However, sustained-release naltrexone implant use in humans has resulted in cases of inadvertent foetal exposure. Here, we used clinically relevant dosing to examine the effects of maternally administered sustained-release naltrexone on the rat brain by examining offspring at birth and in adulthood. Maternal treatment  started before conception and ceased during gestation, birth or weaning. Morphometry was assessed in offspring at birth and adulthood. Adult offspring were evaluated for differences in locomotor behaviour  and opioid neurochemistry, propensity to self-administer morphine and cue-induced drug-seeking after abstinence. Blood analysis confirmed offspring exposure to naltrexone during gestation, birth and weaning. Naltrexone exposure increased litter size and reduced offspring birth-weight but did not alter brain morphometry. Compared to placebo, basal motor activity of naltrexone-exposed adult offspring was lower, yet they showed enhanced development of psychomotor sensitization to morphine. Developmental naltrexone exposure was associated with resistance to morphine-induced down-regulation of striatal preproenkephalin mRNA expression in adulthood. Adult offspring also exhibited greater operant responding for morphine and, in addition, cue-induced drug-seeking was enhanced. Collectively, these data show pronounced effects of developmental naltrexone exposure, some of which persist into adulthood, highlighting the need for follow up of humans that were exposed to naltrexone  Naltrexone is a non-selective opioid receptor antagonist Limited data from humans treated with oral or sustained-release naltrexone suggest no major adverse neonatal outcomes with respect to birth-weights, APGAR scores and head circumferences Previous studies show that rat neonates directly receiving low-dose naltrexone (1 mg/kg/day) are growth retarded Furthermore, it has been shown that perinatal exposure to naltrexone (1–50 mg/kg/day) can alter aspects of adult offspring behaviour pertaining to emotionality, exploratory drive and analgesic response to morphine Experiments adhered to the Prevention of Cruelty to Animals Act, 1986 and the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia. There were 3 cohorts. Procedures for cohort 1 were approved by the Animal Ethics Committee of the University of Western Australia, Perth, Australia  and the Florey Neuroscience Institutes, Melbourne, Australia . Procedures for cohorts 2 and 3 were approved by the Animal Ethics Committee of the University of Western Australia, Perth, Australia  and the Florey Neuroscience Institutes, Melbourne, Australia .ad libitum. All females were nulliparous and 9–11 weeks of age upon arrival. In cohort 1, there were 16 dams (180–270 g), in cohort 2 there were 12/treatment group (197–315 g) and in cohort 3 there were 6/treatment group (240–290 g). Ten males (330–460 g) were used for husbandry.Three cohorts  of SpragMorphine hydrochloride: Glaxo, Australia, in sterile 0.9% saline. Ketamine: Parnell Laboratories, Australia; meloxicam: Boeringher Ingleheim, Germany; neomycin sulfate: Delta Veterinary Laboratories, Australia; and carprofen: Norbrook Laboratories, Australia. Heparinised saline was diluted at 90 IU/ml.in vitro (data not shown) and in vivo experiments Custom-sized naltrexone implants comprised the same formulation as for humans  microspheres, average molecular weight 700,000 Mv, inherent viscosity 0.5 dl/g, specific rotation of –2° to +2°) compressed into a tablet  surrounded by a single coat of poly(DL-lactide) Three cohorts were used in the current study to evaluate the extent of naltrexone exposure in offspring and dams during pregnancy (cohort 1) and before/after pregnancy (cohorts 2 and 3), evaluate gross anatomical outcomes in offspring at birth and adulthood and changes in neurochemistry in adulthood (cohort 2), as well as evaluate behavioural outcomes in adult offspring (cohorts 2 and 3). An overview of the experimental design and timing of events/outcome measures for cohorts 1, 2 and 3 is presented in Dams had a single naltrexone or placebo implant inserted subcutaneously under general anaesthesia (2–3% isoflurane). Carprofen  and neomycin  were injected post-operatively. Dams in cohort 1 were implanted with naltrexone and those in cohorts 2 and 3 with naltrexone or placebo implants.Blood was collected into lithium-heparinised tubes and frozen at −80°C. For cohort 1, maternal blood was obtained from a lateral tail vein Initiation of timed mating took place 12 days after surgical insertion of implants. Timed-matings were undertaken by placement of nulliparous rat dams with 2–3 males overnight; mating was confirmed the following morning by the presence of spermatozoa in vaginal smears. In most cases, conception occurred within 3–5 days of initial presentation to males.All 16 dams received a naltrexone implant, were mated, and sampled at either gestation day 13, 15 18 or at birth, following euthanasia (n = 4 per time-point). Offspring blood was collected at gestation day 18 and at birth, and was pooled into one sample per litter. At gestation days 13 and 15, amniotic fluid was collected and also pooled into a single sample for each litter (n = 4 per time-point). Naltrexone and 6,β-naltrexol levels in all samples were measured as above. The sampling time-points for maternal rats and offspring from cohorts 1, 2 and 3 are summarised in Cohort 2 females were randomly allocated to receive either a naltrexone or placebo implant, were impregnated and gave birth (n = 12/treatment group). Blood was obtained from all maternal rats 1 day prior to mating and at birth. Offspring were either euthanised, sampled for blood and examined at birth , or were raised to 4 weeks, weaned, and raised until 10 weeks (using the remaining 6 dams per group), whereupon they were transferred to the Florey Neurosciences Institute, for evaluation of locomotor behaviour and neurochemistry. Maternal rats fostering these offspring had a blood sample taken at weaning. Individual blood samples were collected from one male and one female from each litter at weaning and at 8 weeks.Duration of pregnancy, litter size and gender were recorded. At birth and 8 weeks, offspring body-weight was recorded and heel-toe, occipital-snout and biparietal lengths were measured using vernier callipers. At 8 weeks, one male and female per litter were perfused transcardially with heparinised saline (5000 IU/L), followed by 250 ml 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.2–7.4) for brain morphometry. Brain weight was recorded and cerebrum length, width, height, as well as brain length (i.e. cerebrum+cerebellum) were measured using vernier callipers, and brain volume was calculated by water displacement as described previously Upon arrival of offspring at the Florey Neuroscience Institutes, animals were acclimatised for 2 weeks and no procedures were undertaken. At 12–14 weeks of age, sensitization to locomotor responses induced by repeated morphine treatment was performed as previously described Testing proceeded as follows. Habituation to the test environment was established using 3 daily 60 min sessions within locomotor chambers. On days 4–8, rats were placed in the locomotor chambers 30 min after an injection of saline (1 ml/kg) or morphine  and activity recorded for 60 min. From days 9–15, animals underwent a period of home-cage abstinence (i.e. they received no injections). On day 16, rats were challenged with saline or morphine (5 mg/kg) and returned to the locomotor chambers . TreatmeFor these experiments, we did not observe differences between males and females. Therefore, for logistical reasons relating to Animal Ethics approval , only males were used for the subsequent neurochemical and operant self-administration experiments.At 14 weeks, after behavioural sensitization testing , male rats were terminally anaesthetised  and brains removed and frozen. Serial sagittal brain sections 5-ol enkephalin  was used to label high affinity µ-opioid receptors 125I]FK33,824, 2000 Ci/mmol) for 60 min. Non-specific binding was determined by addition of 10 µM naloxone to the incubation medium. Slides were washed, dried and apposed to Biomax AR film (Kodak XAR-5) with [14C]microscales. Films were developed automatically (100 Plus Automatic x-ray film processor: All-Pro Imaging).[33P]d-adenosine triphosphate using terminal deoxynucleotidyl transferase. Labelled probes  and applied to adjacent sections in hybridisation buffer . Controls used a 100-fold molar excess of unlabelled oligonucleotide. Sections were hybridised in sealed humidified chambers at 42°C overnight, rinsed , dehydrated in ethanol and air-dried. Dry sections were apposed to x-ray film with [14C]microscales and then developed.Expression of PPE and PPD mRNA has previously been described Scion Image software was used to convert optical densities to radioactivity per unit area  by means of a calibration curve derived from autoradiograms of the simultaneously-apposed [14C]microscales.Autoradiographs showing binding of radioligand to µ-opioid receptor, and binding of oligonucleotide probes to PPE and PPD mRNA, were captured using a Sony XC-77CE CD Video Camera with an attached Nikon Micro-Nikkor 55 mm lens. Sufficient contrast to background intensity allowed manual delineation of brain regions Dams from cohort 3 were randomly allocated to receive either a naltrexone or placebo implant, were mated and gave birth. At birth, litters were reduced to 10  and culled neonates from each litter had blood pooled into one sample per litter. Surviving offspring were weaned at 4 weeks, at which point maternal blood was collected, and offspring were reared until 10 weeks of age, whereupon they were transported to the Florey Neuroscience Institutes for assessment for operant self-administration of morphine. Upon arrival, animals were acclimatised for 2 weeks and no procedures undertaken.via a jugular cannula to an intravenous line : 0.6 mm, outer diameter (OD): 1.2 mm) connected to a 22-gauge swivel  and a syringe held in an infusion pump  with Bcoex-T22 tubing . A light  came on for 20 s, contingent with an active lever press and reward delivery. In addition, a drop of vanilla essence provided an olfactory cue (S+) for the location of the active lever. The chambers were housed in sound attenuated boxes and ventilated with fans. Med-PC IV software (Med Associates) was used to record lever presses.Self-administration of intravenous morphine (0.1 or 0.3 mg/kg/infusion) was assessed using operant chambers  with two levers. Rats were connected The cannulae consisted of a 10 cm length of silastic tubing  sleeved over a 22-gauge cannula . All cannulae were autoclaved. At 12 weeks, rats were anaesthetised (2–3% isoflurane) and cannulae implanted into the jugular vein and flushed with 0.15 ml of antibiotic (1.2 mg trimethoprim and 6 mg sulfadoxin) and heparinised saline. Meloxicam  was given for pain relief. The day after surgery, cannulae were flushed with heparinised antibiotic; thereafter, cannulae were flushed twice daily with heparinised saline. Starting on the day of cannulation, rat daily food intake was restricted to 15 g as this is known to facilitate self-administration of abused drugs via the jugular cannula to the intravenous line. The injection volume was 48 µl per infusion and duration of injection was 2.3 s. A maximum of 50 drug infusions was set, with a 20-s timeout period after an infusion. All post-training sessions (except for cue-induced drug-seeking session at the very end) were 2 h in length, unless 50 infusions were obtained, and were held just before the dark phase of the photoperiod. The active lever remained the same for both oral sucrose training and intravenous self-administration of morphine experiments, as did the position of the olfactory cue and contingent light stimulus.After 3 days of recovery in the home cage, rats underwent operant self-administration training to respond for oral sucrose (5%) at a fixed-ratio of 2 (FR2) schedule. Each of these training sessions were 15 h in duration and took place once daily until lever discrimination was demonstrated (after 3 days in most cases). Subsequently, rats were connected to morphine Rats responded for intravenous morphine (0.3 mg/kg/infusion) at a fixed-ratio of 1 (FR1) schedule (3 weeks), with subsequent assessment on a progressive-ratio (PR) 9–4 schedule The patency of the cannulae was evaluated periodically using ketamine (5 mg). If loss of righting reflex was not apparent within 3 s of infusion, the rat was excluded. In addition, rats were excluded if any cannula blockage or leakage was identified during daily flushing. During assessment of FR1 responding for morphine, criteria were set at a lever discrimination of ≥65% and <25% variation between the numbers of infusions over 3 days.SPSS for Windows and two-tailed tests. Results for free naltrexone and 6,β-naltrexone, in blood or amniotic fluid, were examined with Pearson Correlation coefficient between maternal and offspring levels, stage of pregnancy and time post-implant. Where data failed tests of normality, a Kruskal-Wallis nonparametric ANOVA was employed. Results are for male and females combined unless mentioned otherwise.Unless otherwise indicated, statistical analysis was undertaken using p<0.0125 and p<0.008 were used for measures of body and brain morphometry, respectively. Morphometric data are reported separately for male and female offspring ; duration of morphine exposure; and ligand /probe .Neurochemistry data  were incorporated into generalised linear mixed models for statistical analysis using p<0.05, and for pair-wise comparisons, if p<0.05 after adjustments using the Dunn-Sidák procedure.Operant self-administration data were obtained for male offspring only. For time course analyses (PR and drug-seeking cumulative responses), and analyses of the effect of implant treatment and dose on morphine self-administration, a two-way ANOVA was performed. Comparisons of the PR data between the offspring groups were analysed using an unpaired t-test. Drug-seeking data were analysed by one-way ANOVA, with Student-Newman-Keuls post-tests. Differences were deemed significant if F = 13.53; p<0.01; F = 2.70; p>0.05; 2 = 3.29; p>0.05; Data from cohort 2 offspring were combined for analysis of litter size, length of gestation and gender ratio. In cohort 2, there were 10 naltrexone-exposed litters and 10 placebo-exposed litters which, respectively, produced 144, and 117 live pups. There were ∼23% more pups per litter following maternal naltrexone treatment compared to placebo .In both dams and offspring, free 6,β-naltrexol blood-levels were low with many below the limit of detection (>0.1 ng/mg) and a maximum blood concentration of 0.3 ng/ml found in two maternal samples. Nevertheless, a positive correlation existed between naltrexone and 6,β-naltrexol maternal levels: r <0.001, N = 15 (p<0.01) and offspring (p<0.05) levels correlated with time post-implant . The weight-adjusted naltrexone concentration in maternal blood was 1.19±0.19 ng/ml; raw offspring naltrexone concentration in blood or amniotic fluid was 1.23±0.25 ng/ml. Further analysis revealed that offspring levels correlated with maternal levels: , N = 15 . Further-implant .For cohort 2, naltrexone was detected in maternal and offspring blood at birth and at weaning . Naltrexone was not detected at postnatal day 56 (adulthood). In some offspring at weaning, naltrexone was not detected (n = 8) and these data were excluded from the calculation of means. Unadjusted maternal naltrexone-blood levels are reported elsewhere r <0.01, N = 54; r <0.01, N = 42; mean ± SEM: 0.90±0.12 ng/ml; At weaning, the weight-adjusted mean ± SEM for free naltrexone in blood from implanted females (cohort 3) was 0.95±0.32 ng/ml (n = 6). Raw data for naltrexone in blood from offspring culled at birth (pooled into one sample per litter) was 3.17±1.65 ng/ml. Weight-adjusted maternal values for free naltrexone-blood concentration with respect to the number of days post-implant are represented for all mothers  in Total litter size, as well as maternal weights from each group, were used as covariates. Covariates appearing in the model of offspring at birth were evaluated at 239.42 (mothers’ weight) and 12.66 (litter size). For offspring evaluated in adulthood, values were 272.22 mothers’ weight and 13.67 for litter size.F = 14.93; p<0.001; F = 3.81; p = 0.05; After adjusting for covariates, naltrexone had a significant effect on offspring body weight at birth  = 1.73; p>0.05; F = 14.26; p<0.001; F = 3.28; p = 0.04; In adult offspring, there were no effects from drug treatment on body weight  = 8.10; p = 0.01; F = 7.70; p = 0.01; For brain morphometry, only adult offspring were examined. Each variable was assessed separately with drug group and gender as fixed factors. None of the measures showed a significant main effect of drug group or a significant group by gender interaction. Each variable showed a significant main effect of gender at the adjusted alpha level except for brain width  = 5.70; p<0.05; 32.6%; F = 6.00; p<0.05; 52.3%; F = 12.90; p<0.001; 49.1%; F = 20.45; p<0.001; 42.6%). Horizontal plane activity (ambulation) did not differ on day 1  = 1.80; p>0.05; F = 6.00; p<0.05; 25.3%; We did not observe differences between males and females; accordingly, we combined them into a single data set. After initial placement in locomotor chambers, naltrexone-exposed offspring  exhibited fewer vertical plane entries (rearing) compared to placebo-exposed offspring  = 4.19; p<0.05; F = 5.68; p<0.05), females  = 9.12; p<0.01), and males and females combined  = 14.98; p<0.001; F = 0.29, p>0.05; Given the difference in horizontal activity between treatment groups following habituation by day 3, subsequent data for morphine treatments were calculated as a function of basal activity. While there was no difference between groups on the first day of morphine treatment, from day 4 to 5 (i.e. morphine injection day 1 to 2) there was a significantly greater increase in ambulation of naltrexone-exposed offspring which was 11.5 fold greater than placebo-exposed offspring  or morphine-morphine  compared to expression in morphine-naïve counterparts   compared to placebo-exposed counterparts , revealed a two-way interaction between maternal treatment group , treatment received during behavioural testing  and PPE mRNA expression. Namely, reduced expression was associated with placebo offspring given morphine, either as saline-morphine  . Howeverterparts A and B.terparts . No signterparts .F = 35.94, p<0.001; post hoc comparisons revealed significant preference for the active lever over the inactive lever in placebo-  = 5.97, p = 0.035) and naltrexone-exposed rats  = 13.54, p = 0.004). Significant discrimination was also evident at 0.3 mg/kg/infusion  = 18.68, p<0.001; post hoc comparisons revealing significant preference for the active lever over the inactive lever in both placebo-  = 17.58, p<0.001) and naltrexone-exposed rats  = 33.68, p<0.001).Both naltrexone- and placebo-exposed offspring reliably self-administered morphine . AnalysiF = 6.17, p = 0.025). This difference between groups was not observed over a similar period at 0.1 mg/kg/infusion  = 0.90, p>0.05). At 0.3 mg/kg/infusion, there was a significant escalation in morphine self-administration by naltrexone-offspring, with greater consumption on day 7 compared to day 1 (t(14.63) = 2.57, p = 0.022; t(16.16) = 0.75, p>0.05; F = 0.47, p>0.05) or 0.3 mg/kg/infusion  = 3.50, p>0.05) (data not shown).Analysis of the number of morphine infusions received over 7 days revealed an overall significantly augmented response by naltrexone-exposed offspring at 0.3 mg/kg/infusion over this period (t(10) = 0.83, p>0.05), breakpoint (t(10) = 0.85, p>0.05), drug infusions (t(10) = 0.83, p>0.05), or overall cumulative response  = 0.38, p>0.05) (data not shown). However, at 0.3 mg/kg/infusion, naltrexone-exposed rats displayed a higher breakpoint compared to placebo offspring on a PR3-4 schedule. Unpaired t-tests revealed a significant difference between offspring groups with respect to the final ratio obtained (breakpoint) (t(12) = 2.91, p = 0.013; t(12) = 2.82, p = 0.015; t(12) = 3.01, p = 0.011; No difference was seen between placebo- and naltrexone-exposed offspring for 0.1 mg/kg/infusion assessed on the PR3-4 schedule with respect to active lever presses  = 9.24; p = 0.012; F = 44.06; p<0.001), and a significant interaction between the two factors  = 6.89, p = 0.012). Dunn-Sidák post test comparisons revealed no difference between placebo- and naltrexone-exposed offspring in the first 10 min time bin  = 2.29, p = 0.043) and infusions (t(11) = 2.30, p = 0.042). Cumulative responses were significantly greater in naltrexone-, compared to placebo-exposed rats, in the 30, 40 and 90 min time bins .For the PR9-4 schedule (data not shown) at 0.3 mg/kg/infusion, groups differed with respect to breakpoint  = 4.77, p = 0.023; post hoc test comparisons revealed no difference between placebo- and naltrexone-exposed offspring in the first 10 min time bin (t(14) = 1.41, p>0.05; t = 2.68, 2.94, 2.73, 2.73 and 2.70, respectively, p<0.05; Both groups exhibited robust cue-conditioned drug-seeking behaviour after a period of abstinence . NonetheF = 16.62, p<0.001; post hoc analyses for placebo-  and naltrexone-exposed offspring , confirming enhanced responding in naltrexone-exposed offspring on the active lever on test day compared to the FR1 schedule. Moreover, compared to placebo, there was augmented active lever responding by naltrexone-exposed offspring on the cue-induced drug-seeking test day versus baseline day  = 11.48, p = 0.005; 73%; p<0.001; 124%; F = 0.046, p>0.05).Active lever presses were significantly higher on test day (1-h session) compared to baseline responding (2-h session), despite the absence of drug infusions during the session. This significant cue-conditioned drug-seeking was revealed by two-way ANOVA  compared to placebos, when first introduced to a novel environment. During subsequent re-exposure to the same environment, less rearing (i.e. habituation) occurred in both groups and remained lower in the naltrexone-exposed group, consistent with other studies Moderate morphine doses in rats produce a biphasic locomotor response via networks known to regulate motor behaviour. Treatment of placebo-exposed offspring with morphine was associated with decreased striatal PPE mRNA expression relative to saline-treated placebo rats. However, this effect did not occur in naltrexone-exposed offspring, indicating that morphine-induced regulation of enkephalin was a normal process Interestingly, this was mirrored by resistance of striatal enkephalin mRNA expression to down-regulation following morphine treatment compared to placebos. Enkephalin appears to mediate morphine-induced tolerance and sensitization Although an effect was observed for PPE mRNA, no such changes were seen for PPD mRNA or μ-opioid receptor expression. Other studies have shown changes to opioid receptors following naltrexone exposure Persistent behavioural effects occurred in the absence of substantial morphological changes in adult offspring but were preceded by decreased birth-weights. Because litter size impacts on growth and nutritional intake The sustained-release naltrexone preparation comprised the same formulation used in humans and yielded comparable release- (∼0.4 mg/kg/day) and blood-levels (∼2–10 ng/ml) caecal period” rat: 36 days; human: 182 days). When expressed as a percentage of the caecal period, neurodevelopmental milestones start and finish at approximately the same value In relative terms, brain development in rats and humans occurs to a common timetable defined by the time between conception and eye opening  during labour and within 10 hours of birth increased the relative risk of offspring subsequently becoming addicted to opiates in adulthood via a sustained-release implant impacts both behaviour and neurochemistry in adult offspring, but without obvious morphological effects. Our data are consistent with perturbations that are reported to result from naltrexone exposure during development with significant implications for alterations in morphine-induced neuroplasticity, and increased risk of opioid-abuse later in life. Our findings highlight the need for greater information on the safety of using naltrexone during human pregnancy.The current study demonstrates that chronic, low-dose maternal naltrexone delivered


T, was isolated from mine soils of Dabaoshan in South China, which were acidic (pH 2–4) and polluted with heavy metals. The isolation was Gram-negative, aerobic, non-spore-forming, and rod-shaped bacteria having a cellular width of 0.5−0.6 µm and a length of 1.3−1.8 µm. They showed a normal growth pattern at pH 4.0–9.0 in a temperature ranging from 5°C to 40°C.The organism contained ubiquinone Q-8 as the predominant isoprenoid quinine, and C16∶0, summed feature 8 (C18∶1ω7c and C18∶1ω6c), C18∶0, summed feature 3 (C16∶1ω7c or iso-C15∶0 2-OH), C17∶0 cyclo, C18∶1ω9c, C19∶0 cyclo ω8c, C14∶0 as major fatty acid. These profiles were similar to those reported for Burkholderia species. The DNA G+C % of this strain was 61.6%. Based on the similarity to 16S rRNA gene sequence, GIMN1.004T was considered to be in the genus Burkholderia. The similarities of 16S rRNA gene sequence between strain GIMN1.004T and members of the genus Burkholderia were 96−99.4%, indicating that this novel strain was phylogenetically related to members of that genus. The novel strain showed the highest sequence similarities to Burkholderia soli DSM 18235T (99.4%); Levels of DNA-DNA hybridization with DSM 18235T was 25%. Physiological and biochemical tests including cell wall composition analysis, differentiated phenotype of this strain from that closely related Burkholderia species. The isolation had great tolerance to cadmium with MIC of 22 mmol/L, and adsorbability of 144.94 mg/g cadmium,and it was found to exhibit antibiotic resistance characteristics. The adsorptive mechanism of GIMN1.004T for cadmium depended on the action of the amide,carboxy and phosphate of cell surface and producing low-molecular-weight (LMW ) organic acids to complex or chelated Cd2+.Therefore, the strain GIMN1.004T represented a new cadmium resistance species, which was tentatively named as Burkholderia dabaoshanensis sp. nov. The strain type is GIMN1.004T ( = CCTCC M 209109T =  NRRL B-59553T ).Heavy-metal-tolerant bacteria, GIMN1.004 Burkholderia species is an ubiquitous, microbe that are highly resistant to heavy metals (HMs). Many novel species of the genus Burkholderia have been described in recent years, certain species of Burkholderia have proved to be highly efficient in biocontrol, bioremediation Activities such as mineral excavation, or transportation, smelting, refining, disposal of the tailings and waste waters around mines are important causes of heavy metals contamination 2+ 2SO4 0.2%; K2HP04 0.025%; Glucose 0.1%; Yeast extract 0.01%;1 mM Cd2+; Agar 2.0%) (Difco). The plates were incubated at 28°C for 4 days and all colonies were isolated. Among the isolation, a strain of purple color was isolated, designated as strain GIMN1.004T.Soil samples were collected from Dabaoshan Mine. 25 g soil samples were serially diluted with 225 mL 0.85% NaCl (w/v) and suitable 10-fold dilutions were plated onto MGY agar with Cd2O2, and oxidase activity was determined using 1% (w/v) tetramethyl-p-phenylenediamine after incubation at 28°C for 4 day on MGY agar. Growth after 5 days incubation in MGY liquid medium was assessed at different temperature  and various pH conditions , respectively. For the pH experiment, three different buffers were used : acetate buffer for pH 4.0–5.5; phosphate buffer for pH 6.0–8.0; Tris buffer pH 8.5–10.0. Salt tolerance was tested in MGY supplemented with 1–10% (w/v) NaCl after 5 days of incubation at 28°C Anaerobic growth was assessed using incubation at 28°C for 5 days in 10 ml rubber-stoppered, screw-capped tubes containing MGY medium (9 ml) covered with liquid paraffin. Indole production and the Voges–Proskauer reaction were tested by using standard proceduresGram reaction was determined according to the method described by Smibert & Krieg Other physiological characteristics and the utilization of various substrates as sole carbon sources were determined using the Biolog GN2 MicroPlates (Biolog Identification System) assay as recommended by the manufacturer. Sample preparation and analysis were performed according to the directions of the manufacturer (Biolog GN2 System). The triplicate microplates were read, after 4 h and 24 h incubation, using Microstation hardware (Biolog). The data were analyzed using MICROLOG 3 software (Biolog). All the experiments were carried out in triplicate.T was compared with available 16S rRNA gene sequences from GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/) to determine the approximate phylogenetic affiliation and was aligned with closely related members using CLUSTALW software Sequencing and assembly of 16S rRNA genes were carried out as described by Bakermans & Madsen Burkholderia 16S rRNA gene sequences was constructed using the neighbour-joining method of Saitou & Nei T with certain members of other Burkholeria species.A phylogenetic tree of et alTo measure the G+C content of the chromosomal DNA, genomic DNA from the novel strain was extracted and purified as described by Moore & Dowhan http://www.midi-inc.com/) and analyzed by GC  using the Microbial Identification software package Isoprenoid quinones were extracted from lyophilized cells which cultivated on MGY medium for 7 days at 28°Cwith chloroform/methanol , evaporated under vacuum conditions and reextracted in n-hexane/water . The crude n-hexane-quinone solution was purified using Sep-Pak Vac silica cartridges (Waters) and subsequently analyzed by HPLC  as described by Xie & Yokota T and Burkholderia soli GP25-8T which was obtained from the KACC. DNA relatedness studies were conducted by using the fluorometric microdilution plate method DNA–DNA hybridization was performed to evaluate the genomic DNA–DNA relatedness between strain GIMN1.0042+ and Pb2+ for strain GIMN1.004T and its closest phylogenetic neighbours (B. soli GP25-8T and Burkholderia caryophylli ATCC 25418T) were determined by the plate dilution method as adopted by Summers and Silver et al. 2+ and Pb2+ compared with non-Cd2+ and Pb2+ -supplemented controls under the same conditions. The experiments were carried out in triplicate. Cultures were incubated on NA media at 30°C for 7 d.The MIC of the CdT were used as inocula in the antibiotic resistance tests. Plates containing an antibiotic, as well as a nonselective control plate, were streaked with portions (approximately 10 µl) of inoculum, and growth was scored after 5 days. Resistance to a particular concentration of antibiotic was defined as the ability of a strain to form colonies at that concentration. Stock solutions of kanamycin , streptomycin , ampicillin  and cefetamet  were prepared in distilled water; All solutions were filter-sterilized using 0.45-µm membrane filters. Antibiotics were added to molten agar after sterilization and cooling to 50°C. The NA agar medium without antibiotics was used as controls. The experiments were carried out in triplicate. Cultures were incubated at 28°C for 7 d.The NA agar medium was used for the antibiotic resistance experiments. Stationary-phase broth cultures of GIMN1.0042+ content of bacterial cells grown in NA supplemented with 185.56 mg/L CdCl2 for 48 h cells were harvested, rinsed three times using TSB/10 and dried at 55°C for 24 h. Following addition of 5 ml HNO3 (70% ), mineralization was carried out in a microwave oven. Metal content was determined using an inductively coupled plasma atomic emission spectrometry (ContrAA700). The experiments were carried out in triplicate.To determine the CdT was incubated in MGY medium at 0, 2 or 8 mM Cd2+ at 150 r/min and 30°C for 24 h. After centrifugation at 12000 g for 10 min, precipitated cells were washed with deionized water for 3 times and then vacuum-dried. One mg dried samples were thoroughly mixed with 100 mg KBr and pressed into slice at 10 t/cm2 for 1 min. The slice was analyzed with FTIR spectroscopy Stain GIM1.004Analysis of variance and the Student–Newman–Keuls test (p<0.05) were used to compare treatment means. All the statistical analyses were carried out using SPSS 10.0.T was found to be Gram-negative, non-spore-forming, aerobic, weakly motile. In solid MGY agar medium (pH 4.0), the isolate formed round, light pink to white, opaque, wrinkled, and umbonate colonies with fuzzy boundaries. The cells of GIMN1.004T were rod−shaped, 1.3−1.8 µm in length and 0.5−0.6 µm in diameter (2+ (8 mM), the isolate produced purple crystal particle. Strain GIMN1.004T grew at pH 4.0–9.0 at 28°C, but optimally at pH 6.5. It grew at a temperature ranging from 5 to 40°C at pH 6.5, with an optimum growth at 30°C. It grew well at NaCl concentrations of 0–7% (w/v), but was slightly inhibited by NaCl concentration over 7%. The isolate was resistant to streptomycin (20 µg ml−1), kanamycin (20 µg ml−1), ampicillin (200 µg ml−1), and cefetamet (20 µg ml−1); The physiological and biochemical characteristics, metabolic properties and substrate-utilization results obtained for strain GIMN1.004T were presented in Strain GIMN1.004diameter . In MGY N-acetyl-D-glucosamine, adonitol,L-arabinose, D-arabitol,D-fructose, L-fructose, D-galactose, α-D-glucos,m-inositol,D-mannitol, D-mannose,D-raffinose, D-sorbitol,sucrose, maltose, xylitol, pyruvic acid methylester,succinic acid mono methylester, cis-aconitic acid, formic acid, D-gluconic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, p-hydroxy phenylacetic acid, α-keto butyric acid, D,L-lactic acid, propionic acid, quinic acid, D-glucosaminic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, L-alaninamide,L-alanine,L-alanylglycine, L-asparagines, L-aspartic acid, L-glutamic acid, glycyl-L-glutamic acid, L-histidine, L-phenylalanine, L-proline, L- pyroglutamic acid, L-serine, L-threonine, γ-amino butyric acid, 2- aminoethanol,glycerol, D-glucose-6-phosphate as sole carbon sources. it was negative for the utilization of α-cyclodextrin, N-acetyl-D-galactosamine, i-erythritol, gentiobiose, D-melibiose, β-methyl-D-glucoside, D-trehalose, D-cellobiose, turanose, γ- -hydroxybutyric acid,α- ketoglutaric acid,malonic acid, glucuronamide, glycyl-L-aspartic acid, D-serine, D,L-carnitine, inosine, thymidine, phenyethylamine, putrescine, α-D-glucose-1-phosphate (Biolog GN2 Microplate system).The isolate was positive for utilization of Tween 40 and 80,T. A BLAST search Burkholderia. The 16S rRNA gene sequence of strain GIMN1.004T showed a similarity level of 99.4% (over 1428 bases) to that of Burkholderia soli strain GP25-8T (GenBank accession No. DQ465451), and of 96–98% to that of other Burkholderia species and Burkholderiaceae bacteria. Comparison of the culture characteristics of strain GIMN1.004T and its closest phylogenetic neighbours, however, revealed significant differences from Burkholderia soli GP25-8T  was found with respect to Burkholderia soli DSM 18235T, the corresponding level of DNA–DNA relatedness as determined by hybridization was 25.0%. The values were well below the 70% cut-off point for species classification, as recommended by Burkholderia.In the phylogenetic tree , strain T were in agreement with the results of the phylogenetic inference, confirming that the novel bacterium belongs to the genus Burkholderia. The novel bacterium clearly differed from the related species in terms of cell-wall composition. The major quinones of GIMN1.004T were ubiquinone Q-8, as in the case of other species of the genus Burkholeria16∶0 (25.05%), summed feature 8 (17.76% C18∶1ω7c and C18∶1ω6c), C18∶0 (10.57%), summed feature 3, C17∶0 cyclo (5.52%), C18∶1ω9c (5.23%). while that of Burkholderia soli was C18∶1ω7c (33.5%), summed feature 3 , C16∶0 (15.3%), summed feature 2  (Twas 61.6 mol%.The chemotaxonomic characteristics of strain GIMN1.0041 iso I) . The DNAT grew well in NA agar medium supplemented with ≤18 mM Cd2+ and visible growth was observed in the presence of 19–22 mM Cd2+. The MIC of Cd2+ for strain GIMN1.004T was 22 mM. Strain GIMN1.004T grew well in NA agar medium supplemented with ≤4 mM Pb2+ and visible growth was observed in the presence of 5–6 mM Pb2+. The MIC of Pb2+ for strain GIMN1.004T was 6 mM. B. soli GP25-8T and B. caryophylli ATCC 25418T couldn’t grow in NA agar medium supplemented with ≤1 mM Cd2+ and Pb2+ under the same conditions. The testing of antibiotic resistance showed that stain can grow well at Amp, Kn, Cat and Str.Strain GIMN1.004T was 144.94 mg/g by atomic absorption, while the adsorbability of another 8 strain cadmium-tolerant bacteria which isolated under the same condition was 60 mg/g. The adsorptive mechanism of GIMN1.004T for cadmium depended on the action of the amide,carboxy and phosphate of cell surface and producing low-molecular-weight (LMW ) organic acids to complex or chelated Cd2+.The testing results showed that the adsorbability of GIM1.0042+. Based on the tolerant ability and higher adsorbability of the Cd2+, bacterial strain GIM1.004T have potential implications cleaning up of detoxifying metal-contaminated soils in the future.Cadmium is an ubiquitous toxic metal that was capable of modulating immune responses T represented a new species of the genus Burkholderia, which has been tentatively named as Burkholdria dabaoshanensis sp. nov. (da.bao.shan.en'sis. N.L. fem. adj. dabaoshanensis pertaining to dabaoshan which the strain was isolated ) with the strain type GIMN1.004T ( =  CCTCC 209109T =  NRRL B-59553T ).Therefore, based on the physiological, biochemical, chemotaxonomic, and molecular genetic results described above, GIMN1.004


F. boothii (formerly F. graminearum strain DK21), to F. graminearum, F. asiaticum, F. oxysporum f. sp. lycopersici, and Cryphonectria parasitica. Relative to virus-free strains, the FgV1-DK21 recipient strains had reduced growth rates, altered pigmentation, and reduced virulence. These results indicate that protoplast fusion can be used to introduce FgV1-DK21 dsRNA into other Fusarium species and into C. parasitica and that FgV1-DK21 can be used as a hypovirulence factor and thus as a biological control agent.There is increasing concern regarding the use of fungicides to control plant diseases, whereby interest has increased in the biological control of phytopathogenic fungi by the application of hypovirulent mycoviruses as a possible alternative to fungicides. Transmission of hypovirulence-associated double-stranded RNA (dsRNA) viruses between mycelia, however, is prevented by the vegetative incompatibility barrier that often exists between different species or strains of filamentous fungi. We determined whether protoplast fusion could be used to transmit FgV1-DK21 virus, which is associated with hypovirulence on  Although fungicides successfully control many diseases caused by plant-pathogenic fungi, fungal pathogens remain a major source of plant disease. Because of the development of fungicide-resistant strains, and increasing public concern regarding environmental and food safety, there is renewed interest in biological control based on application of hypovirulent mycoviruses.Cryphonectria parasiticaThe potential of mycoviruses for managing plant-pathogenic fungi was first demonstrated for C. parasitica, and five related mycoviruses have been completely sequenced Cryphonectria hypovirus 1 (CHV1) was successfully used as a biological control agent of C. parasitica in Europe, i.e., CHV1-infected strains exhibited reduced virulence, reduced asexual and sexual sporulation, and reduced pigment production. CHV1 was unsuccessful as a biological control agent in North America, however, because the host fungus in North America has multiple vegetative compatibility groups (VCGs) that limit the spread of the virus Double-stranded RNA mycoviruses have been described in yeasts, mushrooms, and filamentous fungi AspergillusF. poaeRosellinia necatrixThe failure of mycovirus transmission caused by vegetative incompatibility can be overcome in the laboratory by using protoplast fusion F. graminearum species complex (Fg complex) comprises 13 phylogenetically distinct species based on DNA sequences from 13 independent genetic loci F. boothii  were chosen (6 protoplasts/ml) were fused by the 60% polyethylene glycol (PEG 3350)-mediated method, virus-infected strains were finally selected on hygromycin B-containing PDA .We first determined whether FgV1-DK21 can overcome the VCG barrier in other e chosen . To impre chosen  and 2. AF. asiaticum and F. graminearum were similar to those of strain DK21. Like strain DK21, FgV1-DK21 recipient strains of F. asiaticum and F. graminearum had reduced growth rates and increased pigmentation relative to virus-free strains  and F. graminearum  were also present in each virus-infected strain  by protoplast fusion. Four strains produced by the fusion procedure were selected and compared with virus-free EP155 and CHV1-infected EP155. CHV1-infected colonies (UEP) were smaller than virus-free colonies and lacked the orange pigment of virus-free colonies  when few molecular genetic tools are available C. parasitica and measured in apple inoculations was greater with FgV1-DK21 dsRNA than with CHV1  at 25°C at 150 r.p.m. for 5 days while strains of C. parasitica were grown in 50 ml of EP complete medium All strains used in this study  were sto4Cl containing 10 mg/ml of driselase (InterSpex Products), as described previously 2) to protoplast suspensions. Transformants with resistance to hygromycin B were obtained by transforming the fungal protoplasts with the plasmid pUCH1 F. boothii. Following the transformation, FgV1-DK21 was transmitted by anastomosis and screened on PDA containing 50 µg/ml of geneticin (Duchefa). For genomic DNA extraction, fungal strains of the Fg complex were grown in 50 ml of CM at 25°C, 150 r.p.m. for 5 days. The mycelia were harvested by filtration through sterile Whatman no. 2 filter paper, ground in liquid nitrogen using a mortar and pestle, and then suspended in CTAB buffer ∶2-mercaptoethanol (100∶1). Genomic DNA was extracted sequentially with chloroform∶isoamyl alcohol (24∶1), precipitated with isopropanol. The extracted genomic DNA was extracted twice with phenol∶chloroform∶isoamyl alcohol (25∶24∶1), treated with RNase A (20 µg/ml) for 1 h at 37°C, precipitated with isopropanol, and then finally suspended in distilled water. For the Southern hybridization of hygromycin B-resistant mutants, the extracted genomic DNA was digested with Kpn I for 12 h, and the Southern hybridization of G418-resistant mutants, it was digested with Spe I and Kpn I for 12 h at 37°C. A 10-µl of the digested DNA was separated on 0.8% agarose gel for 8 h. The gel was submerged twice in denaturation solution (1.5 M NaCl and 0.5 N NaOH) for 20 min at room temperature and capillary blotted onto a positively charged nylon transfer membrane  in 0.4 N NaOH. Probe labeling reactions were performed in 20 µl of 10 mM Tris-HCl pH 7.5, 7 mM MgCl2, 0.1 mM DTT, 30 µCi [α-32P] dCTP, 3 mM dNTP mix, 10 pmoles of random primers and 2 U klenow fragment (TaKaRa). After hybridization, unhybridized probe is removed by washing in low stringency wash buffer (2×SSC and 0.1% SDS) and high stringency wash buffer (0.1×SSC and 0.1% SDS). Hybridization signal intensities were measured using a Bio-imaging Analyzer system .Protoplasts of fungal strains were prepared by treatment of fresh mycelia grown on YPG liquid medium  for 3 h at 30°C with 1 M NHFg complex were extracted from an agarose gel with QIAquick® gel extraction kit (Qiagen) by following the manufacturer's instructions. DNA sequencing was performed at the National Instrumentation Center for Environmental Management of the Seoul National University with an ABI Prism 3730 XL DNA Analyzer (Applied Biosystems) according to manufacturer's instructions. The sequence data were analyzed using a BLAST search tool and were aligned using Clustal W PCR of TEF and histone H3 gene region was performed as described with modification 4Cl containing 5 mg/ml of driselase and 8 mg/ml of lysing enzyme . Protoplasts were harvested by centrifugation at 2,544× g at 4°C for 10 min, washed twice with STC , and suspended in 300 µl of MMC buffer . Equal volumes of the two protoplast suspensions (100 µl of 1×107 protoplasts/ml) were mixed and placed on ice for 30 min. After 500 µl of PEG solution  was added to the protoplast suspension, the mixture was incubated at 20°C for 20 min. Protoplast fusants were regenerated in 700 µl of potato dextrose broth  for 7 days in the dark, plated on 15 ml of YCDA , and then selected on PDA containing 50 µg/ml of hygromycin B and 50 µg/ml of geneticin. Antibiotic-resistant colonies were screened again on hygromycin B-containing PDA.Protoplast fusion was performed according to a previously described method with modifications 5′-TGTGGGAGAAGAAGTATGGCCT-3′ and 5′-ATCAGGAACCATTGAAAGAGTCC-3′ (RdRp region) or 5′-ATGGACACCAAGGATATTTA-3′ and 5′-TTAGGGGTGCAAGGCCCTTTTC-3′ (ORF2 region)). PCR reactions were performed using the following conditions: one step at 94°C for 3 min; 35 cycles at 93°C for 45 sec, 60°C for 40 sec, and 72°C for 1 min 30 sec; and finally one step at 72°C for 10 min. PCR products were analyzed by 1% agarose gel electrophoresis.Total RNA was isolated with extraction buffer according to a previously described method F. boothii, F. asiaticum, and F. graminearum were performed as described 4•7H2O, 0.1% NH4NO3, and 0.1% KH2PO4) at 25°C and 150 r.p.m. for 5 to 7 days. Conidia were collected by filtering through six layers of sterile cheese cloth. A 10-µl volume of the spore suspension (105 conidia/ml) in 0.01% (v/v) Tween-20 was injected into one floret of each flowering wheat head. Wheat plants inoculated with 0.01% (v/v) Tween-20 alone served as a control. For each treatment, 10 replicate wheat heads were inoculated. Inoculated plants were placed in a growth chamber . Wheat heads were examined for symptoms 14 days post-inoculation.Virulence assays with F. oxysporum f. sp. lycopersici strains was measured with a Fusarium wilt assay as described previously 5 microconidia/ml of the F. oxysporum f. sp. lycopersici strains in distilled water. Twenty seedlings per treatment were planted in pots containing sterile soil and maintained in a growth chamber at 28°C with 14/10 h light/dark cycle. Severity of disease symptoms was calculated using an index from 0  to 4 (dead plant).Virulence of C. parasitica strains was assayed as described previously with minor modifications Virulence of Figure S1Alignments of histone H3 (A) and translation elongation factor 1α (B) sequences from F. boothii strains. Green boxes indicate uniquely fixed nucleotide characters. The following nucleotides are fixed for F. boothii: histone H3 positions 148 (C), 252 (C), 300 (T), and 404 (T); translation elongation factor 1α position 616 (C). GenBank accession numbers of nucleotide sequences (histone H3/translation elongation factor 1α) used are as follows: NRRL 26916 (AY452827.1/AF212444.1), NRRL 29011 (AY452828.1/AF212445.1), NRRL 29020 (AY452829.1/AF212443.1), NRRL 29105 (AY452838.1/AF212446.1).(TIF)Click here for additional data file.Figure S2Detection of FgV1-DK21 RNAs in recipient virus-infected strains. Total RNAs extracted from Fusarium graminearum species complex (A) and Cryphonectria parasitica strains (B). Equal amounts (3 µg) of total RNA were electrophoresed through a 0.8% agarose gel in TAE buffer system. Fb: F. boothii; Fg: F. graminearum; Fa: F. asiaticum; Cp: Cryphonectria parasitica. Lanes 1, 3, 5, and 7 and 2, 4, 6, and 8 represent virus-free and virus-infected strains, respectively in panel A, while in panel B, lanes 1–6 represent virus-free F. boothii, virus-infected F. boothii, EP155, and four FgV1-DK21-infected strains, respectively. (C) Quantitative RT-PCR analysis of virus-infected strains. Presence of viral dsRNA was confirmed by RT-PCR amplification with a primer pair designed from the RdRp coding region of FgV1-DK21. PCR products were separated on 1% agarose gel. Lanes 1–6 represent negative control (no DNA template), F. boothii, F. graminearum, F. asiaticum, F. oxyspocum f. sp. lycopersici, and C. parasitica (#4), respectively. Lane M denotes 1-kb ladder DNA size marker.(TIF)Click here for additional data file.Figure S3AFLP fingerprints of genomic DNAs of virus-free and virus-infected fungal strains. (A) Fusarium strains. Fb, F. boothii; Fg, F. graminearum; Fa, F. asiaticum; Fo, F. oxysporum f. sp. lycopersici; Lane M, λ DNA; lanes 1, 3, 5, and 7, virus-free strains; lanes 2, 4, 6, and 8, virus-infected strains. (B) C. parasitica (Cp) strains. Lane M, λ DNA; lane 1, virus-free F. boothii; lane 2, virus-infected F. boothii; lane 3, EP155; lane 4, UEP; lanes 5 to 8, EP155 infected with FgV1-DK21. Genomic DNAs of λ DNA and fungal strains were amplified with the primer combinations EcoR I +0/Mse I +0 and EcoR I +CA/Mse I +GC, respectively. . The molecular weight size range of the fingerprints is 100–500 nucleotides.(TIF)Click here for additional data file.Text S1AFLP fingerprints of genomic DNAs of virus-free and virus-infected strains.(DOC)Click here for additional data file.


Mannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. β-Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above.Tth man5) of 1,827 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 608 amino acid residues, and was over-expressed in Escherichia coli BL21 (DE3). The results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5 is a novel β-mannosidase of glycoside hydrolase family 5. The optimal activity of the Tth Man5 β-mannosidase was obtained at pH 5.5 and 85°C and was stable over a pH range of 5.0 to 8.5 and exhibited 2 h half-life at 90°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 4.36±0.5 mM and 227.27±1.59 μmol min-1 mg-1, 58.34±1.75 mg mL-1 and 285.71±10.86 μmol min-1 mg-1, respectively. The kcat/Km values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 441.35±0.04 mM-1 s-1 and 41.47±1.58 s-1 mg-1 mL, respectively. It displayed high tolerance to mannose, with a Ki value of approximately 900 mM.A β-mannosidase gene (This work provides a novel and useful β-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes. These β-mannosidases from different GHFs possess considerable industrial applications in many fields, such as food, feed and pulp/paper industries [Mannans are complex polysaccharides representing one of the major components of hemicellulose, consisting of four types: linear mannan, glucomannan, galactomannan, and galactoglucomanan . Each ofdustries . In addidustries ,8.Pyrococcus furiosus, Thermotoga maritima, and Thermotoga neapolitana belonged to the GHF1, GHF2, and GHF2, respectively [Thermotoga thermarum, isolated from continental solfataric springs at Lac Abbe , is an anaerobic hyperthermophilic bacteria that grows at 80°C [During the last two decades, thermostable enzymes from thermophilic or hyperthermophilic microorganisms have become the hotspots of researches in many fields . The amiectively -13. Ther at 80°C . And it E. coli.The biotechnology industry is essential in modern societies , which iTth man5 gene isolated from the T. thermarum genome was 1,824 bp in length coding 608 amino acids and it was predicted as an endo-β-mannanase (Theth_0949) available at NCBI and CAZy sites  . As shown in Figure Sorangium cellulosum So ce56, 32% identity to putative β-mannosidase from Actinosynnema mirum DSM 43827 and 32% identity to the glycoside hydrolase from Streptomyces flavogriseus ATCC 33331. The results of alignments also revealed that Glu141, Glu237, Glu238, Glu292 and Glu591 were conserved amino acids among these GHF5 β-mannosidases. According to the CAZy database, two glutamic acids are the acid/base and the nucleophile, respectively. Against the similar catalytic domain of GHF5 endoglucanase (EXPDB No: 1TVP_A) from Pseudoalteromonas haloplanktis, it was presumed that active amino acids of Tth Man5 β-mannosidase were Glu141 and Glu238 [The d Glu238 .T. thermarum for expression, the protein production was very difficult to detected (data not shown). Thus, in order to increase the expression level of Tth Man5 β-mannosidase in Escherichia coli, rare codons were replaced by optimal codons without change of amino acid sequence (data not shown). The mature protein without the signal peptide, allowing the insertion of a His6-tag at the C-terminus, was successfully expressed in E. coli BL21 (DE3), after induction with IPTG for 5 h at 37°C. The recombinant protein in the cell-free extract was purified by a heat treatment followed by a nickel affinity column  and 1,4-β-D-mannan. However, no activity was detected towards konjaku powder, guar galactomannan and galactan . However, the enzyme activity was apparently inhibited by 1 mM Cu2+ and Zn2+, 0.05% Tris and 0.1% SDS. Enzyme kinetic studies in the addition of pNPM and 1,4-β-D-mannan as the substrate at optimum temperature and pH allowed the determination of the Michaelis-Menten parameters  and Maximum-Parsimony (MP) methods. Both trees displayed almost the same topological structures (NJ tree was not shown). It revealed the presence of three well-supported clades and each clade consisting of a separated monophyletic group Figure . Clade IT. thermarum was cloned and was finally defined as a β-mannosidase through the biochemical characterization.β-Mannosidase is an important hydrolytic enzyme which attacks the non-reducing end of the β-linked mannooligosaccharides to release mannoses . It playT. thermarum β-mannosidase is distinct from the other glycosyl hydrolases from T. thermarum. The substrate specificity and amino acid sequence of the β-mannosidase are apparently different from those of the xylanase and β-xylosidase from T. thermarum previously reported [. Based on sequences similarity, the Tth Man5 β-mannosidase belongs to GHF5. It has a homologous relationship with those from S. cellulosum (33%), A. mirum (32%) and S. flavogriseus (32%) , the acid/base and the nucleophile are identified as Glu141 and Glu238 residues, respectively [The reported ,20. Base) Figure . Compares Figure . This suectively . It is dT. thermarum DSM 5069 exhibits the highest activity at temperature 85°C  from Thermotoga thermarum DSM5069 was purchased from DSMZ; German Culture Collection of Microorganisms and Cell Cultures . Escherichia coli Top10 (Novagen) was used for routine molecular cloning work and E. coli BL21 (DE3) (Novagen) as the host for expression the recombinant β-mannosidase. The vectors pET-20b and pET-28a were used for cloning and expression. Cells of recombinant strain (E. coli Top10 or E. coli BL21 (DE3)) harboring pET-20b-Tth man5 and pET-28a-Tth man5 were grown in Luria-Bertani (LB) medium with addition of ampicillin (100 μg ml-1) and kanamycin (50 μg ml-1), respectively. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added with the concentration of 0.5 mM.DNA extraction was carried out as standard methods . RestricTth man5 gene was amplified by PCR of genomic DNA using primer 1 and primer 2 as shown in Table NcoI and XhoI at the 5’ and 3’ end, respectively. Primers 3 and 4 used NdeI and XhoI restrictions sites. PCR was performed as follows: 94°C, 5 min; 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 100 s; and 72°C, 10 min. The amplified DNA fragments were digested with the corresponding restriction endonucleases, and inserted into the corresponding sites in pET-28a and pET-20b (Novagen) to produce recombinant plasmids. The two plasmids encode a recombinant β-mannosidase bearing a C-terminal His6-tag, under the control of a T7 inducible promoter. The sequence of the inserts in pET-28a and pET-20b was confirmed by DNA sequencing.The coding sequence of E. coli BL21 (DE3) cells in 200 mL of LB with appropriate antibiotic selection harbouring recombinant plasmids were grown at 37°C and 200 rpm. When the OD600 reached 0.4 to 0.5, the expression of β-mannosidase was induced by the addition of 0.5 mM IPTG and the culture was incubated at 37°C and 200 rpm for 5 h. Cells were harvested by centrifugation at 4°C , washed twice with 20 mM Tris–HCl buffer (pH 8.0), and re-suspended in 5 mL of 5 mM imidazole, 0.5 mM NaCl, and 20 mM Tris–HCl buffer (pH 7.9). The cell extracts after sonication were heat treated at 70°C for 30 min, cooled in an ice bath, and then centrifuged . The obtained supernatants were loaded on to an immobilized metal affinity column (2 mL)  with a flow rate 0.2 mL min-1. Finally, 1 mL fractions were collected by eluting with 0.4 M imidazole, 0.5 M NaCl, and 20 mM Tris–HCl buffer (pH 7.9). The fractions containing β-mannosidases were dialyzed overnight against storage buffer  and then kept at -80°C until further use. The analysis of production, purity and molecular mass of the enzymes were determined by SDS-PAGE, using broad range molecular weight markers purchased from Thermo Fisher Scientific Inc.  as standards. The protein content was determined using Bradford reagent with albumin from bovine serum as standard. Oligomerization state of Tth Man β-mannosidase was determined by size exclusion chromatography on a AKTAFPLC™  system with a Superdex 200 10/30 GL column as described by Zhang et al. [g et al. .pNPM  was used for β-mannosidase activity analysis. Under standard assay condition, the purified enzyme (0.1 μg) was incubated with 10 μL of 20 mM substrate pNPM in 50 mM imidazole-potassium buffer (pH 5.5) for 10 min at 85°C. The total reaction volume was 0.2 mL. Subsequently, 600 μl of 1 M Na2CO3 was added to stop the reaction. The p-nitrophenol absorbance (pNP) was measured at 405 nm. One unit of enzyme activity was defined as the amount of enzyme necessary to liberate 1 μmol pNP per min under the assay conditions. All assays were performed in triplicate.Substrate The optimum pH for β-mannosidase was determined by incubation at various pH conditions (pH 4.5-8.5) at 85°C for 10 min in 50 mM imidazole-potassium buffer. The optimum temperature for the enzyme activity was determined by standard assay ranging from 60°C to 90°C in 50 mM imidazole-potassium buffer at pH 5.5. The results were expressed as relative activity to the value obtained at either optimum temperature or optimum pH. The maximum activity detected for pH optimum and temperature optimum were defined as 100%. PH stability assays were determined by measuring residual β-mannosidase activity after pre-incubation of enzymes in the pH rang of 4.5 to 8.5. Thermostability assays were determined by measuring residual β-mannosidase activity after pre-incubation of enzymes at 75°C, 80°C, 85°C, and 90°C for 30 min, 60 min, 90 min and 120 min. The activity of the enzyme without pre-incubation was defined as 100%.2+, Zn2+, Mn2+, Ca2+, Al3+, Ni2+, Cu2+ and Co2+ were assayed at concentrations of 1 mM in the reaction mixture. The chemical reagents EDTA (1 mM), Tris (0.05%), Tween 60 (0.05%), and SDS (0.1%) in the 0.2 mL reaction mixture were assayed. The enzyme was incubated with each reagent for 1 h at 85°C before the addition of pNPM to start the enzyme reaction. The activity of the enzyme without the chemical reagents or metal cations was defined as 100%.The effects of metal ions and chemical reagents on β-mannosidase activity of purified enzyme (0.1 μg) were determined. MgpNPM (1 to 50 mM) under standard reaction conditions described as above. The influence of various mannose concentrations range from 0.1 M to 1.3 M on the β-mannosidase activity was investigated using pNPM as substrate. The Ki value of mannose was determined defined as amount of mannose required for inhibiting 50% of the β-mannosidase activity. All assays were performed in triplicate.Kinetic constant of β-mannosidase was determined by measuring the initial rates at various concentrations of -1 to 60 mg mL-1 under standard reaction conditions described as above. All assays were performed in triplicate.The substrate specificity of the enzyme was determined by using following substrates, such as konjaku powder , guar galactomannan (medium viscosity), 1,4-β-D-mannan and galactan . The enzyme activities were assayed using the dinitrosalicylic acid (DNS) method . The rean-butanol, acetic acid, and water , and detected using the orcinol/concentrated sulfuric acid reagent [The 1,4-β-D-mannan was treated with purified Tth Man5 β-mannosidase, and the degradation was subjected to analysis on thin-layer chromatography (TLC). The reaction mixture (200 μL) contained 5% 1,4-β-D-mannan (wt/vol) and 4 μg of enzyme in 50 mM imidazole-potassium buffer (pH 5.5). The reaction was carried out for various times  at 80°C, and stopped in a water bath (4°C). After centrifuged for 10 min at 12,000 rpm, the supernatants of the reaction mixtures were applied on silica gel TLC plates . Sugars on the plates were separated with a solvent system consisting of  reagent .T. thermarum and against the CAZy database. Clstal X2 was used for multiple sequence alignment [A BLAST engine was used to search the amino acid sequences related to the β-mannosidase from lignment . Phylogelignment .T. thermarum DSM 5069 is AEH51033.The GenBank accession number of β-mannosidase from The authors declare that they have no competing interests.HS carried out the cloning, expression and drafted the manuscript. YH and YZ helped to purified and characterized the Tth Man5 β-mannosidase. WL and XL helped to analyze the data. FW directed the over-all study and revised the manuscript. All authors read and approved the final manuscript.


Lactobacillus, Lactobacillus farraginis JCM 14108T and Lactobacillus composti JCM 14202T, isolated from the compost of distilled shōchū residue. Their genome information will be useful for studies of ecological and physiological functions of these Lactobacillus species.Here, we report the draft genome sequences of two type strains of  Lactobacillus strains have been isolated from various environments and are frequently used as probiotics. Strains NRIC 0676 and NRIC 0689  were isolated from the compost of distilled shōchū  residue as the type strains of the novel species L. farraginis and L. composti, respectively  into 129 and 94 contigs, with N50 lengths of 51,335 and 87,613 bp, respectively. These assemblies resulted in draft genome sequences of 2,884,682 bp for JCM 14108T and 3,449,704 bp for JCM 14202T, with 46.2× and 28.2× redundancies and G+C contents of 42.1 and 43.9%, respectively. A total of 3,079 and 3,863 protein-coding genes and 61 and 49 RNA-coding sequences for JCM 14108T and JCM 14202T, respectively, were identified using the RAST server  and GH3 families, whereas L. farraginis JCM 14108T has only those of GH3. Both strains have genes encoding α-galactosidases of GH36. L. composti JCM 14202T has genes encoding β-galactosidases of GH2 and GH42, whereas L. farraginis JCM 14108T has only those of GH2. In addition, L. composti JCM 14202T has genes encoding xylanase of GH43, α-mannosidase of GH38, and α-N-arabinofuranosidase of GH51. Detailed analyses of the genomes of these strains will facilitate studies on the functions of Lactobacillus spp. in the compost of distilled shōchū residue.The phosphoenolpyruvate:carbohydrate phosphotransferase (PTS) system is involved in the transport and phosphorylation of numerous carbohydrates , 6. L. cL. farraginis JCM 14108T and L. composti JCM 14202T have been deposited at DDBJ/EMBL/GenBank under accession no. BAKI01000001 to BAKI01000129 and BAMK01000001 to BAMK01000094, respectively.The genome sequences of


Langerhans cells constitute a special subset of immature dendritic cells localized in the epidermis that play a key role in the skin's immune response. The production of cytokines is a key event in both the initiation and the regulation of immune responses, and different drugs can be used to remove or modify their production by DC and, therefore, alter immune responses in a broad spectrum of diseases, mainly in human inflammatory and autoimmune diseases. In the present study, we examined the effects of prednisone, thalidomide, cyclosporine A, and amitriptyline, drugs used in a variety of clinical conditions, on the production of TNF-α, IL-10, and IL-12 by purified epidermal Langerhans cells and peritoneal macrophages in BALB/c mice.All drugs inhibited TNF-α production by Langerhans cells after 36 hours of treatment at two different concentrations, while prednisone and thalidomide decreased IL-12 secretion significantly, amitriptyline caused a less pronounced reduction and cyclosporine A had no effect. Additionally, TNF-α and IL-12 production by macrophages decreased, but IL-10 levels were unchanged after all treatments.Our results demonstrate that these drugs modulate the immune response by regulating pro-inflammatory cytokine production by purified epidermal Langerhans cells and peritoneal macrophages, indicating that these cells are important targets for immunosuppression in various clinical settings. Dendritic cells (DC) are professional antigen-presenting cells (APC) that possess the unique ability to stimulate naïve T cells and initiate a primary immune response . In the In medicine, LC are often studied due to their role in numerous skin diseases, including psoriasis and contact and allergic dermatitis , and theDifferent drugs may be used to modify cytokine production by DC and thus alter the initiation and regulation of immune responses to a broad spectrum of diseases, such as human inflammatory and autoimmune diseases . Immunosin vitro.LC and macrophages (MΦ) are effective APC whose secretion of immunoregulatory and pro-inflammatory cytokines plays a critical role during T-cell priming . To gain-2 M stock solutions. ELISA kits for TNF-α, IL-12 (p40/p70), and IL-10 were purchased from BD Pharmingen .Prednisone, thalidomide, cyclosporine A, amitriptyline and LPS were purchased from Sigma-Aldrich  and were dissolved in dimethyl sulfoxide (DMSO) or methanol to make 10Female BALB/c mice were provided by the Evandro Chagas Institute, where they were maintained under specific pathogen-free conditions until use at the age of 8-12 weeks. All procedures were carried out under the Brazilian Law 1153-A, which regulates animal research in Brazil, and were approved by animal ethics committee of Pará Federal University.2. The epidermis was then incubated with DNAse enzyme  for 20 min at room temperature, after which an epidermal cell suspension was obtained by vigorous pipetting of the epidermal sheets. Next, the cell suspension was treated with mouse anti-mouse Iad  monoclonal antibody  for 45 min on ice. The cells were then incubated in plates coated with goat anti-mouse IgG  (1:100) for an additional 45 min at 4°C. After washing away floating cells, adherent LC were collected and resuspended in complete medium, consisting of RPMI-1640 supplemented with 10% fetal calf serum , 10000 U/ml penicillin/streptomycin solution (Sigma), and 50 μM β-mercaptoethanol , dispensed into 96-well flat bottom plates and incubated in humidified 5% CO2 at 37°C. These cells are cultured in suspension and can be maintained in culture flasks that are not tissue-culture treated.LC were prepared using the previously described panning method, resulting in a purity of over 95% . BrieflyAfter exposure to prednisone, thalidomide, cyclosporine A, or amitriptyline for 36 h, LC or MΦ were rinsed three times in phosphate-buffered saline (PBS) and incubated with propidium iodide (10 μg/ml) immediately prior to flow cytometric analysis  or before mounting in dilute medium on a glass slide with coverslip to assess cell viability.2 at 37°C for 1 h to allow MΦ adherence. The non-adherent cells were removed by three washes with RPMI-1640 medium. The purified MΦ were incubated for an additional 24 h with 1 ml RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin/streptomycin solution and 50 μM β-mercaptoethanol and 10 ng/ml lipopolysaccharide  [MΦ were isolated from the peritoneal cavities of female BALB/c mice. Briefly, 10 ml of cold PBS was injected into the peritoneal cavity of each mouse and the resultant exudate was immediately collected, washed, and resuspended in RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin/streptomycin solution and 50 μM β-mercaptoethanol. The cell suspension was then dispensed into 24-well flat bottom plates and incubated in humidified 5% CO; Sigma) .5 cells/well) or peritoneal MΦ (5 × 104 cells/well) were incubated with or without prednisone, thalidomide, cyclosporine A, or amitriptyline at varying concentrations  in RPMI-1640 medium supplemented with 10% fetal calf serum, 10000 U/ml penicillin/streptomycin solution and 50 μM β-mercaptoethanol. Drug concentrations were chosen based on the results of preliminary studies which showed that these concentrations had no effects on the functions of other cell types [In the culture experiments, purified LC  or pg/ml × 104 cells .Culture supernatants were collected after 36 h, centrifuged, stored at -20°C and subjected to protein quantification at the indicated time-points by ELISA, using mouse TNF-α, IL-12, and IL-10 immunoassay kits according to the manufacturer's instructions (BD Pharmingen). Protein levels were assessed using a microplate reader at 450 nm , and each sample was tested in triplicate. Data are expressed in pg/ml × 10Data obtained from three independent experiments are presented as mean ± SD and were compared using the Student's t test for single comparisons or analysis of variance (ANOVA) for multiple comparisons. Differences were considered significant at p < 0.05.-6 M or 10-8 M . TNF-α production (21.8 ± 1.4 pg/ml × 105 cells) decreased to 10.8 pg/ml × 105 cells (p < 0.05) after treatment with 10-6 M prednisone and to 9.5 pg/ml × 105 cells (p < 0.05) after treatment with 10-8 M prednisone, corresponding to an almost 50% reduction  and to 4.4 ± 3.8 pg/ml × 105 cells at 10-8 M   and to 7.6 ± 0.5 pg/ml × 105 cells at 10-8 M   and by 44% at 10-8 M . IL-12 production (9.4 ± 0.5 pg/ml × 105 cells) decreased to 2.9 ± 0.7 pg/ml × 105 cells (p < 0.01) after treatment with 10-6 M prednisone and to 4.0 ± 1.1 pg/ml × 105 cells (p < 0.01) after treatment with 10-8 M prednisone, corresponding to a 69% and 57% reduction, respectively  and to 3.5 ± 1.8 pg/ml × 105 cells at 10-8 M  , while the lowest dose of the same drug did not have a statistically significant effect. Additionally, no IL-10 release was detected in the culture supernatants over the 36-hour incubation period (data not shown).The purified LC acquires mature phenotypes during culture even without exogenous stimulation . We utilAfter exposure to prednisone, thalidomide, cyclosporine A, or amitriptyline for 36 h, we assessed the cell viability of cultured MΦ by PI staining. None of the immunomodulatory drugs used affected the cell viability of cultured MΦ , corresponding with a 56% or 53% (129.6 ± 33.0 pg/ml × 104 cells or 138.2 ± 6.0 pg/ml × 104 cells) reduction. Thalidomide at 10-6 M downregulated TNF-α secretion by 65.6%, to 100.9 ± 9.0 pg/ml × 104 cells (p < 0.01), while LPS-stimulated MΦ incubated with 10-8 M thalidomide exhibited a slight, but statistically insignificant, reduction in TNF-α release. Similarly, cyclosporine A reduced TNF-α secretion significantly (117.4 ± 59.7 pg/ml × 104 cells) but only at the highest dose, while the lowest dose of the same drug did not have a statistically significant effect. When LPS-stimulated MΦ were incubated with 10-6 M amitriptyline, TNF-α release was reduced from 339.3 ± 82.3 pg/ml × 104 cells to 120.6 ± 16.9 pg/ml × 104 cells , while 10-8 M amitriptyline caused no significant reduction , thalidomide at 10-6 M (139.0 ± 9.8 pg/ml × 104 cells), cyclosporine A at 10-8 M (149.7 ± 9.1 pg/ml × 104 cells), and amitriptyline at both 10-6 M and 10-8 M (150.0 ± 5.4 pg/ml × 104 cells and 148.6 ± 8.6 pg/ml × 104 cells), all corresponding to about a 40% reduction. Meanwhile, IL-10 levels by LPS-stimulated MΦ were not altered by any of the drugs tested  by prednisone at 10Epidermal DC are believed to be involved in allergic and irritant contact dermatitis , as wellin vitro TNF-α and IL-12 production by unstimulated cultured LC was reduced by prednisone. Previous data demonstrated that DC derived from human monocytes were similarly suppressed by dexamethasone [in vitro data summarized above appear to disagree with our findings, since we did not see a consistent change in IL-10 production due to prednisone treatment. This discrepancy may be due to differences in the experimental setup, including the fact that previous studies added glucocorticoids before or together with LPS and used human cells. Thus, the type of stimulus and cell source may influence the effects of glucocorticoids on IL-10 secretion.We found that ethasone . Our resethasone . Other rethasone . All of Prednisone also inhibited the IL-12 secretion by LPS-stimulated MΦ after 36 hours of treatment. This finding corroborates published data showing that MΦ treated with dexamethasone for 18 hours, followed by stimulation with Listeria antigen for two days, exhibited significantly reduced IL-12 production .-6 M), supporting previous studies showing that thalidomide inhibits IL-12 production by LPS-stimulated monocytes [Thalidomide has been shown to profoundly inhibit the ability of LC to present skin-purified antigens and to produce TNF-α . Our datonocytes . Anotheronocytes , what maWe next examined the effects of cyclosporine A on unstimulated cultured LC. TNF-α production by LC was inhibited at both concentrations of cyclosporine A, suggesting that the effects of this drug are similar to those of prednisone and thalidomide. However, we observed no significant changes in the IL-12 secretion by cyclosporine A-treated LC, despite previous observations that the drug blocked IL-12 production by CD40-stimulated monocyte-derived DC . It is pAlthough the mechanism underlying cyclosporine A effects on LC remains to be elucidated, our results support the hypothesis that cyclosporine A inhibits unstimulated cultured LC TNF-α secretion.Other investigators have also observed decreased basal TNF-α secretion by the monocyte cell line U936  culturedWe also examined the effects of amitriptyline on LC and MΦ cytokine secretion. Despite recent work showing that amitriptyline plays an immunomodulatory role, little is known about its mechanism of action and target immune cells. It was previously reported that similar tricyclic antidepressants, such as clomipramine, imipramine, and citalopram, cause reduction in TNF-α release by LPS-stimulated peripheral blood monocytes . Recentlin vitro and in vivo studies are necessary to substantiate these findings and to provide further information on the mode of action of prednisone, thalidomide, cyclosporine A and amitriptyline on a cellular and molecular level.In summary, the study indicates that there are differential regulation by immunosuppressive drugs on TNF-α and IL-12 production by LC and MΦ, which constitute important targets for immunomodulatory drugs. Further APC: antigen-presenting cells; DC: dendritic cells; ELISA: enzyme-linked immunosorbent assay; IL-10: interleukin-10; IL-12: interleukin-12; LC: Langerhans cells; LPS: lipopolysaccharide; MΦ: macrophages; NF-κB: nuclear factor-kappa B; TNF-α: tumor necrosis factor-α.The authors declare that they have no competing interests.SRC participated in the design of the study, carried out some of the literature searches, conducted the experiments and drafted the manuscript. MBS participated in the design of the study, conducted the experiments and synthesized the findings. JLFV provided some drugs for the study. JPS participated in the design of the study and carried out some of the literature searches. CGS participated in the design of the study, synthesized the findings and edited the manuscript. All the authors read and approved the final manuscript.


This study was conducted to investigate the effect of flavonoids, a major family of antioxidants contained in foods, on retinal ganglion cell (RGC) death induced by hypoxia, excessive glutamate levels, and oxidative stress. Moreover, to assess the structure-activity relationships of flavonoids, three types of flavonoids with different numbers of hydroxyl groups and varieties of sugar chains were studied.2, 5% CO2, 37 °C) for 12 h, 25 µM glutamate over three days, or oxidative stress by depleting antioxidants from the medium for 24 h. RGC survival rates were calculated under each condition and compared with vehicle cultures. Modification of cell death signaling after stress-induced apoptosis and necrosis by flavonoids was assessed using caspase-3 and calpain immunoreactivity assays.Three kinds of flavonoids—nicotiflorin, rutin, and quercitrin—were used. The death of neonatal rat purified RGCs was induced by hypoxic conditions  may enhance neuroprotective activity. Hypoxia, glutamate, and oxidative stress are important factors in ocular disease states affecting retinal ganglion cells (RGCs). Retinal ischemic and inflammatory diseases such as retinal vascular occlusion or diabetic retinopathy elicit hypoxic and/or oxidative stress–induced RGC death -3. LikewFlavonoids comprise a large family of plant-derived compounds widely distributed in fruits and vegetables ,15. TherO-rutinoside (nicotiflorin), quercetin 3-O-rutinoside (rutin), and quercetin 3-O- rhamnoside (quercitrin)—from more than 7,000 known flavonoids, and investigated their neuroprotective potential using rat primary-isolated RGCs cultured under three kinds of stress conditions: hypoxia, excessive glutamate levels, and oxidative stress. Since these three compounds have quite similar structures, excluding the number of hydroxyl groups and sugar side chains, the structure-activity relationships for neuroprotective activity were also considered.In the current study, we selected three types of flavonoid compounds—kaempferol 3-Six- to eight-day-old Wistar rats were purchased from Saitama Laboratory Animal Supply Inc. . All animal experiments were conducted with approval from the Animal Care and Use Committee and under the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.O- rutinoside (nicotiflorin) was purchased from Extrasynthese ; quercetin 3-O-rhamnoside (quercitrin) was from Synthese ; and quercetin 3- O-rutinoside (rutin) was purified by one of the authors, Tsukasa Iwashina, from the flowers of Astrophytum ornatum (Britton & Rose) [Cell culture reagents were obtained from Gibco . A cell-dissection kit of papain was obtained from Worthington Biochemical . Calcein-AM was obtained from Sigma . Poly-L-lysine, BSA, L-glutamine, and recombinant neurotrophic factors (human brain-derived neurotrophic factor and rat ciliary neurotrophic factor) were obtained from Sigma. Monoclonal antibodies (OX-41) against rat macrophages and monoclonal antibodies (OX-7) against rat and mouse Thy-1.1 were obtained from Chemicon . B-27 supplement minus antioxidants (AO-) was obtained from Gibco. The origins of the flavonoids were as follows: kaempferol 3- & Rose) . All fla2 and 95% air at 37 °C for 3 days.RGCs were purified by a two-step immunopanning procedure, as described previously . Briefly2, 90% N2, and 5% O2 mixture) [2, 95% air, at 37 °C) [After culturing for three days, RGC death was induced by the following three conditions. For hypoxia, the plates were transferred to a controlled-atmosphere incubator and incubated for 12 h, during which oxygen levels were reduced (hypoxic condition: 5% COmixture) . For exct 37 °C) . For oxit 37 °C) .Nicotiflorin and rutin were added at final concentrations of 0.1 nM, 1 nM, and 10 nM, and quercitrin at 10 nM, 100 nM, and 1 µM. Culture medium with flavonoids were made by a serial dilution with B27 neurobasal medium. In the present study, a surviving RGC was defined as a cell with a calcein-AM-stained cell body and a process extending at least three cell diameters from the cell body ,26,29. AIn our previous studies, we determined the cell death patterns of purified RGCs where death was induced by hypoxia, glutamate, and oxidative stress under our culture conditions, and found that both hypoxia- and glutamate-induced death mainly involved mitochondrial-dependent and Bax-mediated apoptosis , while ot test or one-way ANOVA followed by Dunnett’s test were used in this study. All data are indicated as mean±SD.The Student After 12 h of hypoxia stress, the RGC survival rate without nicotiflorin was reduced to 56.0±3.1%. The RGC survival rates of the nicotiflorin-treated group were 54.3±3.2%, 62.3±1.9%, 73.8±2.0%, 71.4±4.9%, and 56.1±3.2% at final concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1 µM, respectively . The RGCAfter 72 h of glutamate stress, the RGC survival rate without nicotiflorin was reduced to 69.8±7.4%. The RGC survival rates in the nicotiflorin-treated group were 71.4±9.2%, 80.9±7.8%, 89.1±5.2%, 85.7±3.4%, and 72.2±2.6% at final concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1 µM, respectively . The RGCAfter 24 h of oxidative stress, the RGC survival rate without nicotiflorin was reduced to 44.4±8.1%. The RGC survival rates of the nicotiflorin-treated group were 45.4±12.6%, 57.3±15.8%, 71.2±9.9%, 70.8±4.1%, and 48.1±2.9% at final concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1 µM, respectively . The RGCAfter 12 h of hypoxia, caspase-3 activity in RGCs without rutin was 0.80±0.14, whereas caspase-3 activity without hypoxia was 0.55±0.11. Caspase-3 activities with rutin were 0.62±0.1, 0.58±0.21, and 0.59±0.09 at final concentrations of 1 nM, 10 nM, and 100 nM, respectively (arbitrary units). Rutin showed a significant reduction of caspase-3 activity under hypoxia . Rutin showed a significant reduction of caspase-3 activity by glutamate stress . There was no significant change of caspase-3 activity by oxidative stress n=6; .After 12 h of hypoxia, calpain activity in RGCs without rutin was 0.59±0.03, whereas caspase-3 activity without hypoxia was 0.51±0.03. Calpain activities in RGCs with rutin were 0.60±0.4, 0.58±0.02, and 0.58±0.05 at final concentrations of 1 nM, 10 nM, and 100 nM, respectively (arbitrary units). There was no significant change of calpain activity under hypoxia n=6; .After 72 h of glutamate stress, calpain activity in RGCs without rutin was 0.60±0.03, whereas caspase-3 activity without glutamate was 0.51±0.03. Calpain activities in RGCs with rutin were 0.62±0.02, 0.58±0.04, and 0.59±0.03 at final concentrations of 1 nM, 10 nM, and 100 nM, respectively (arbitrary units). There was no significant change of calpain activity under hypoxia n=6; .After 24 h of oxidative stress, calpain activity in RGCs without rutin was 0.78±0.19, whereas caspase-3 activity without oxidative stress was 0.56±0.14. Calpain activities in RGCs with rutin were 0.59±0.13, 0.58±0.23, 0.57±0.13 at final concentrations of 1 nM, 10 nM, and 100 nM, respectively (arbitrary units). Rutin showed significant reduction of calpain activity by oxidative stress . In our previous studies using the same hypoxic culture system, the effective doses of lomerizine, a Caectively ,27. Thusll death .In previous reports about flavonoids using the RGC-5 cell line, the effective doses (over 10 µM) were quite a bit higher than our results, although the examined flavonoids and the conditions to induce RGC-5 cell death were different from those used in our experiments -23. Anot2+ levels despite high levels of ROS [So far, there have been no reports comparing flavonoid structure-activity relationships. This is the first study to indicate the differences in biologic activities among three kinds of flavonoids with a similar chemical structure shown in . Althougs of ROS ,19. In tOxidative stress has been implicated in many types of ocular diseases. Several previous reports have shown that RGC damage under oxidative stress is mainly linked with increased intracellular ROS and calcium influx ,28. DuriRutin, as a representative of the three flavonoids, caused a significant inhibition of both apoptotic and necrotic pathways in RGCs. In this study, we investigated the effect of rutin on the activities of caspase-3 and calpain, which are the representative proteases in apoptotic and necrotic pathways, respectively. As a result, rutin suppressed these activities, but there was no dose-dependency. These data suggest that other protective signals in addition to caspase-3 or calpain may be present. There is a limitation to investigating the signaling molecules related to apoptosis and necrosis using primary cultures of RGCs, because a large number of primary cells is required for biochemical assays targeting various death signals. Further experiments are required to clarify the mechanism of these flavonols.Nicotiflorin and rutin, with only one hydroxyl group difference between them structurally, are contained mainly in fruits and vegetables. Quercitrin, with a different sugar side chain than rutin, is contained mainly in tea and some medicinal plants. The beneficial effects of dietary plant substances containing these flavonoids has been recorded since ancient times and passed on for generations. Buckwheat and tartary buckwheat, which are commonly ingested in Japan and other Asian countries, are both rich in rutin; amazingly, rutin accounts for as high as 1% of the total weight of buckwheat and tartary buckwheat ,38. A feIn summary, we studied the neuroprotective effects of three kinds of flavonoids using purified rat RGC cultures, and found that nicotiflorin and rutin had beneficial effects on hypoxic, glutamate, and oxidative stress at concentrations as low as 1 nM. The structure-activity relationships suggested that the sugar side chain of flavonoids might be important for neuroprotective activities. Moreover, rutin showed inhibitory effects on both apoptotic and necrotic pathways. Nicotiflorin and rutin have the potential to be promising neuroprotective drugs in the future.