Bacillus anthracis spores germinate and the subsequent vegetative growth results in bacteremia and toxin production. Anthrax toxin is tripartite: the lethal factor and edema factor are enzymatic moieties, while the protective antigen (PA) binds to cell receptors and the enzymatic moieties. Antibiotics can control B. anthracis bacteremia, whereas raxibacumab binds PA and blocks lethal toxin effects. This study assessed plasma PA kinetics in rabbits following an inhaled B. anthracis spore challenge. Additionally, at 84 h post-challenge, 42% of challenged rabbits that had survived were treated with either levofloxacin/placebo or levofloxacin/raxibacumab. The profiles were modeled using a modified Gompertz/second exponential growth phase model in untreated rabbits, with added monoexponential PA elimination in treated rabbits. Shorter survival times were related to a higher plateau and a faster increase in PA levels. PA elimination half-lives were 10 and 19 h for the levofloxacin/placebo and levofloxacin/raxibacumab groups, respectively, with the difference attributable to persistent circulating PA-raxibacumab complex. PA kinetics were similar between untreated and treated rabbits, with one exception: treated rabbits had a plateau phase nearly twice as long as that for untreated rabbits. Treated rabbits that succumbed to disease had higher plateau PA levels and shorter plateau duration than surviving treated rabbits.Inhaled  Bacillus anthracis causes anthrax [B. anthracis infection [ anthrax , and its anthrax . Human i anthrax ,2,3. Altnfection ,5.1 monoclonal antibody against PA [Clinical presentation of inhalational anthrax is similar in non-human primates, rabbits, and humans ,7,8,9. Aainst PA , and is ainst PA . Justifiainst PA ,14 requiPrior studies have shown that raxibacumab is efficacious in the treatment of inhalation anthrax in the post-exposure prophylaxis setting, as well as when administered at the onset of disease . In thosThe in-life portions of this good laboratory practice study were conducted at the Battelle Biomedical Research Center . The study was approved by Battelle’s institutional animal care and use committee. The assays for plasma PA concentrations and PA kinetic analyses were performed at Human Genome Sciences, Inc. .Pathogen-free New Zealand white rabbits surgically implanted with vascular access ports (VAP) were supplied by Covance Research Products, Inc. . Of the 210 rabbits  supplied, 180 were required for the study, with the extra animals available as replacements before the spore challenge, if required. No rabbits were replaced after the spore challenge. After a quarantine period (minimum of 7 days), rabbits that were in good health, free of malformations, and exhibited no signs of clinical disease were randomly selected for the study. Rabbit age was not a criterion for placement in this study.® Oral Solution  was administered intragastically (IG) as supplied, without dilution.LevaquinRaxibacumab  and raxibacumab vehicle  were produced at HGS as ready-to-use sterile liquid formulations and were stored at 2 to 8 °C prior to use. Raxibacumab vehicle contains 0.13 mg/mL citric acid, 2.8 mg/mL sodium citrate, 10 mg/mL sucrose, 18 mg/mL glycine, and 0.2 mg/mL polysorbate 80 at pH 6.5.Prior to administration, raxibacumab or vehicle control were aseptically transferred to individual dosing vials according to a randomization scheme, and the vials were stored at 2 to 8 °C prior to dosing. The study director and technicians were blinded to raxibacumab or placebo administered to each rabbit.The study was conducted in 3 sets of challenge days, with 70 rabbits representing a challenge set. Rabbits from each challenge set were randomized to 1 of 2 challenge days such that 30 rabbits  were spore challenged on each day, and the remaining 10 rabbits were a replacement group. The challenge order within each challenge day was also randomized.50, 105,000 colony forming units [B. anthracis (Ames strain) spores in a plethysmography chamber and a Class III cabinet system. The aerosol challenge duration was based upon an estimated aerosol challenge concentration and a cumulative minute volume gathered “real” time throughout the exposure.On Study Day 0, rabbits were individually challenged with a targeted aerosol 200 times the median lethal dose (LDng units ) of B. apost the median challenge time for a group of animals spore challenged on the same day. Levofloxacin was administered by gastric intubation, followed by intravenous (IV) injection of raxibacumab or placebo (raxibacumab buffer), such that there were 2 treatment groups: levofloxacin alone or levofloxacin in combination with raxibacumab.Treatments were initiated at a fixed time of 84 h (±4 h) The 76 rabbits that survived to the designated treatment time were randomly assigned to the 2 treatment groups. All treated rabbits were administered 50 mg/kg of levofloxacin IG once daily (qd) for 3 consecutive doses (2 mL/kg). Immediately after administration of the first levofloxacin dose, rabbits also received either a single IV 40 mg/kg raxibacumab or placebo dose (0.8 mL/kg) via the VAP or a marginal ear vein. post raxibacumab dose, as well as terminal specimens, were not collected from the VAP. Blood specimens were centrifuged, and the plasma was harvested. The plasma was filtered, tested for sterility, and stored at ≤−70 °C prior to shipment to HGS for assay. Bacteremia was assessed by culture at all collection times, with the exceptions of 5 min and 8 h post raxibacumab dose. Quantitative bacteremia was determined at 24 h post-challenge, immediately prior to the first treatment (84 h), 24 h after the first levofloxacin dose (prior to the second levofloxacin dose), and 2 days post the third levofloxacin dose.Blood samples for plasma PA analysis were taken from the VAP, the medial auricular artery, or the marginal ear vein into tubes containing ethylenediaminetetraacetic acid as an anticoagulant. Blood specimens were collected from all rabbits prior to spore challenge, as well as from all surviving rabbits at 12, 24, 36, 48, 60, and 72 h after spore challenge. For all treated rabbits, additional blood specimens were collected just prior to the first levofloxacin dose; at 5 min and 8 h after the raxibacumab dose; at 2 h after each levofloxacin dose; at 24 h after the first and second levofloxacin doses; at 2 and 3 days after the third levofloxacin dose; and at 7, 14, 21, and 28 days after the raxibacumab dose. When feasible, a terminal blood sample was taken just prior to euthanasia for animals that were judged to be moribund. Blood specimens collected at 7, 14, 21, and 28 days et al. [Samples were stored at ≤−70 °C at HGS. Just prior to assay, samples were thawed and working aliquots taken. Plasma total (free and raxibacumab-bound) PA concentrations were determined using an electrochemiluminescence (ECL)-based bridging assay. In brief, diluted plasma samples in duplicate wells were combined with rabbit polyclonal antibody (pAb) anti-PA-biotin (capture) and rabbit anti-PA pAb labeled with Meso Scale Discovery SULFOTAG™, an ECL label (detector), and allowed to equilibrate in a streptavidin-coated assay plate. Following equilibration, plates were washed and read for ECL counts. The concentration of PA in plasma samples was interpolated from a reference standard curve. The assay run had to meet system suitability criteria for the results to be considered valid. Samples were diluted 1:4 or 1:500, depending on the expected PA concentration. If samples were out of range at either dilution, they were then repeated at the alternative dilution. For each dilution, 3 quality controls (QC) were run twice on each plate . For the 1:4 dilution, 820, 24, and 1.5 ng/mL were used. For the 1:500 dilution, 102500, 3000, 187.5 ng/mL were used. Four out of the six QC samples had to recover between 75% and 125% with a CV ≤ 20%. At least one QC of each QC level must have met this criterion. Individual samples had to have a CV ≤ 20% to be accepted. The lower and upper limits of quantitation were 0.5 ng/mL and 136,500 ng/mL of PA in 100% rabbit plasma, respectively. Accuracy and precision was measured at 0.5, 1.5, 24, and 820 ng/mL. Accuracy values of 96%, 115%, 108%, and 102%, respectively, were observed for each spike. Precision values of 13%, 11%, 6%, and 8%, respectively, were observed for each spike. Both the reference standard curve and QC were prepared with recombinant PA (83 kDa) with an estimated purity of 97% (based on reverse phase high performance liquid chromatography). PA was prepared based on the method of Laird post spore challenge, actual magnitude of spore challenge, and actual dose times were used for the analyses. PA concentrations below the lower limit of quantitation were treated as zero. The PA concentration-time data was divided into 2 subgroups for analysis. The first subgroup comprised those rabbits that were subjected to the spore challenge but did not survive until the 84 h post-challenge treatment time . The other subgroup comprised those rabbits that were subjected to the spore challenge, survived until the 84 h post-challenge treatment time, and were administered levofloxacin and either raxibacumab or placebo . The plasma PA concentration-time data for both subgroups were analyzed using the NONMEM software (Version VI 1.3).Actual times B. anthracis septicemia, reflecting both the onset and magnitude [y is the number of bacteria present at time t, and a, b, and c are constants.Systemic PA levels have been shown to correlate with agnitude ,17. In aagnitude , which det al. [0) and A = ln(N∞/N0). N is the number of bacteria present at time t, 0N is the number of bacteria initially present at time 0, ∞N is the number of bacteria present at infinite time (the asymptotic phase), μm is the maximum specific growth rate, and λ is the lag time.Zwietering et al.  publisheB. anthracis septicemia [et al. [αw and βw are weights applied to the 2 functions; the sum of the 2 weights is constrained to equal 1.Surrogate measures for bacterial numbers, such as absorbance of light at 620 nm by liquid bacterial growth medium, have been used in the Gompertz model . Since spticemia ,17, plaspticemia . Liquori [et al.  modeled AT, μmT, and λT are the factors by which A, μm, and λ in the β phase differ from those in the α phase.That approach was applied to describe diauxic plasma PA concentration-time profiles , applyinm,2 is the maximum specific growth rate for the second rising phase and λ2 is the lag time for the second rising phase.In the present study, untreated rabbits exhibited at least 1 or more of the following phases of the diauxic Gompertz model: initial lag phase, first rising phase, plateau phase, and second rising phase, but no rabbits had evidence of the terminal plateau phase in their profile. Since there was no evidence of a terminal plateau phase, a composite modified Gompertz/second exponential growth model was used to describe plasma PA kinetics. That model consisted of the modified Gompertz model combined with a second exponential rising phase that occurs after a second lag phase, and is described by the following set of equations:Body weight, sex, age, size of spore challenge, duration of spore challenge, and time to first bacteremia by culture (TBAC) were evaluated as potential covariates.In the present study, it was observed that plasma PA concentration-time profiles in treated animals generally showed an initial rise, but before or shortly after the second rising phase was attained, plasma PA concentrations began to decline, with the decline of plasma PA levels generally coinciding with the sterilization of bacteremia. Hence, it was expected that, for treated rabbits, PA levels would initially follow the same composite modified Gompertz/second exponential growth model described by Equation 7 through 9, but with the addition of a monoexponential elimination phase that starts after treatment. This modified model can be expressed as follows:t ≤ txt, Equation 7 through 9 apply; For times t > txt;txt), and kelm is the first order elimination rate constant. Although this model assumes that PA elimination occurs only after treatment, that assumption allows direct comparison of the composite modified Gompertz/second exponential growth model parameters between untreated and treated rabbits.For times versus levofloxacin plus raxibacumab) was assessed as a possible covariate in the model. Body weight, sex, age, size of spore challenge, duration of spore challenge, and TBAC were also evaluated as potential covariates.It should be noted that measured plasma PA (free plus raxibacumab bound) levels are decreased in the presence of raxibacumab, with the expected peak raxibacumab level resulting in measured plasma PA concentrations about 30% to 64% of the actual value. Hence, treatment  spore dose (range: 86 to 348 × LD50). There were no significant differences in spore exposure between treatment groups or among challenge days. Of the 180 rabbits that were challenged, 104 rabbits  died prior to the 84 h post-challenge treatment time (the untreated group), while 76 rabbits (42%) remained alive and were randomized to treatment with levofloxacin and placebo  or levofloxacin and raxibacumab . At 28 days after the last levofloxacin dose, there were 24 survivors (65%) in the levofloxacin/placebo group and 32 survivors (82%) in the raxibacumab/levofloxacin group. Although the 17% absolute difference in survival rate between the groups did not reach statistical significance (p = 0.0874), that difference in mortality rate is clinically meaningful, since there were about half as many deaths (18% versus 35%) when raxibacumab was administered. There was no evidence of any relationship between survival time or outcome and body weight, sex, age, size of spore challenge, duration of spore challenge, or time to first bacteremia by culture.A total of 180 rabbits were challenged with a target 200 × LDIt should be noted that the survival rate for rabbits that were treated with levofloxacin alone (65%) approximates the 55% survival rate for humans in the 2001 anthrax attack ,24. IndeThe mean (with SD error bars) observed plasma PA concentration-time profiles for the untreated animals included in the analyses are illustrated in As shown in 0N), lag time for the first growth phase (λ), maximum specific growth rate for the first phase (μm), or lag time for the second growth phase (λ2). Given the complexity of this model, the substantial variability in the data, and the varying contribution of each animal to the model, inability to adequately model inter-individual variability for all parameters in the model could be expected. Rather than revealing any deficiency in the model, this outcome may be a qualitative reflection of the data being modeled. Since the purpose of this modeling exercise was to provide a description of the observed data, the modeling results can be considered adequate for the intended purpose, but should be interpreted with caution due to the poor definition of the variability components.Modeling of inter-individual variability for all of the model parameters was attempted, but was not successful. In the final model, inter-individual variability could not be estimated for PA concentration at time 0  or the maximum specific growth rate for the second phase  were assessed  phase to assessed . SurvivaThe mean (with SD error bars) observed plasma PA concentration-time profiles for the treated animals are illustrated in elm) and no delay time.The data for treated animals were consistent with the composite modified Gompertz/second exponential growth model with a monoexponential elimination phase. Alternate parameterizations using clearance and volume of distribution terms and/or a delay time between treatment and the start of PA elimination were unsuccessful due to apparent over-parameterization of the model. In particular, the estimate for the delay time was very small, implying that the data did not support estimation of that parameter. The final model was parameterized with a first-order elimination rate constant  were significantly higher (33%) than those for rabbits that survived, and duration of the plateau phase was significantly shorter (29%) than for rabbits that survived. These findings are consistent with the hypothesis that death due to toxemia tends to be associated with PA levels that are higher, and progress to higher levels earlier.Possible differences in PA kinetics for treated rabbits that died and survived were assessed . The 95%m was associated with sex, and some variability in kelm was associated with treatment group. No other associations between PA kinetics and the covariates assessed were found. For females, λ was about 80% of that for males, and μm was only about 40% of that in males. Despite these differences, there was not a difference in survival rates between males and females, suggesting that the differences were not clinically meaningful. For rabbits, administered levofloxacin plus raxibacumab, λ was 7% longer than that for rabbits administered levofloxacin alone. This small difference would not be expected to be clinically meaningful. PA elimination was 45% slower in rabbits administered levofloxacin with raxibacumab than in rabbits administered levofloxacin alone. This may reflect systemic persistence of measurable PA resulting from the PA-raxibacumab complex. However, λ and kelm were not significantly different between the survivor and non-survivor subgroups  in this study were 10 and 19 h for rabbits administered levofloxacin alone and levofloxacin with raxibacumab, respectively, with the difference likely reflecting persistence of the PA-raxibacumab complex in circulation. That is, when raxibacumab is not present to bind PA, the PA 1/2.elmt is 10 h. However, when raxibacumab is present , PA is bound in PA-raxibacumab complexes. Although the t1/2.elm of the complex has not been measured, it is very likely extremely long (many days) relative to that for PA alone (less than 0.5 day). In essence, the PA-raxibacumab complex probably acts as a sink which slows PA elimination. The formation of the complex also clears plasma of the toxin, leading to efficacy.Based on the ki.e., those that died prior to the 84 h post-spore challenge treatment time; 2, which essentially represents the duration of the plateau phase of the serum PA profile. This suggests that although an inhaled B. anthracis spore challenge can be lethal, survival to the 84 h post-challenge treatment time was dependent on the duration of the plateau phase, rather than other PA kinetics. Since λ2 was not associated with the covariates assessed in this study, survival to the 84 h post-challenge treatment time was likely dependent on other factors. Most important, for those animals that were treated in the late stages of toxemia, both levofloxacin alone and levofloxacin plus raxibacumab were highly effective in attaining high survival rates.There was similarity in PA kinetics between the untreated rabbits . However, that difference was clinically meaningful, with about half as many deaths (18% versus 35%) when raxibacumab was administered.The 17% absolute difference in survival rate between the treated groups did not reach statistical significance . For that group, TBAC was associated with elm, while sex was a significant covariate for λ and μm. None of these effects were clinically meaningful, since they were not associated with differences in survival outcome.Rabbits that survived had plasma PA concentrations that tended to decrease at times coincident with negative bacteremia. In treated rabbits, treatment group was a significant covariate for λ and kt1/2 for PA was 10 and 19 h for rabbits administered levofloxacin alone and levofloxacin plus raxibacumab, with the difference likely reflecting persistence of the PA-raxibacumab complex in the circulation. For the treated rabbits that died, plateau PA levels were higher than in surviving rabbits and duration of the plateau phase was shorter than in surviving rabbits, consistent with death due to toxemia being associated with PA levels that are higher, and progress to higher levels earlier.In surviving rabbits, the i.e., those that died prior to the 84 h post-spore challenge treatment time) and the treated rabbits that survived, with one exception: the duration of the plateau phase, reflected by λ2, was nearly twice as long in treated than untreated rabbits.PA kinetics were similar between untreated rabbits (


Ficus deltoidea, is a perennial herb that is used to assist labor, firm the uterus post-delivery and to prevent postpartum bleeding. In view of its claimed uterotonic action, the mechanisms underlying plant’s effect on uterine contraction were investigated.2) to synchronize their oestrous cycle. A day after injection, uteri were removed for in-vitro contraction studies. The dose dependent effect of Ficus deltoidea aqeous extract (FDA) on the tension produced by the isolated rat’s uteri was determined. The effects of atropine (2×10-8 M), atosiban (0.5 IU), THG113.31 (10 μM), oxodipine (0.25 mM), EDTA (1 mM), 2-amino-ethoxy-diphenylborate (2-APB) (40 mM) and thapsigargin (1 mM) on the maximum force of contraction (Emax) achieved following 2 mg/ml FDA administration were also investigated.Adult female SD rats were injected with 2 mg/kg 17β-oestradiol  and was dependent on the extracellular Ca Uterotonic plants, are plants that stimulate uterine contraction and have been used since the ancient times to assist labor, remove the retained placenta, treat post partum bleeding and as an abortifacient . Despite, which belongs to the family Moraceae [One of the plants that is popularly used among the Southeast Asian women to induce uterine contraction is Ficus deltoideaMoraceae . Ficus dMoraceae . DiffereMoraceae . All parMoraceae . AdditioMoraceae . RecentlMoraceae .in-vitro using isolated uteri from rodents. Bafor et al. [in-vitro study using isolated strips of mouse uteri to investigate the effect of the active constituent of Ficus exasperata leaf extract on uterine contraction. They have found that metabolites generated from this extract possess both tocolytic and uterotonic activities. Earlier, in-vitro contraction studies using isolated rat’s uteri revealed both stimulatory [in-vitro contraction of the uteri isolated from oestrogenized rats. Another Ficus species, Ficus capensis was shown to inhibit in-vitro contraction also in the isolated rat’s uteri [The notion that Ficus deltoidea affects uterine contraction was based on several previous studies involving other Ficus species which also indicate their effect on the uterine contractility. Most of these studies were conducted r et al.  conductemulatory  and inhimulatory  effects mulatory  demonstr’s uteri . In view’s uteri ,17,18,20F deltoidea from female sub-species used in this study were supplied by Delto Medicama Plantation, Kuala Selangor, Malaysia. The plant sample was deposited at the Herbarium in Rimba Ilmu, University of Malaya, Kuala Lumpur for authentication and identification purposes with Herbarium number: KLU 46469. The leaves were air-dried, cut into small pieces and grounded into powder form. Each of the pulverized parts were weighed (100 grams) and boiled twice in 1 litre distilled water for 4 hours. The aqueous extract was then concentrated by heating at 60°C and was later subjected to freeze- drying  and was stored in a container until further use. Stock solution was obtained by dissolving small aliquots of this extract in water.Leaves of ad libitum. Cleanliness of the housing environment was maintained daily. Shredded recycled paper was used as bedding. Each group consists of six rats (n = 6). All experimental procedures were approved by the University of Malaya Medical Center Animal Ethics Committee (Ethics Reference No: FIS/ 01/12/2008/ NS (R)). Intact, non-ovariectomised female WKY rats were treated with high dose of E2 at 2 mg/kg/day to synchronize their oestrous cycle [2 (1.0 mM), CaCl2 (2.0 mM) and D- glucose (10 mM) while the pH was maintain at 7.40 with NaOH. The temperature of the organ bath was maintained at 37°C. 95% O2 and 5% CO2 was continuously delivered into the bathing solution. Each uterine strip was placed under optimum resting force of 1 g and was allowed to equilibrate for 30 minutes prior to drug administration. During this period, the strips were washed with 10 ml fresh physiological solution every 15 minutes according to the method by Oropeza et al., [Adult female Wistar Kyoto (WKY) rats weighing 250 grams were purchased from the Animal House, Faculty of Medicine, University of Malaya, Kuala Lumpur. The rats were housed in a controlled environment with temperature kept at 25°C, relative humidity between 30–70%, 12 hours light–dark cycle and had free access to rodent food pellet and tap water us cycle . A day a et al., . Each ex-2 M Ach (Sigma-Aldrich), 7 I.U. oxytocin (Sigma-Aldrich) and 5 μg/ml PGF2α (Sigma-Aldrich) produced maximum force of contraction (Emax), which values differ between the respective agonists. Meanwhile, 2 mg/ml FDA also resulted in maximum contraction (Emax), however with a lower Emax than other tested agonists. Atropine (Sigma-Aldrich) (2 × 10-8 M), a muscarinic receptor antagonist; atosiban (Fluka Co) (0.5 IU), an oxytocin receptor antagonist, THG113.31 (Theratechnologies) (10 μg/ml), a PGF2α receptor antagonist, oxodipine (Sigma-Aldrich) (0.25 μM), an L-type Ca2+-channel blocker, 2-APB (Sigma Aldrich) (40 μM), an IP3 receptor (IP3R) blocker, thapsigargin (Sigma Aldrich) (1 μM), a sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor and EDTA (Sigma Aldrich) (1 mM), a Ca2+ chelator were administered to investigate the mechanism underlying FDA effect on uterine contraction. In order to observe the effect of these inhibitors, 2 mg/ml FDA was initially added into the bathing solution and once contraction was stable at Emax, these inhibitors were either individually or simultaneously added and their effects on the Emax were then recorded.FDA was added in a dose-dependent manner (0.125-4.0 mg/ml) and the dose- response effect was then recorded. Preliminary investigation revealed that 1 × 10t- test. p < 0.05 was considered to be statistically significant.Results were expressed as mean ± SEM. Data was analyzed using Student’s In Figure In Figure -2 M Ach, 7 I.U oxytocin and 5 μg/ml PGF2α were 2.98 ± 0.25, 3.51 ± 0.47 and 3.43 ± 0.19 g respectively.In Table 2+ channel antagonist into the bathing solution containing isolated uterine tissue pre-exposed to 2 mg/ml FDA resulted in the Emax to decrease by 88.5%. Meanwhile, administration of EDTA into this solution which resulted in depletion of extracellular Ca2+ caused the Emax to decrease by a greater percentage (96.8%). Lesser degree of inhibition by oxodipine and EDTA in isolated uterine tissue pre-exposed to oxytocin indicated that this effect of oxytocin was not solely dependent on the extracellular Ca2+.In Figure .In Figure 2+. These mechanisms were confirmed from inhibition of the maximum tension (Emax) produced by 2 mg/ml FDA following administration of the antagonists to these receptors and inhibitors to the Ca2+ channels. FDA is 1.43 times less potent than oxytocin, which is a gold standard uterotonin [To the best of our knowledge, this study is the first to display uterotonic effect of Ficus deltoidea, which justifies the claim that this plant assists in uterine contraction. We have shown that FDA effect is mediated via muscarinic, oxytocin and PGF2α receptors and is dependent on the extracellular Caerotonin . Apart ferotonin ,22 and F2 and in the late pregnancy particularly at term [2 administration to the rats prior to the experiment can result in an increase in the number of these uterotonin receptors, potentiating the effect of FDA on uterine contraction.Our findings suggested that FDA-induced uterine contraction was mediated mainly via the oxytocin receptor as evidenced by the highest degree of inhibition of the Emax by atosiban. Moderate inhibition of the Emax by THG113.31 suggested that FDA binding to PGF2α receptor produced moderate degree of contraction while the lowest inhibition by atropine suggested that FDA binding to the muscarinic receptor produced the least degree of contraction. The cumulative inhibitory effect observed following concomitant administration of atropine, THG113.31 and atosiban confirmed the involvement of all three receptors in mediating FDA-induced uterine contraction. The presence of muscarinic, oxytocin and PGF2α receptors in the uterus has been previously reported -25. Thes at term . In view2 and at term [2[2+ dependent pathway, while activation of the muscarinic receptor which is coupled to G protein alpha(i) potentiates contraction through inhibiting the cAMP production [There is a possibility that the greatest effect produced following FDA binding to the oxytocin receptor (as evidenced by the greatest inhibition of the Emax by atosiban) was due to high number of this receptor expression in the uterus. Meanwhile, lesser inhibition by THG113.31 and atropine suggested that the number of PGF2α and muscarinic receptors expressed was lower than the number of oxytocin receptor expression. Up-regulation of oxytocin receptor by E at term ,27 has b at term , rat [25 at term  uterus w at term -33, rabb at term  and huma at term  uterus wt term [2. Previout term [2,36, suppt term [2. Oxytocit term [2,39. Actioduction . Besidesoduction .2+, which is essential for smooth muscle contraction [2+ enters the cell via the voltage-gated dihydropyridine channels at the myocyte plasma membrane [2+ enters down its concentration gradient. This will then trigger the release of Ca2+ from the intracellular stores [2+ in myometrial contraction was investigated following oxytocin and 2 mg/ml FDA administration. Our findings indicate that oxytocin-induced uterine contraction depends mostly on the extracellular Ca2+ while intracellular Ca2+ is also required for contraction. Following binding of oxytocin to its G protein-coupled receptor, phospholipase C (PLC) will be activated which causes an increase in inositol trisphosphate (IP3) and diacylglycerol (DAG) levels. IP3 activates the IP3R receptor at the sarcoplasmic reticulum membrane which causes the release of stored Ca2+ into the cytosol. Increased cytosolic Ca2+ will further induced extracellular Ca2+ influx [2+ level. Ca2+ will then binds to calmodulin, which activates the myosin light chain kinase leading to phosphorylation of myosin light chains, triggering contraction [Catraction , can be traction ,43. Extrmembrane . Followir stores . In this+ influx , resultitraction .2+. This could be similar to the contraction induced by wild ginger (Costus speciosus) rhizome [2+. In this study, FDA binding to the muscarinic, oxytocin and PGF2α receptors may trigger the extracellular Ca2+ influx prior to contraction. Although FDA has been shown to mediate its uterotonic effect, mostly via oxytocin receptor binding, the contraction produced however does not depend on the intracellular Ca2+ as evident from the lack of inhibition on the Emax by 2-APB. This is in contrast to oxytocin-induced uterine contraction, whereby its dependency on the intracellular Ca2+ was evidenced from the inhibition of Emax by 2-APB. We speculated that the inability of FDA to induce the release of Ca2+ from the internal stores could be due to its inability to provide adequate stimulus to trigger the intracellular cascade leading to the release of Ca2+ from the intracellular stores, despite of its binding to the oxytocin receptor. On the other hand, FDA may also bind at lower affinity to other uterotonin receptors, which may explain lesser potency of FDA as uterotonin as compared to oxytocin, PGF2α and Ach.A marked decrease in the Emax following oxodipine and EDTA administration suggested the dependency of FDA-induced uterine contraction on the extracellular Ca rhizome  and pome rhizome  extracts2+ influx could also involve other agonists’ receptor binding. This includes the PGF2α receptor; which was found to mediate uterine contraction in the laying hens via inducing the influx of extracellular Ca2+[2+ by thapsigargin which inhibit the reuptake of cytosolic Ca2+ into the sarcoplasmic reticulum. The consistently high cytosolic Ca2+ will activate extracellular Ca2+ entry which would further enhance uterine smooth muscle contraction [In addition to the binding to the oxytocin receptor, FDA-induced extracellular Caular Ca2+. Our fintraction .in-vitro model, our study has provided the first scientific evidence to support the claim that Ficus deltoidea stimulates uterine contraction. The active compound that is responsible in mediating this effect is currently unknown, although Ficus deltoidea has been reported to contain flavonoids isovitexin, vitexin [in-vitro study using isolated rodent’s uteri therefore provides preliminary evidence which could be used to further explore the in-vivo effect of this plant compound on uterine contraction.Using  vitexin , proantr vitexin . Phytoch vitexin . This inAuthors have nothing to disclose.NS is the project leader who planned this study, was involved in data interpretation and prepared the manuscript for publication. Meanwhile, VNA conducted all experiments. Both authors have read and approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/359/prepub


The resulting [Ca2+]i accumulation further enhances Ca2+-induced Ca2+ release, generating the positive inotropic effect. However, cardiac glycosides have some toxic and side effects such as arrhythmogenesis, confining their extensive clinical applications. The mechanisms underlying the proarrhythmic effect of glycosides are not fully understood. Here we investigated the mechanisms by which glycosides could cause cardiac arrhythmias via impairing mitochondrial energetics using an integrative computational cardiomyocyte model. In the simulations, the effect of glycosides was mimicked by blocking NKA activity. Results showed that inhibiting NKA not only impaired mitochondrial Ca2+ retention (thus suppressed reactive oxygen species (ROS) scavenging) but also enhanced oxidative phosphorylation (thus increased ROS production) during the transition of increasing workload, causing oxidative stress. Moreover, concurrent blocking of mitochondrial Na+/Ca2+ exchanger, but not enhancing of Ca2+ uniporter, alleviated the adverse effects of NKA inhibition. Intriguingly, NKA inhibition elicited Ca2+ transient and action potential alternans under more stressed conditions such as severe ATP depletion, augmenting its proarrhythmic effect. This computational study provides new insights into the mechanisms underlying cardiac glycoside-induced arrhythmogenesis. The findings suggest that targeting both ion handling and mitochondria could be a very promising strategy to develop new glycoside-based therapies in the treatment of heart failure.Cardiac glycosides have been used for the treatment of heart failure because of their capabilities of inhibiting Na The resultant greater Ca2+-induced Ca2+ release (CICR) allows for more powerful contractions by cross-bridge cycling in response to stimulations Glycosides are capable of inhibiting sarcolemmal Na2+ stores become too high, some Ca2+ might be released spontaneously through ryanodine receptors (RyRs), causing early or delayed afterdepolarizations or triggered activity 2+ overload is not the only root cause of glycoside-induced cardiac arrhythmias + accumulation caused mitochondrial Ca2+ deficiency, NADH imbalance, and increased reactive oxygen species (ROS) accumulation. Since many ion channels/exchangers underlying the action potential (AP) or involved in Ca2+ handling  are redox and/or ATP sensitive, ouabain-induced mitochondrial dysfunction can disturb Ca2+ cycling and elicit erratic action potentials. Indeed, Liu et al. have demonstrated that ouabain caused mitochondrial oxidative stress and DADs in guinea pig ventricular myocytes +/Ca2+ exchanger, a.k.a. mNCE, inhibitor) with ouabain retained mitochondrial Ca2+ and NADH levels, suppressed ROS production and prevented ouabain-induced DADs 2+ and NADH homeostasis in glycoside-induced oxidative stress and cardiac arrhythmogenesis.A classical hypothesis on the proarrhythmic effect of glycosides is that when SR Ca2+ is regulated not only by mNCE but also by mitochondrial Ca2+ uniporters (MCU), the primary pathway of mitochondrial Ca2+ uptake. While the molecular identity of MCU has been revealed recently 2+ uptake are still incompletely understood 2+ cycling and excitation-contraction (E-C) coupling remains controversial. The major restriction of mitochondrial Ca2+ uptake is attributed to the low affinity of MCU to Ca2+. Specifically, the Ca2+ concentration for half-maximal mitochondrial Ca2+ uptake via MCU was reported as ∼10–20 μM in isolated mitochondria 2+ concentration during E-C coupling (0.5–1.5 μM). Therefore, it has been argued that mitochondria may have a minor role in regulating cellular Ca2+ cycling and E-C coupling a.k.a mfn2) 2+ microdomain, facilitating dynamic interorganellar coupling  similar to experimental observations. In addition, the impaired mitochondrial function could be significantly alleviated by inhibiting mNCE but barely ameliorated by enhancing MCU. Finally both Ca2+ and AP alternans could be elicited when severe ATP depletion occurred as the result of high frequency pacing and concurrent NKA inhibition, implicating a novel mechanism underlying the arrhythmogenic effect of cardiac glycosides.In this study, we incorporated a detailed antioxidant system into a recently published computational model of the cardiomyocyte to quantitatively investigate the mechanisms underlying cardiac glycoside-induced mitochondrial energetic dysfunction and glycoside- mediated abnormal Ca+ ([Na+]i) on the interaction between mitochondrial energetics and cellular electrophysiology. The model was based on our recently published ECME-RIRR model 2+ handling, as well as mitochondrial energetics, ROS-induced ROS release (RIRR) and intramitochondrial Ca2+ regulation. To replicate mitochondrial Ca2+ and Na+ handling, a refined mitochondrial model +, H+ and Pi+ channels was incorporated. Moreover, a detailed antioxidant subsystem developed by Kembro et al. A multiscale guinea pig cardiomyocyte model was developed in order to systematically examine the effects of glycoside-induced high cytosolic Na2+ in the cardiomyocyte. Some studies suggest that mitochondria take up Ca2+ solely from the cytoplasm 2+ microdomain 2+ from both the cytosol and the microdomain. The kinetics formulas and parameters of Ca2+ uptake remained the same as those in the ECME-RIRR model 2+ uptake flux equals the flux in the ECME-RIRR model: uniV, uni,1V and uni,2V are the flux of the total Ca2+ uptake, the Ca2+ uptake from the cytosol, and the Ca2+ uptake from the microdomain, respectively. p1 and p2 represent the percentages of Ca2+ uptake flux from each pool, and their values are determined by the distribution of MCU proteins on the mitochondrial membrane.It is still debated how mitochondria take up Ca++ . The nonlinear ordinary differential equations were integrated numerically with CVODE as described previously The formulas of other processes, such as membrane potential, ion channels and metabolic reactions, and model parameters were the same as those in the ECME-RIRR model + concentration in 3 mins. This amount of Na+ increase is comparable to that caused by 1 μM ouabain in the experimental study 2+ uptake, energy production and ROS metabolism during the transition of increasing workload. Various p1:p2 ratios (ranging from 1∶0 to 0∶1) were evaluated to match the experimental data 2+ transients and AP under low  and relatively high (i.e. shunt = 2.5%) O2− production conditions. We also explored whether blocking mNCE or enhancing MCU alleviates NKA inhibition-induced mitochondrial energetics impairment. Finally we examined whether NKA inhibition and associated mitochondrial dysfunction are capable of triggering Ca2+ alternans in a paced cardiomyocyte. The simulation results were post-processed and plotted using Origin 8.6 .The cardiomyocyte was stimulated at 0.25 Hz until the steady state was reached. The steady state values were then used as initial conditions for all simulations. In the model, the effect of cardiac glycosides was mimicked by inhibiting NKA activity by 50%, which caused a 2.4-fold increase in cytosolic Na2+ uptake pools (i.e. cytosol vs. mitochondria-SR microdomain or p1:p2 ratio) on Ca2+ dynamics during the transition of increasing workload with or without NKA inhibition. As expected, increasing pacing frequency (from 0.25 Hz to 2 Hz) caused significant increase in both cytosolic and mitochondrial [Ca2+] (2+ completely from the cytoplasmic pool (i.e. p1 = 1 & p2 = 0), [Ca2+]i had the smallest elevation (e.g. systolic [Ca2+]i increased by 62.5%) in response to the increasing workload (2+ uptake from the cytosol to the microdomain (or reducing p1:p2 ratio) enhanced [Ca2+]i accumulation. When p1 = 0 & p2 = 1 (i.e. completely from the microdomain), the increase of [Ca2+]i was the greatest (2+ ([Ca2+]m) was similar to that of [Ca2+]i, except when [Ca2+]m was completely taken from the microdomain (i.e. p1 = 0 & p2 = 1), in which case [Ca2+]m increased first and then decreased slightly . NKA inhibition started to suppress the pacing-induced [Ca2+]m elevation with the pool shifting to the microdomain (i.e. reducing p1:p2 ratio). Specifically, when p1:p2 = 1∶3, NKA inhibition caused about 15% reduction in [Ca2+]m accumulation, which was comparable to the experimental data (18% reduction in systolic [Ca2+]m and 16% in diastolic [Ca2+]m)  monotonically enhanced the accumulation of [Caake pool . The effp2 ratio . As summ[Ca2+]m) . Therefoshunt = 1%). Increasing pacing frequency from 0.25 Hz to 2 Hz caused a 1.6-fold elevation in [Na+]i and a 1.5-fold increase in mitochondrial Na+ concentration ([Na+]m), respectively (m)  , [ATP]i d [ATP]m . Inhibitenuation . The ele study m  and ATP  study m . These sshunt = 2.5%). As shown in shunt per se did not significantly alter ion concentrations and mitochondrial energetics under control condition. However, simultaneously blocking NKA (by 50%) and increasing energy demand (from 0.25 Hz to 2 Hz) caused detriment on mitochondrial energetics under the higher ROS production condition. Specifically, NKA inhibition triggered sustained mitochondrial oscillations, including NADH  significantly ameliorated NKA inhibition-induced mitochondrial dysfunction, without abolishing the inotropic effect of NKA inhibition  (data not shown). At the higher ROS production rate condition (e.g. shunt = 2.5%), blocking mNCE by 60% significantly delayed NKA inhibition-induced mitochondrial oscillations  significantly increased [Ca2+]m .Another way to compensate NKA inhibition-induced mitochondrial Ca [Ca2+]m . Howevernificant . Enhancinificant , ROS stinificant , and ΔΨm dropped . Further2+]i elevation  slightly enhanced NKA inhibition-induced m uptake (and resultant reduced NADH production) was not the sole reason causing oxidative stress during NKA inhibition, because ROS accumulation could occur even when NADH did not decline significantly  NKA inhibition blunting [Ca2+]m accumulation, which reduces NADH production and therefore ROS removal, and (ii) NKA inhibition increasing [Ca2+]i and ATP hydrolysis, which produces a large amount of ADP that stimulates mitochondrial respiration and therefore ROS production.In cardiomyocytes, mitochondria are the major sites of ROS production, and NADH plays a vital role in regulating both the production and scavenging of ROS. Previous studies showed that inhibiting mitochondrial Caficantly . It is k2+ (up to 40-fold) and enhanced ATP consumption in cultured chick ventricular cells via increasing the synthesis of ubiquinone While the role of NKA inhibition in regulating mitochondrial ROS is well appreciated, the effect of ouabain on mitochondrial ATP synthesis remains elusive. A recent study showed that ouabain suppressed glucose-induced mitochondrial ATP production in rat pancreatic islets 2+ overload and associated abnormal RyR Ca2+ release et al. suggested that mitochondrial dysfunction, particularly ouabain-induced ROS production, might also contribute to the arrhythmogenic effect of glycosides 2+ in the cytosol, mitochondria and SR, as well as the profiles of metabolic intermediates such as ATP and NADH. Our results showed that NKA inhibition caused a significant ROS accumulation during the transition of moderate workload increment. The mitochondria-derived ROS may lead to abnormal Ca2+ handling and erratic APs, as demonstrated in 2+ and AP alternans by reducing ATP. It is worthy to point out that although in our simulations Ca2+ alternans could be induced only at higher pacing rate (i.e. 4 Hz), the mechanism underlying the genesis of this type of alternans is different from that of classical alternans induced by increasing pacing rate. Actually, in our hands pacing at 4 Hz per se did not induce Ca2+ alternans. Further analysis showed that the genesis of Ca2+ alternans was attributed to NKA inhibition-induced mitochondrial dysfunction. Particularly, severe ATP depletion impaired SERCA activity, reduced the SR Ca2+ content, and thereby generated SR release and uptake mismatch, causing intracellular Ca2+ alternans. A similar mitochondrial dysfunction-induced Ca2+ alternans has been reported by several experimental studies 2+ alternans was further supported by the mNCE inhibition simulation. As shown in 2+ and suppressing the genesis of alternans. Thus, our in silico study revealed a new plausible mechanism responsible for glycoside-induced cardiac arrhythmias, which is different from the traditional paradigm of SR Ca2+ overload and associated abnormal RyR Ca2+ release. Our model also suggested that the increase of workload could potentiate the mismatch between energy supply and demand and increase the risk of NKA inhibition-mediated arrhythmias. Thus, digitalis toxic effects could be more problematic at increased heart rate.In previous animal and clinical studies, the arrhythmogenic effects of glycosides have been mainly attributed to NKA inhibition-induced Ca2+]m accumulation that preserves mitochondrial NADH homeostasis and reduces ROS production. Our simulations are quantitatively comparable to these experimental results. Moreover, we showed that the mNCE inhibition attenuated cytosolic Ca2+ accumulation, which contributed to the anti-arrhythmic effect of CGP-17157. In another word, the therapeutic effect of mNCE inhibitors was attributed to not only preserving [Ca2+]m retention and mitochondrial function, but also attenuating glycoside-induced intracellular Ca2+ overload. One should be aware though that it is the [Ca2+]i elevation that is primarily responsible for the  inotropic effect of glycosides treatment. Therefore, the dosage of the mNCE inhibitor is likely to be critical to counteracting the adverse effects of glycosides. Actually, we showed that too much mNCE inhibition, such as by 90%, completely abolished NKA inhibition-induced Ca2+ accumulation, although it significantly improved mitochondrial function and suppressed oxidative stress.Studies have shown that CGP-17157, a mNCE inhibitor, mitigated ouabain-induced adverse effects on mitochondrial function and lowered the occurrence of cardiac arrhythmias in guinea pigs 2+ uniporters (MCU) activity was not evident, which might be caused by the substrate compartmentation of MCU. Our model analysis showed that MCU need to take up most of Ca2+ (>70%) from the microdomain in order to reproduce NKA inhibition-induced [Ca2+]m attenuation observed in the experimental studies 2+ taken up by MCU is from the microdomain instead of cytoplasm. Thus, enhancing MCU activity increased [Ca2+]m uptake and retention, but it did not substantially reduce NKA inhibition-induced [Ca2+]i overload and ROS production. These in silico results also imply that MCU are not uniformly distributed across the mitochondrial membrane, but rather are concentrated at mitochondria-SR contacting sites. The non-homogenous localized expression of ion channels on the sarcoplasmic or intracellular membranes has been reported previously. For instance, studies showed that L-type Ca2+ channels are concentrated in the neighborhood of the junctional SR at T-tubules 2+ uptake in response to increased energy demand. With the molecular identity of MCU emerging While inhibiting mNCE efficiently relieved NKA inhibition-induced adverse influence on mitochondrial energetics, the effect of increasing mitochondrial Ca2+ compartmentation for MCU, therefore providing advantages over existing cardiomyocyte models 2+ wave propagation plays a critical role in intracellular Ca2+ regulation and genesis of Ca2+ alternans 2+ microdomain” notion proposed in this study (and by others elsewhere) may be involved in regulating Ca2+ wave propagation. Incorporating spatial resolution of local Ca2+ distribution in the microdomain would allow examination of its role in regulating cellular electrophysiology and mitochondrial energetics. (iii) The current model did not incorporate the direct link between mitochondrial ROS and redox sensitive SR Ca2+ proteins. It has been shown that oxidative stress can modulate SR Ca2+ handling proteins and cause abnormal electrical activities in cardiomyocytes ATP), as our main focus is on mitochondrial dysfunction and Ca2+ handling. It is expected that incorporating KATP would exacerbate the deleterious effect of NKA inhibition-induced mitochondrial dysfunction on cardiac action potentials. As shown before, depletion of ATP activates KATP currents, resulting in AP shortening or even cellular inexcitability The model developed in this study incorporates subcellular Ca2+ alternans, contributing to its proarrhythmic effect. Finally, the study verifies the role of mitochondria-SR Ca2+ microdomain in regulating mitochondrial Ca2+ uptake and energetics. Taken together, this work underscores the importance of targeting both intracellular ion handling and mitochondrial energetics in developing new glycoside-based heart failure therapies.In summary, the present study provides a novel computational tool to quantitatively investigate the proarrhythmic effects of glycosides (and resultant NKA inhibition) in the cardiomyocyte. The simulations reveal that NKA inhibition can cause mitochondrial oxidative stress through two separate and concomitant pathways, which involve an intricate interplay between cardiac electrophysiology and mitochondrial energetics. Moreover, the model suggests that under more severe conditions, NKA inhibition can induce CaFigure S12− production conditions with or without concurrent mNCE inhibition.Effects of blocking NKA on cytosolic and mitochondrial ATP under low O The cell was paced at 0.25 Hz for 1 min then at 2 Hz for 3 mins. Black: control; Red: 50% NKA inhibition; Dark green: 50% NKA inhibition+60% mNCE inhibition. (A): [ATP]i; (B): [ATP]m. shunt = 1% and p1:p2 = 1∶3.(TIF)Click here for additional data file.Figure S22+ and mitochondrial energetics changes.Effect of blocking MCU or enhancing mNCE on NKA inhibition-induced Ca Black: control; Red: 50% NKA inhibition; Dark green: 50% NKA inhibition+60% MCU inhibition; Blue: 50% NKA inhibition+100% mNCE enhancement. (A): [Ca2+]i; (B): [Ca2+]m; (C): NADH; (D): ROS. shunt = 1% and p1:p2 = 1∶3.(TIF)Click here for additional data file.File S1Supplemental Information model equations.(DOCX)Click here for additional data file.File S2Supplemental Information model parameters.(DOCX)Click here for additional data file.


A hereditary postprandial hypertriglyceridemic rabbit (PHT rabbit) is a new dyslipidemic model showing remarkably high plasma triglycerides with only limited elevation of plasma total cholesterol. In PHT rabbits, plasma triglyceride was markedly elevated postprandially compared with healthy Japanese white (JW) rabbits. In physiological experiments, the ring preparation of the thoracic aorta was suspended in an organ bath filled with modified Krebs-Henseleit solution, and the developed tension was recorded. Endothelial function was evaluated by acetylcholine-induced vasorelaxation in each preparation with intact endothelium. The acetylcholine-induced endothelium-dependent relaxation was diminished in PHT compared with JW rabbits, suggesting endothelial dysfunction in PHT rabbits. Histological examination was carried out in adipose tissue, liver and aorta. They were fixed in formaldehyde and embedded in paraffin. The tissues were sliced (4 μm) and stained using hematoxylin-eosin solution. In the adipose tissue, the visceral fat accumulated, and the size of adipose cells was enlarged in PHT rabbits. The liver of the PHT rabbit was fatty and degenerated. In aorta, increased intimal thickness was observed, suggesting the progression of atherosclerosis in the PHT rabbit. This study suggests the important role of postprandial hypertriglyceridemia in atherosclerosis. By using PHT rabbits, the effects of hypertriglyceridemia on health and diseases could be evaluated precisely. Dyslipidemia, especially hypercholesterolemia, is a risk factor for a variety of diseases such as hypertension, ischemic heart disease and cerebrovascular events. In those diseases, vascular atherosclerotic changes exist as a common pathophysiological mechanism. Atherosclerosis is considered to be related strongly to dyslipidemia such as hypercholesterolemia, where high plasma concentration of oxidized low density lipoprotein (LDL) may play a pivotal role in promoting vascular intimal thickening.In addition to hypercholesterolemia, hypertriglyceridemia may be another factor for diseases. The strong relationship between serum triglyceride level and the mortality of ischemic coronary heart diseases was originally reported in the epidemiologic research carried out in 1985 as the Stockholm Prospective Study ,3,4, andBecause hypertriglyceridemia is reported to decrease the serum level of high-density lipoprotein (HDL) while it increases the remnant lipoproteins and small dense LDL, these effects would induce thrombogenesis, intimal proliferation and promote atherosclerosis . One of in vivo.In spite of the implication from many clinical and physiological findings, the direct effect of triglyceride on the onset of atherosclerosis is not yet clarified from the pathological studies because triglyceride itself could not be detected in the atherosclerotic region, in contrast to the oxidized LDL. In addition, the problem that there were no adequate animal models of hypertriglyceridemia without hypercholesterolemia made it difficult to explore the precise role of triglyceride For the purpose of investigating the effect of triglyceride, a new animal model named PHT rabbit has been segregated in Yamagata University Animal Center to examine the link between triglyceride and cardiovascular diseases ,15. The In this study, we investigated the role of triglyceride in the fat accumulation within the adipose tissue, in the fatty liver degeneration, and in atherosclerosis by using the postprandial hypertriglyceridemic PHT rabbits with different ages. The progression of atherosclerosis was evaluated by a histopathological method in combination with physiological examination of the age-related vascular endothelial dysfunction.n = 4, six-month-old PHT; n = 4, 40-month-old PHT; n = 4) were used to examine the age-related changes in the endothelium-dependent relaxation of PHT rabbit .Experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals by the US National Institute of Health  and undad libitum by automatic watering system.All the animals used for the experiments were bred and maintained under the conventional housing condition, and were clinically healthy. All animals were housed individually in the animal room controlled at 22 ± 2 °C, humidity at 55% ± 15% and a light: dark-cycle of 12 h:12 h (light on at 6:00). Each animal was fed standard rabbit chow  at 12:00 daily. The nutritional composition and energy value of the diets were as follows: crude protein, 17.1%; crude fat, 5.4%; crude fiber, 17.1%; crude ash, 9.6%; crude nitrogen-free extract, 43.5%; water 7.4%, and energy, 2087 kcal/kg. Water was supplied Body weights and abdominal circumference were measured under the anesthesia of pentobarbitone sodium (30 mg/kg). Then, rabbits were euthanatized with an intravenous overdose of pentobarbitone sodium (300 mg/kg) and exsanguination, and liver, thoracic aorta and visceral adipose tissues were isolated and weighed.Acetylcholine chloride , pentobarbitone sodium and phenylephrine  were used.The rabbits were fed 120 g of standard diet daily at noon. For analysis of fasting triglyceride, the diet was withdrawn at 18 evening until next noon (fasting period: 18 h). Then rabbits were fed a standard diet again and started eating. The plasma triglyceride elevated gradually and reached maximum level after 15–18 h. Blood from each rabbit was collected via marginal ear artery after 18 h of fasting and at 18 h after the start of feeding. Then, blood was centrifuged at 3000 rpm  and supernatants were collected. Plasma concentration of triglyceride and cholesterol were measured by enzymatic methods using Triglyceride E- and Cholesterol E-test .Each tissue was fixed overnight in 10% formaldehyde at 4 °C. Tissues were embedded in paraffin and cut into 4 μm cross-sections. Microscopic examination was performed with hematoxylin-eosin stained sections to assess fatty changes in liver, adipose tissues and atherosclerotic changes in aorta.2 and 5% CO2 . The solution contained 118 mM NaCl, 4.7 mM KCl, 24.9 mM NaHCO3, 1.18 mM MgSO4, 1.18 mM KH2PO4, 11.1 mM glucose, 1.8 mM CaCl2, and 0.057 mM ascorbic acid. The vascular developed tension was recorded by the isometric force transducer  for measurement of changes in the contractile force. The preparations were stretched to a resting tension of 2.0 g, and the solution was changed every 15 min. After an equilibration period of 1 h, each preparation was contracted with 66.7 mM KCl repeatedly until reproducible contraction was obtained. Then, preparations were precontracted with phenylephrine (1 μM) to reach maximal tension, and acetylcholine was added cumulatively to measure the relaxation response  with pairwise comparison by Tukey-Kramer test or by unpaired The body weights of the PHT rabbits were 3.0 ± 0.1 kg (12-month-old) and 3.1 ± 0.1 kg (33-months-old), respectively. The weights of the visceral fats (per body weight) were 84.79 ± 3.59 (in 12-month-old PHT) and 63.75 ± 10.99 g/body weight (kg) (in 33-month-old PHT), respectively. The visceral fats were increased in PHT rabbits of both ages compared with 12-month-old Japanese white (JW) rabbits . In addiThe fasting  triglyceride concentration in the normal Japanese White rabbit (JW) was <50 mg/dL (0.5 mmol/L) . In JW rTo clarify the morphologic changes in visceral fat of PHT rabbits, the histopathological examination was carried out . The adiIn the mesenteric adipose tissue, the size of fat cell was measured. As shown in In the normal JW rabbit, liver did not show any pathological changes . In contJW rabbits  did not show atherosclerotic lesions , while hThe atherosclerotic changes shown in In this study, the role of postprandial hypertriglyceridemia was investigated by using a newly segregated line of PHT rabbits. Although the preprandial triglyceride levels were low, plasma triglyceride concentrations reached to several times greater concentration of 800–1000 mg/dL after feeding, and the levels were not different between 12-month-old and 33-month-old PHT rabbits. In addition, the hypertriglyceridemia continued for a long period, at least up to 33-month-old. Previous studies showed that the triglyceride levels of normal 10- to 17-month-old Japanese white rabbits were 35.4 mg/dL in fasting and 62.8 mg/dL in postprandial conditions . Previouet al. [The weights of visceral fats in PHT rabbits were increased in 12-month-old, compared with 6-month-old PHT rabbits as well as that of JW rabbits, and the accumulation of fat was not different between 12-month-old and 33-month-old rabbits . In accoet al.  reportedIn PHT liver, there was a remarkable fatty degeneration in the hepatocyte around the central veins. It is known that fatty liver is caused by obesity, especially by visceral fat accumulation . RecentlIn this study, the progression of atherosclerosis was evaluated by physiological as well as histopathological experiments. The intimal thickening of the thoracic aorta was detected most frequently in 33-month-old PHT rabbits, and the region extended widely. These observations indicate that the progression of atherosclerosis in PHT rabbits is age-dependent.et al. [Whether triglyceride can trigger the atherosclerosis is controversial. Triglyceride is water-insoluble and delivered in blood vessels as lipoprotein-bound form. Hypertriglyceridemia is usually accompanied by high levels of chylomicron and VLDL. Chylomicron is metabolized and the chylomicron remnant is suggested to be able to promote atherosclerosis . Becauseet al.  reportedIn human and WHHL rabbit, apoproteins B48 and B100-containing lipoptoreins were detected in the aortic intimal lesions and implicated to promote atherosclerosis ,25. Theset al. reported the coronary angiography data suggesting that small chylomicron remnants were implicated in the progression of coronary artery diseases in patients with type 2 diabetes mellitus [Mero mellitus . In accomellitus .et al., showed that blood pressure measured by a telemetry system was significantly higher in PHT than JW rabbits [Endothelial dysfunction was evaluated by a physiological method using isolated thoracic aorta. The endothelium-dependent relaxation by acetylcholine occurred concentration-dependently in aortic ring preparations. The relaxation response was diminished in 12-, 33- and 40-month-old PHT rabbits compared with that of 6-month-old PHT rabbits, suggesting the endothelial dysfunction in older PHT rabbits, probably due to the decreased production of nitric oxide which plays an important role in vascular relaxation, inhibition of platelet adhesion and aggregation. These results are consistent with the histopathological changes observed in PHT aorta. Together with these results, it could be implicated that endothelial cells might be injured and atherosclerosis could be accelerated in hypertriglyceridemia. In addition, the atherosclerotic vessels would induce hypertension. In fact, Fukuda  rabbits ,28.This study showed that the marked elevation of postprandial plasma triglyceride, visceral fat accumulation, fatty degeneration of liver, early atherosclerotic intimal thickening and rapid onset of vascular dysfunction in endothelial cells in a hypertriglyceridemic model of PHT rabbits. These results suggest that postprandial hypertriglyceridemia may be an important risk factor for promoting atherosclerosis.


Total hip replacement (THR) is considered to be the most effective treatment option for advanced osteoarthritis of the hip in large breed dogs. However, a proportion of post-THR patients suffer prosthesis dislocation for various reasons, which may be addressed by a constrained acetabular prosthesis design. The study proposed a new THR with constrained acetabular component that aimed to decrease the incidence of postoperative dislocation while maintaining the necessary range of motion (ROM); and, through computer-simulated implantations, evaluated the ROM of the THR with and without malpositioning of the acetabular component.A new THR with a constrained acetabular component that had an inward eccentric lining and a 60° cut-out on the dorsal side was designed, and its computer-aided design models were implanted into the pelvic and femoral models reconstructed from the computed tomography data of six healthy Labrador Retriever dogs. The allowable and functional ROM of the implanted THR were determined via computer simulations. The contact patterns between the bone or the prosthetic components at extreme positions of the THR were analyzed. Influence of malpositioning of the acetabular component on the ROM was assessed.The means (SD) of the functional ranges for flexion, extension, adduction, abduction, internal rotation and external rotation were 51.8° (6.6°), 163.3° (7.3°), 33.5° (5.7°), 74.0° (3.7°), 41.5° (8.3°) and 65.2° (9.9°), respectively. Malpositioning of the acetabular component by 20° in one direction was found to reduce ROM in other directions .From the computer-aided surgical simulations, the new THR was found to have sufficient functional ranges for flexion, extension, abduction, adduction and external rotation for Labrador Retrievers. Analysis of the malpositioning of the acetabular component suggests that accurate placement of the acetabular component is critical for achieving desirable ROM for daily activities. Total hip replacement (THR) is considered to be the most effective treatment option for advanced osteoarthritis of the hip in large breed dogs, primarily subsequent to dysplasia of the joint and other disabling conditions. Previous studies have shown that it is effective in pain relief and restoration of the joint function -4. HowevAmong the postoperative complications, prosthesis dislocation has a severe impact on the function of the joint and can lead to the final failure of the primary THR, requiring revision surgery. With the existing unconstrained acetabular prosthesis design, it has been reported that as high as 4.7% to 11% of canine post-THR patients suffered prosthesis dislocation ,9,14. SeThe problem of postoperative dislocation has been investigated in human THR, which may lend useful information to the resolution of the same problem in the design of canine THR. An efficient approach to reducing the incidence of dislocation is a constrained acetabular prosthesis design, the lining of which covers more than half of the surface area of the femoral head . The conThe purposes of this study were to propose a new THR with constrained acetabular cup design for dogs which aims to decrease the incidence of postoperative dislocation while maintaining the necessary ROM by adopting an inward eccentric lining and a partial cut-out on the dorsal side of the acetabular component; and, through computer-simulated implantations, to evaluate the ROM of the THR in a group of Labrador Retriever dogs with and without malpositioning of the acetabular component.A new THR with a constrained acetabular cup design with an eccentric inner surface was developed Figure . The aceA prototype of the current THR design was manufactured with the acetabular cup made of high-density polyethylene as a unitary component and the femoral component made of titanium alloy. The prototype was tested for its mechanical ROM, defined as the maximum rotation of the femoral component within the acetabular component with respect to the coordinate system embedded to the acetabular component called the acetabular prosthesis coordinate system (APCS), without being implanted in a dog. The Z-axis of the APCS was normal to the ventral opening surface, the X-axis was parallel to a line connecting the mid-points of the junctions between the ventral and dorsal opening surfaces, and the Y-axis was obtained as the cross-product of the Z-axis and X-axis Figure . The maxSix healthy client-owned Labrador Retriever dogs  without any neuromusculoskeletal diseases at the hip joints participated in the current study, with written informed consents from the owners as approved by the Institutional Animal Care and Use Committee. The subjects were recruited from different families (different owners) and were not genetically related within 5 generations. Body height, defined as the distance between the level ground and the top of the shoulder while the animal was standing erect in the neutral position, was measured using a tape measure. Similarly, body length was defined and measured as the distance between the front of the chest and the base of the tail. The pelvis and the femur of the dogs were scanned by computed tomography  from the cranial border of the ilium to the mid line of the knee joint with a CT slice thickness of 0.7 mm, a pixel size of 0.725 mm × 0.725 mm and an averaged dose length product (DLP) of 154 mGycm. During the CT data collection, the dogs were maintained anesthetically using intravenous propofol and were positioned in dorsal recumbency with the hind limbs caudally extended. From the CT data the bony parts, namely the pelvis and right femur, were semi-automatically segmented from all image slices to obtain subject-specific surface models of the bones using a commercially-available software package .With the subject-specific models of the bones, computer-simulated implantations of the new total hip were performed with the computer-aided design (CAD) models of the prosthesis components, namely the acetabular component, femoral head and the femoral stem, via a commercial reverse engineering software package . With the assistance of the graphic user interface of the software, a senior surgeon (WCH) incorporated the acetabular component of the THR into the right pelvis, replacing the natural acetabulum to simulate the standard surgical procedure  Figure a. The acFor the description of the motion of the hip, each model pelvis and femur was embedded with an anatomical coordinate system (ACS) Figure . For theThree types of ROM were determined for the new THR: mechanical, allowable and functional ROM. The mechanical ROM of the THR indicated the maximum angles of the three rotations of the femoral component within the acetabular component outside the body, and was determined with respect to the APCS Figure . The othIn order to assess the effects of the malpositioning of the acetabular component on the functional ROM of the THR, the acetabular component was systematically deviated from its neutral orientation simulating common positional errors during surgery, namely rotational deviations in the lateral opening and retroversion. The simulated malpositions ranged from -20° to 20° at increments of 5°, which were based on the possible range of mal-alignment during surgery reported in the literature . For eacThe mechanical ROM of the THR obtained from the computer models were 117.3°, 81.8° and 360° for rotations about the X-axis, Y-axis and Z-axis, respectively. The results agreed well with those measured from the prototype with only small differences in the X-axis and Y-axis rotations, which were less than 1.9° and 1.8°, respectively. The allowable and functional ranges of flexion, adduction and internal rotation were identical, while the allowable ranges of extension, abduction and external rotation were slightly greater than the corresponding functional ROM Table .The collision points on the acetabular component by either the femoral bone or the prosthetic component were found to be predominately on the anterior side (around the 3 o’clock position) for flexion and internal rotation, and around the 5 and 7 o’clock positions on the ventral side for adduction and external rotation, respectively Figure . Those fWith decreasing lateral opening angles of the acetabular component, the ROM in the transverse and the horizontal planes was decreased, but slightly increased in the sagittal plane Figure . In contThe current study described a newly-developed THR that had a constrained acetabular component with an inward eccentric lining design and a 60° cutaway region on the dorsal side. The design was aimed to increase the coverage of the femoral head while retaining the necessary ROM for daily activities. With computer-aided surgical simulations, the new THR was found to have sufficient functional ranges for flexion, extension, abduction, adduction and external rotation for Labrador Retrievers and similar breeds. The associated bone/prosthesis contact patterns suggest that an additional cutaway region on the anterior side of the component may be helpful for further increasing the ranges for flexion and internal rotation. Analysis of the malpositioning of the acetabular component suggests that accurate placement of the acetabular component is critical for achieving desirable ROM for daily activates.Computer simulations of THR implantation were used in the current study to assess the ROM of the new THR, which had several advantages over traditional methods such as measuring the ROM directly on living subjects using either goniometers -28 or phThe allowable and functional ROM measured in the current study provided useful data for the baseline (or maximum) ROM the new THR could achieve, although physiological ROM was not determined in the current study because of the difficulty in simulating soft tissues including the ligaments, joint capsules and muscles. In general, the functional ranges of the flexion, extension, adduction and external rotation of the current THR were comparable to those of the native hip reported in the literature Table . In the For the ROM in the horizontal plane, the range of external rotation in the current study appeared to be well-sufficient for dogs Table , but comThe positions of the contact between the acetabular and the femoral components occurred primarily on the opening surface of the acetabular component for all kinematic components except abduction Figure . WithoutMalpositioning of the acetabular component affected the ROM of the replaced hip. Deviations in the lateral opening or retroversion from the neutral position will affect the ROM of all motion components: some increased but others decreased. This was not unexpected because the ventral rim of the native acetabulum and the opening surface of the acetabular component were essentially 3D in shape and in space. Increased ROM in one anatomical plane will naturally reduce the ROM in one or more of the other components. The current analysis showed that any inaccuracy of up to 15 degrees of retroversion will reduce the range of abduction by about 10 degrees. Since an increase in the retroversion of the acetabular component will reduce the overall ROM in all anatomical planes, malpositioning with an excessive increase in retroversion should be avoided. In contrast, a decrease of 5° in retroversion  may be beneficial for increasing the ranges of internal rotation and flexion with a slight compromise of ranges of extension and external rotation. This is helpful for improving the current THR design that had slightly smaller internal rotation but greater ranges of extension and external rotation. From the current results of the malpositioning analysis of the THR, it is suggested that the acetabular component be implanted according to the native orientation with a slight decrease in the retroversion to provide ranges of motions required by Labrador Retrievers. For this purpose, a well-designed surgical guiding device is critical for accurate component placement for optimizing the performance of the THR.In the assessment of the functional ROM of the proposed constrained acetabular component, the current study was limited to healthy Labrador Retrievers. The acetabular component was placed with reference to the native orientation of the acetabular geometry of the individual dog. However, in practice the THR are implanted to treat degenerated or diseased hip joints with deformed anatomy and osteophytes, which may affect the accurate positioning of the cup following the current surgical guideline, and thus the final functional ROM. Therefore, further study on dogs with hip dysplasia or degeneration will be needed to test whether the functional ROM would be different from the current results. Further study is also needed to investigate the stress and wear behavior of the proposed constrained cup THR design as limited ROM has been associated with risks of increased stress between interfaces, accelerated wear and eventual component failure in human constrained THR .A total hip replacement having a constrained acetabular component with an inward eccentric lining design and a 60° cutaway region on the dorsal side was developed in the current study. With computer-aided surgical simulations, the new THR was found to have sufficient functional ranges for flexion, extension, abduction, adduction and external rotation for Labrador Retrievers. Further increases in ranges of flexion and internal rotation may be achieved by an additional cutaway region on the anterior side of the component, which may be helpful for its application in other breeds. Analysis of the malpositioning of the acetabular component suggests that accurate placement of the acetabular component is critical for achieving desirable ROM for daily activities. Further study should be conducted on the design of a surgical guiding device for accurate component placement for optimizing the performance of the THR.THR: Total hip replacement; ROM: Range of motion; CT: Computed tomography; CAD: Computer-aided design; ACS: Anatomical coordinate system; APCS: Acetabular prosthesis coordinate system; PPP: Posterior pelvic plane; SD: Standard deviation.The authors declare that they have no competing interests.C-HW carried out the studies of design and evaluation of the canine THR, participated in the CT data collection and data analysis, and drafted the manuscript. C-CL developed the software programs, performed data analysis and helped draft the manuscript. T-WL participated in the design of the THR, the methodological development, the interpretation of the results and the drafting the manuscript. S-MH participated in the design of the THR, CT imaging, and the revision of the manuscript. C-CH participated in data analysis, engineering drawing and manuscript drafting. L-SY participated in the design of the THR, methodological development and drafting of the manuscript. All authors read and approved the final manuscript.


DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The “10-23 DNA enzyme” is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs.In this study, we inserted a nonpairing bulge at the 5′ end of the “10–23 DNA enzyme” to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 5′ end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme.Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12–15 bp away from the core catalytic domain. Zamecnik and Stephenson (1978) inhibited the replication of Rous sarcoma virus by the use of a short antisense DNA oligonucleotide (ASON) that was reverse complementary to a particular transcript of the virus in vivo antisense applications due to their stability and nuclease resistance At least four different approaches have been used for gene silencing to date, these include: gene knockout by homologous recombination in vitro selective amplification. The selected molecule was designated the “10–23 DNA enzyme,” and was composed of a catalytic domain of 15 deoxynucleotides flanked by two substrate-recognition domains of ∼8 nucleotides on each side Breaker and Joyce (1994) made the assumption that because RNA and DNA are similar chemical compounds, DNA molecules with enzymatic activity could also be developed as ribozymes. This proposition led to the development of a DNA enzyme identified from a library of >1,000 different DNA molecules by successive rounds of In this study, we designed different sizes of bulges at different positions at the 5′ end of the regular DNAzyme. A non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme. Furthermore, the bulge structure would activate cellular RNases to hydrolyze the mRNA, resulting in increased mRNA transcript degradation. We introduced different sized oligo loops between the DNAzyme core sequence and the ASON sequence .To assess the efficiency of the ASON–Bulge–DNAzyme complex, the ASON–Bulge–DNAzymes were chemically synthesized and co-transfected with pEGFP-C1 plasmid into BHK21 cells. As shown in To examine whether the efficiency of the ASON–Bulge–DNAzyme was site limited, we synthesized another ASON–Bulge–DNAzyme targeting a different site, the eGFP mRNA. As shown in To assess the inhibition effect of different bulge constructs, we tested eGFP expression levels by a GFP fluorescence assay. DNAzymes with three, six, nine and 12 oligo bulges were designed and co-transfected with pEGFP-C1 plasmid into BHK21 cells. The results showed that the six oligo bulge was the most effective . FurtherTo quantitatively assess the inhibition effect of these constructs, we tested eGFP expression levels at different time points by a GFP fluorescence assay. The results further confirmed that the ASON–Bulge structure could not only enhance the efficiency of the DNAzyme, but could also sustain the inhibitory effect for longer compared with the regular DNAzyme .To test whether the ASON–Bulge–DNAzyme decreased eGFP expression by virtue of the mRNA degradation pathway, a semi-quantitative RT-PCR assay was employed to test the mRNA level of eGFP. The results confirmed that the ASON–Bulge structure could enhance the efficiency of the DNAzyme by decreasing the eGFP mRNA level. Western blot analysis further confirmed the efficiency of the ASON–Bulge–DNAzyme .To assess whether the ASON–Bulge–DNAzyme was gene specific, we synthesized an ASON–Bulge–DNAzyme against firefly luciferase. The results showed that this structure was more effective than the regular DNAzyme .5′-GATTCTCCAGAAGGTGGTTTCGATGCCATCATGCAAGTTGCAGTTTGTGGATCACTGATT-3′, β3: 5′-GATGCCCCAGAGGGTGGCTTTGATGCCATCATGCAGGCTACAGTCTGTGATGAAAAGATT-3′). The chemically synthesized DNAzyme was used to transfect HepG2 cells. As shown in To test whether the ASON–Bulge–DNAzyme was effective against endogenous genes, we selected integrins as target molecules. Integrins are a large family of heterodimeric transmembrane glycoproteins that attach cells to extracellular matrix proteins of the basement membrane or to ligands on other cells. There are many types of integrins on the cell surface relating to different functions. We designed an ASON–Bulge–DNAzyme against the endogenous integrin β1  at the 5′ end of DNAzymes to enhance the specificity of targeting to the mRNA substrate. To avoid too high a binding strength between the DNAzyme and the target mRNA, we introduced a nonpairing bulge structure into the DNAzyme. The formation of a bulge structure ensured that the target mRNA was easily hydrolyzed. Our results demonstrated that the incorporation of an ASON and a mispairing bulge in the DNAzyme conferred higher efficiency and specificity in gene silencing.A 15 base ASON was inserted at the 5′ end of the “10-23” deoxyribozyme. To avoid too high a binding strength between the DNAzyme and the target mRNA which might interfere with DNAzyme efficiency, we introduced a nonpairing loop at the 5′ end of the DNAzyme. The oligonucleotides used for this were as follows:Regular DNAzyme-1 against eGFP:5′-CTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′ASON-Bulge-DNAzyme-1 against eGFP:5′-TGCTCAGGGCGGACTGAAGTACCTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′Bulges of differing sizes were incorporated into the complex using the following oligonucleotides:The 3 nt Bulge–DNAzyme:5′-TGCTCAGGGCGGACTGTACCTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′The 6 nt Bulge–DNAzyme:5′-TGCTCAGGGCGGACTGAAGTACCTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′The 9 nt Bulge–DNAzyme:5′-TGCTCAGGGCGGACTGAAGCGATACCTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′The 12 nt Bulge–DNAzyme:5′-TGCTCAGGGCGGACTGAAGTGACGTTAACCTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′The position of the bulge in the complex was altered using the following oligonucleotides:1. Bulge–6 nt–DNAzyme:5′-TGCTCAGGGCGGACTGAAGTACCTCAAGGGCTAGCTACAACGAGGTTGTCGG-3′2. Bulge–9 nt–DNAzyme:5′-TGCTCAGGGCGGACTGAAGTACCTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′3. Bulge–12 nt–DNAzyme:5′-CTTGCTCAGGGCGGAAGTACGGTGCTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′4. Bulge–15 nt–DNAzyme:5′-TCTTTGCTCAGGGATCTACCTGGGTGCTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-3′DNAzymes against Luciferase were constructed using the following oligonucleotides:Regular DNAzyme against Luciferase:5′-AGCATCGAGAGGCTAGCTACAACGACCGTTGT-3′ASON–Bulge–DNAzyme against Luciferase:5′-GGGCAATTTACCACGAGCAATAGCATCGAGAGGCTAGCTACAACGACCGTTGT-3′.DNAzymes against integrin β1 were constructed using the following oligonucleotides:Regular DNAzyme against integrin β1:5′-TTGCATGAGGCTAGCTACAACGAGGCATCG-3′ASON–Bulge–DNAzyme against integrin β1:5′-GTGATCCACAAACTGCAAGTACAACTTGCATGAGGCTAGCTACAACGAGGCATCG-3′Two pairs of specific primers were designed according to the sequence of the eGFP gene and the β-actin gene. The primers were synthesized by Shanghai Generay Biotech Co., Ltd (China).5′-CTGGACGGCGACGTAAACGG-3′eGFP-F1: 5′-CACGAACTCCAGCAGGACCAT-3′eGFP-R1: 5′-GGCTCCGGCATGTGCAAGGC-3′β-actin-F1: 5′-GAAGGTAGTTTCGTGGATGCC-3′β-actin-R1: 2O in a total volume of 20 µl for 1 h at 42°C. The PCR conditions were as follows: 1 cycle at 94°C for 5 min; followed by 30 cycles of 30 s at 94°C, 30 s at 55°C and 60 s at 72°C. A final elongation step of 10 min at 72°C was included.cDNA was synthesized using 4 µl of the total RNA, 4 µl of 5×reverse transcriptase buffer, 1 µl of 10 mM dNTPs, 1 µl of eGFP-R1 primer (10 µM), 1 µl of β-actin-R1 primer (10 µM), 1 µl of RNase inhibitor (20 U/µl), 1 µl of MMLV reverse transcriptase (5 U/µl) and 7 µl of RNase-free HTwo pairs of specific primers were designed according to the sequence of the integrin β1 gene and the integrin β3 gene. The primers were synthesized by Shanghai Generay Biotech Co., Ltd.5′-ATCCCAGAGGCTCCAAAGATAT-3′Integrin β1-F1: 5′-CACCAAGTTTCCCATCTCCAG-3′Integrin β1-R1: 5′-AGGATTACCCTGTGGACATC-3′Integrin β3-F1: 5′-CCGTGATCTTGCCAAAGTCAC-3′Integrin β3-R1: 2O in a total volume of 20 µl for 1 h at 42°C. The PCR conditions were as follows: 1 cycle at 94°C for 5 min; followed by 35 cycles of 40 s at 94°C, 30 s at 55°C and 60 s at 72°C. A final elongation step of 10 min at 72°C was included.cDNA was synthesized using 4 µl of the total RNA, 4 µl of 5×reverse transcriptase buffer, 1 µl of 10 mM dNTPs, 1 µl of integrin β1-R1 primer (10 µM), 1 µl of integrin β3-R1 primer (10 µM), 1 µl of RNase inhibitor (20 U/µl), 1 µl of MMLV reverse transcriptase (5 U/µl) and 6 µl of RNase-free HBHK-21 cells (from ATCC) were cultured in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum (FBS) supplemented with 1% penicillin–streptomycin (GIBCO). Cells were cultured in 24-well plates and transfected by Lipofectamine 2000 . The ratio of DNAzyme to Lipofectamine was 1 µg:2 µl. Cells were co-transfected with 0.5 µg of pECFP-C1 plasmid and 0.25 µg of siRNA expression cassettes in each well.BHK-21 cells were harvested 36 h after eGFP DNAzyme transfection. They were then washed with PBS three times, and lysed with 100 µl of lysis buffer. Total cellular protein was determined using the BioRad Protein Assay reagent (Bio-Rad Laboratories). The eGFP was detected using mouse anti-GFP antibody . The blot was developed using ECL Plus (Amersham Biosciences).BHK-21 cells were harvested and lysed with 200 µl of lysis buffer . The eGFP fluorescence was analyzed using a luminometer .


Computational modeling has played a central role in the understanding of localized Ca2+ signals in neurons and other cell types. Although many models consider only simple, one-to-one Ca2+ buffering stoichiometry, practically all buffers have multiple Ca2+ binding sites which often display cooperative binding . Giv2+ signals that cannot be achieved by any combination of non-cooperative buffers, in particular during a sequence of action potentials or synaptic inputs. Namely, Figure 2+ buffering can potentially serve as a mechanism of short-term synaptic depression, in contrast to the case of non-cooperative buffers (Figure Here we extend recent modeling studies of cooperative buffering -5, and fs Figure , which apost-synaptic Ca2+ dynamics as well, yielding a deeper insight into synaptic transmission and its dynamic regulation, and may also have relevance for Ca2+-dependent processes in other cells such as endocrine cells, myocytes and immune system cells.We explore this phenomenon in detail, demonstrating the dependence of such buffer-induced short-term synaptic plasticity on all relevant buffering parameters. These results may lead to better understanding of