/L× 120 minutes in wildtype animals versus 16.5 ± 3.9 nmol/L × 120 minutes intransgenic animals (β-cell specificoverexpression of human IAPP. It was found that the insulin response to proteinwas impaired in transgenic mice. This confirms that these mice have impairedinsulin secretion, as previously was reported also after oral glucose challenge[α cells as opposed to the reduced β-cell immunostaining inthese animals which is associated with significantly reduced islet insulincontent [This study evaluated the islethormone responses to oral protein ingestion in mice with hallenge. The maicontent . This hycontent –11. Hencβ-cell IAPP expression does not affect gastric emptying, and,therefore, the inhibited glucagon response to oral protein in these mice is notdue to impaired gastric emptying.In this study, we also determinedthe acetaminophen concentration after acetaminophen administration to determinewhether gastric emptying had been altered in the transgenic mice. Previously,inhibition by IAPP of gastric emptying has been demonstrated  and chanβ-cell specific overexpression of human IAPP increases baseline glucagonlevels and impairs the glucagon response to oral protein in association withimpaired insulin response. This shows that a disturbed α-cell function in thesemice is evident in association with the previously described disturbed β-cellfunction [In conclusion, this study has shownthat function , 16.


Progenitor cells in the adult dentate gyrus provide a constant supply of neuronal precursors, yet only a small fraction of these cells survive and develop into mature dentate granule cells (DGCs). A major challenge of current research is thus to understand the stringent selection process that governs the maturation and functional integration of adult-born DGCs. In mature DGCs, high-frequency stimulation (HFS) of the perforant path input elicits robust expression of the immediate early gene Arc/Arg3.1, trafficking of its mRNA to dendrites, and local synthesis of the protein necessary for consolidation of long-term potentiation (LTP). Given the synaptic commitment inherent in LTP consolidation, we considered that HFS-evoked expression of Arc could be used to timemap the functional integration of newborn DGCs. Dividing cells were birthmarked by BrdU-labeling at 1, 7, 14, 21, or 28 days prior to induction of LTP and expression of Arc was examined by confocal microscopy. Contrary to expectation, LTP did not induce Arc expression in newborn cells at any age, suggesting they might be refractory to synaptically-evoked Arc expression for at least one month. Importantly, however, spontaneous expression of Arc was detected in BrdU-labeled cells and strongly associated with the survival and maturation of NeuN-positive DGCs. Moreover, Arc expression at the earliest ages (1 and 7 days), clearly precedes the formation of glutamatergic synapses on new neurons. These results suggest an unexpected early role for Arc in adult-born DGCs, distinct from its functions in LTP, LTD, and homeostatic synaptic plasticity. New neurons continue to be generated in the adult mammalian brain. Neurogenesis, which occurs solely in the subventricular zone (SVZ) of the olfactory bulb and the dentate gyrus of the hippocampal formation, encompasses the proliferation of neural stem and progenitor cells and the differentiation, migration and eventual incorporation of neurons into the pre-existing circuitry Ample evidence supports a role for activity-dependent synaptic plasticity in hippocampal-dependent learning and memory To this end, newly dividing cells were birthmarked by injection of the thymidine analogue BrdU, and LTP was induced 1, 7, 14, 21, and 28 days thereafter. Surprisingly, although Arc was sharply upregulated in the pre-existing granule cell population, LTP-inducing stimulation did not enhance Arc expression in newborn cells of any age. Instead, we found expression of Arc protein in the absence of LTP induction. Furthermore, newborn neurons positive for Arc expression exhibited selective survival and maturation as NeuN-positive DGCs in the granule cell layer. This demonstration of Arc expression at the earliest ages (1–7 days) suggests a function for Arc in newborn cells that might be distinct from its role in LTP consolidation in the pre-existing granule cell population.n = 40: 250–350 g and all between 10–12 weeks of age at the time of BrdU injection) were individually housed with ad libitum access to food and water under climate-controlled conditions (23°±0.5°C). Rats were maintained on a 12-h light/dark cycle (light on at 07:00) and received at least 7 days to acclimatize to their new environmental conditions prior to onset of the experiment. This investigation, designed to minimize the number of animals and suffering, was carried out in accordance with the Norwegian Regulation on Animal Experimentation, the European Convention for the Protection of Vertebrate Animals used for scientific purposes and the guidelines of the Norwegian Animal Research Authority.Male Sprague Dawley rats  was given intraperitoneally (i.p.) 1, 7, 14, 21, or 28 days prior to LTP induction. This design allowed us to ascertain Arc expression in new neurons throughout their proliferative (24 h) phase to their long-term survival (4 weeks) .LTP was induced in the DG, using methodology previously described Subsequently, 195 minutes after completing the HFS protocol, rats were transcardially perfused with 4% paraformaldehyde solution in 0.1 M sodium phosphate buffer (pH 7.4). Brains were removed and post-fixed in the same solution overnight at 4°C, before being transferred to a sodium phosphate buffer  and stored at 4°C. Following cryoprotection of the brains by overnight immersion in a 30% glucose solution, 30 µm coronal serial sections were prepared on a cryostat microtome. Sections were collected in NaPBS with sodium azide and stored at 4°C. Immunohistochemical stainings were performed on the same animals using parallel sets of coronal sections.RNA probes were prepared from a cDNA insert matching the first 2975 nucleotides of the Arc mRNA (GenBank accession number NM_019361) cloned into the pCRII-TOPO vector . Antisense and sense probes were transcribed from linearized plasmids using T7 and SP6 polymerase in the presence of digoxigenin (DIG) labeling mix according to instructions of the manufacturer . Floating sections were placed in PBS for 5 min, permeabilized with proteinase K (10 µg/ml) for 5 min at 37°C, and postfixed . After fixation, sections were treated with 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8, for 10 min, washed twice in 2×SSC, and placed for 10 min in a prehybridization buffer. Probes were applied to the sections and hybridization was performed in a humidified chamber at 60°C for at least 16 hours. Sections were washed twice with 2×SSC at RT for 30 min, once with 50% formamide in 2×SSC at 65°C, rinsed in 2×SSC at 37°C, incubated with 20 µg/ml RNase A at 37°C for 30 min, and incubated in RNase A buffer at 65°C for 30 min. After blocking in 2% blocking reagent for 1 hour at RT, alkaline phosphatase-coupled anti-DIG antibody  was applied for another hour at RT. Visualization was done with the chromogenic substrates nitroblue-tetrazolium-chloride and 5-bromo-4-chlor-indolyl-phosphate (Roche). Hybridization using the Arc sense probe remained unstained and served as a control. Pictures were taken on a Nikon  Eclipse 80i microscope coupled to a Nikon DS-5M camera .For all stainings (unless otherwise specified) every sixth section throughout the rostral/caudal extent of the hippocampus (Bregma −2 to −6) was collected and coded before processing for immunohistochemistry to ensure objectivity. Stainings were performed on free-floating sections under continuous agitation.2O2 for 15 min to reduce endogenous peroxidase activity, before being incubated in the primary polyclonal rabbit anti-Arc antibody raised against amino acids 1–300 of the Arc protein  for 60–72 h at 4°C. Subsequently, sections were washed with NaPBS and incubated at room temperature with biotinylated donkey anti-rabbit IgG . To confirm specificity of results obtained with the applied antibody, comparative staining was performed using an alternative mouse monoclonal antibody for Arc raised against amino acids 264–385 of the Arc protein  (Sections were preincubated in 0.3% H 612603) . Further2O2 for 15 min (Arc) or 30 min (BrdU). Finally, sections were washed, mounted on slides, dehydrated and coverslipped with DPX  together with polyclonal rabbit anti-Arc H300  and monoclonal mouse anti-NeuN  or the latter with polyclonal rabbit anti-GFAP . Sections were then incubated in Alexa-488 donkey anti-rat  followed by Alexa-555 donkey anti-mouse  and Alexa-647 donkey anti-rabbit  antisera , 5a–c.Quantification of BrdU-labeling was conducted using a modified unbiased stereology protocol z-axis and rotating them in orthogonal x-y planes using a 63× oil objective (1 µm steps). To exclude potential cross-bleeding between detection channels, triple-labeling was imaged in sequential scanning mode.For multi-labeling, percentages of BrdU-labeled nuclei coexpressing Arc and NeuN, or GFAP were determined by analyzing 50–100 randomly selected BrdU-labeled nuclei throughout the GCL and SGZ using a Leica TCS SP2 AOBS confocal microscope . Absolute cell numbers of BrdU+/Arc+ and BrdU+/Arc− cells were calculated by multiplying the total number of BrdU+ cells by their estimated percentages. Care was taken to verify double-labeling and control for false positives by analyzing BrdU-positive nuclei in their SigmaStat 3.1 software was used to perform analysis of variance. ANOVA for repeated measures was used for statistical analysis of group effects of electrophysiological data. One- and two-way ANOVA were performed for the analysis of immunohistochemical findings. Results were considered significant when p≤0.05. Pairwise multiple comparison procedures  were applied to more accurately assess the source of variation between groups.1,55 = 237.7; p<10−3)   (F p<10−3) . The mag = 0.37) . HFS-LTP = 0.37)  and immu = 0.37) , respect1,56 = 0.5, p = 0.48; GCL: F1,56 = 0.248, p = 0.62; SGZ: F1,56 = 0.0481, p = 0.83). Data from both hemispheres were thus pooled for further analysis.Before further analysis of Arc expression in newborn cells, we first examined the distribution and total number of BrdU-labeled cells in the dentate gyrus  of young adult male rats which received a single BrdU injection (200 mg/kg) and were sacrificed 1, 7, 14, 21 or 28 days later. BrdU is a thymidine analog which incorporates into the DNA of dividing cells during the S-phase of the cell cycle, rendering it a widely used marker for birth dating dividing cells, monitoring proliferation and determining their fate. HFS-LTP 195 minutes prior to tissue collection had no effect on proliferation and/or survival of BrdU-labeled cells in the hippocampal regions in any age group examined  (−4) and −47.2%  compared to days 1 and 7 respectively. In contrast, one-way ANOVA revealed a progressive increase in the GCL  as well as a time effect in the SGZ  (−4), +161%  and +133%  change in the 7, 14 and 21 day groups compared to day 1, with a respective increase of +57.3%  and +50.0%  in the 7 and 14 day groups compared to day 1 in the SGZ. By 28 days, and in line with expectations, numbers were significantly reduced compared to days 7 , 14  and 21   . By day  p<10−3) . Specifi p<10−5) .4,28 = 6.593, p<10−3) (−5) and +73%  compared to days 1 and 7 respectively. Our results confirm previously established patterns of cell proliferation, survival, and migration during the first month after BrdU injection We also found a significant, time-dependent reduction in the percentage of BrdU+ cells in the SGZ with a corresponding increase in the GCL (F p<10−3) , suggest41,481 = 15.716, p<10−3) and SGZ , but there were no effects of stimulation or a time-stimulation interaction on BrdU+/Arc+ cells in the hilus , GCL  or SGZ  (We expected to find Arc expression solely in older neurons which have reached synaptic maturity and are able to undergo long-lasting strengthening of the synapse in response to patterned activation. Immunofluorescent colabeling of BrdU and Arc  confirme= 0.829) . Time ef4,22 = 7.005, p<0.001), while BrdU+/Arc+ cells increased with time . Specifically, post-hoc analysis revealed a significant increase in BrdU+/Arc+ cells in the GCL in the 14, 21 and 28 day groups compared to days 1  and 7  respectively. BrdU+/Arc− cells in the GCL however, were decreased by 21  and 28  days compared to day 7. Taken together, the results demonstrate a correlation between Arc expression, starting in the youngest newborn cells and survival of these cells in the GCL  was paralleled by a decrease in BrdU+/Arc− cells   and 7 , while a notable decrease occurred from 21 to 28 days . Although still higher than day 1 , levels at 28 days returned to those comparable with day 7, consistent with cell death and/or migration into the GCL. BrdU+/Arc− cells in the SGZ increased by day 7  and from there significantly declined by days 14 , 21  and 28 . Compared to days 1 and 14, levels were significantly reduced by days 21  and 28 . The hilus was included in all analyses of Arc to obtain a complete representation of the hippocampus, although it did not show any outstanding results. No time effect was found on the number of BrdU+/Arc+ cells , and only a slight effect was seen in total BrdU+/Arc− cells . Posthoc analysis revealed reduced numbers at 28 days compared to day 1  as the only significant difference (Given the lack of differences in patterns of BrdU+/Arc+  as well  the GCL . A simil p<10−3) . Specififference .4,22 = 10.508, p<10−3). Specifically, post-hoc analysis revealed a significant increase in BrdU+/Arc+ cells in the GCL in the 14, 21 and 28 day groups compared to days 1  and 7  . Post-hoc analysis revealed a significant increase in BrdU+/Arc+ cells in the SGZ in the 14, 21 and 28 day groups compared to days 1  and 7    . Similar p<10−6) . Again t= 0.653) . When pr4,23 = 29.395, p<10−3)   and 7 , with a corresponding reduction of BrdU+/Arc+/NeuN− cells. Likewise, a similar pattern of expression appeared in the GCL, with a significant time-dependent increase in BrdU+/Arc+/NeuN+ cells  and decrease in BrdU+/Arc+/NeuN− cells . Post-hoc analysis revealed a significant increase in BrdU+/Arc+/NeuN+ cells by days 14, 21 and 28 compared to days 1  and 7 , while a significant decrease appeared in BrdU+/Arc+/NeuN− cells by days 21  and 28  compared to day 7. There were no new cells, positive for Arc, expressed in the GCL after 1 day (irrespective of NeuN expression), giving way to the statistical discrepancy between the otherwise exactly inversely correlated NeuN+ and NeuN− cell populations. Of the entire new DGC population (SGZ and GCL) irrespective of Arc expression, 1.76% (±1.76) assumed the NeuN phenotype by 1 day, 17.6% (±5.6) by 7 days, 62% (±5.7) by 14 days, 74.1% (±5.1) by 21 days and 83.9% (±2.5) by 28 days.Having found that preferential survival seems to occur in newborn cells positive for Arc expression, we proceeded to explore the nature of this specific subpopulation of BrdU cells by performing a triple labeling for BrdU/Arc/NeuN . The res p<10−3)  coupled  p<10−3) . Post-hoThis study examined expression of the immediate early gene Arc/Arg3.1 in dentate gyrus BrdU-labelled cells during the first four weeks of neuronal maturation. Given its pivotal role in late-phase LTP and memory consolidation The dentate gyrus produces new cells throughout adulthood in a variety of mammalian and nonmammalian vertebrates Given this maturational timeline, expression of Arc in 1 and 7 day old cells  and 7, mAnother intriguing property of new neurons (up to 4 weeks of age) seems to be their refractory nature to HFS-evoked Arc induction. This is all the more surprising when considering that neurons between the ages of 1–3 weeks have lower thresholds for LTP induction relative to pre-existing DGCs Ramirez-Amaya et al. (2006) have recently demonstrated enhanced behaviourally-evoked expression of Arc in 5 month old DGCs relative to the older, pre-existing neurons In the current study, a relatively large fraction of newborn cells express Arc at 1 and 7 days  File S1LTP had no effect on BrdU+/Arc− expression. Besides a time effect in the SGZ , there were no effects of time, stimulation or their interaction on BrdU+/Arc− cells in the hilus , GCL  or SGZ . Post-hoc analysis revealed that compared to days 1 and 7, BrdU+/Arc− cell numbers in both hemispheres were significantly reduced by days 14, 21 and 28 with a further decrease from day 14 to 21 and 28 on the stimulated side. The *, #, $ symbols represent significant effects compared to days 1, 7, and 14 respectively. One, two or three symbols represent p<0.05, p<0.005, p<0.0005 respectively.(0.13 MB TIF)Click here for additional data file.