Leea indica is a medicinal plant used traditionally to cure cancer. In this study, the cytotoxic compounds of L. indica were isolated using bioassay-guided approach. Two cycloartane triterpenoid glycosides, mollic acid arabinoside (MAA) and mollic acid xyloside (MAX), were firstly isolated from L. indica. They inhibited the growth of Ca Ski cervical cancer cells with IC50 of 19.21 μM (MAA) and 33.33 μM (MAX). MRC5 normal cell line was used to calculate selectivity index. MAA and MAX were about 8 and 4 times more cytotoxic to Ca Ski cells compared to MRC5. The cytotoxicity of MAA was characterized by both cytostatic and cytocidal effects. MAA decreased the expression of proliferative cell nuclear antigen, increased sub-G1 cells, and arrested cells in S and G2/M phases. This study provides the evidence for the ethnomedicinal use of L. indica and paves the way for future mechanism studies on the anticancer effects of MAA. Leea indica (Burm. f.) Merrill is a traditional Chinese medicine which belongs to the Leeaceae family. It is a perennial shrub which is widely grown in tropical and subtropical countries such as Malaysia, China, India, and Thailand. The leaves and roots of L. indica are used to treat diabetes, cardiac diseases, and various ailments such as fever, headache, dizziness, soreness, eczema, sprain, leprosy, bone fracture, body pain, muscle spasm, diarrhea, and dysentery , C35H56O8. Positive ESI-MS m/z: 627.3851 [M+Na]+. 1H NMR : δ 0.47 , 0.75 , 0.85–1.66 (6 × CH3), 3.39–4.42 , 5.01 , 5.21–5.5 . The 13C NMR data of compound 1 was shown in Compound 2 was identified as 1α, 3β-dihydroxy-cycloart-24-ene-28-oic acid 3-O-[β-D-xylopyranoside], C35H56O8. Positive ESI-MS m/z: 627.3851 [M+Na]+. 1H NMR : δ 0.44 , 0.72 , 0.91–1.68 (6 × CH3), 3.39–4.42 , 5.08 , 5.20–5.48 . The 13C NMR data of compound 2 was shown in Compound μg/mL of MAA and MAX, respectively. As shown in 50 values of MAA and MAX for Ca Ski cells were 11.60 ± 0.29 μg/mL (19.21 μM) and 20.13 ± 0.21 μg/mL (33.33 μM), respectively. When compared to Ca Ski cells, MAA and MAX were less cytotoxic to the normal cells, as revealed by the relatively higher IC50 values on MRC 5 (94.32 ± 0.75 μg/mL for MAA and 79.25 ± 0.66 μg/mL for MAX). In contrast, camptothecin (CPT) displayed comparable IC50 value on both Ca Ski and MRC 5 cells (2.51 ± 0.33 and 4.74 ± 0.33 μg/mL). The small difference in IC50 value led to our deduction that CPT cannot differentiate between normal and cancer cells and killed both cells at almost the same efficiency. This lack of tumor-cell specificity was also reported by other scholar [MAA and MAX were evaluated for their cytotoxic effect on Ca Ski cervical cancer cells and MRC 5 normal cells using MTT assay. A 72 h exposure to Ca Ski cells with MAA or MAX led to a significant dose-dependent reduction in cell viability. According to  scholar .50 values against MRC 5 when compared to CPT. Moreover, we also compared the cytotoxicity of MAA, MAX, and CPT based on their SI values. MAA was about 8 times more cytotoxic to Ca Ski cells compared to MRC 5, while MAX was about 4 times more cytotoxic to Ca Ski cells compared to MRC 5  and TBE (dye exclusion) assays, which measure mitochondria metabolic death and cell membrane integrity, respectively. As mentioned earlier, we showed that MAA caused a conspicuous dose-dependent reduction of formazan formation in Ca Ski cells  is greatly expressed in most of the proliferating cancer cells including cervical cancer , 36. PCNEarlier, we have showed that the cytotoxicity of MAA in Ca Ski cells was derived from both cytostatic and cytocidal effects. Moreover, we also demonstrated that the cytostatic effect was due to antiproliferative effect, as reflected by the decrease of the proliferation marker, PCNA. Subsequently, we checked whether the antiproliferative effect was associated with any cell cycle phase-specific arrest. After treated with MAA for 12 and 24 h, the proportion of S-phase and G2/M-phase in the treated cells was significantly higher compared to the untreated cells. The perturbation of cell cycle progression, caused by the sustained accumulation of cells in the S and G2/M phases may be in part responsible for the cytostatic/antiproliferative effect of MAA. This was in agreement with the retardation of cell proliferation by MAA at 12 and 24 h in the TBE assay . In addi L. indica belong to the group called cycloartane triterpenoid glycoside. Recently, this group of compounds has received considerable attention for their cytotoxic potential [Combretum molle on cancer cells [The mollic acid glycosides (MAA and MAX) isolated fromotential , 40. Forer cells . HoweverTherefore, our findings here warrant the need for further investigation on the anticancer potential of MAA, especially for cervical cancer. Elaborate studies to identify the mechanisms of action are in progress.α-L-arabinoside (MAA) and mollic acid β-D-xyloside (MAX), were isolated form L. indica for the first time through bioassay-guided method. Preliminary studies showed that the cytotoxicity of MAA was associated with decrease of PCNA expression, cell cycle S and G2/M phases arrest, and induction of hypodiploid cells.Conclusively, two cytotoxic cycloartane triterpenoid glycosides, namely mollic acid


Early research with beta-alanine (β-ALA) supplementation has shown increases in muscle carnosine as well as improvements in body composition, exercise performance and blood lactate levels. Creatine monohydrate supplementation has been extensively researched for its effects on anaerobic exercise performance. Recently, studies have examined the combined effects β-ALA and creatine supplementation on anaerobic exercise performance and lactate threshold. The purpose of the present study was to examine the acute and chronic effects of β-ALA supplementation with and without creatine monohydrate on body composition, aerobic and anaerobic exercise performance, and muscle carnosine and phosphagen levels in college-aged recreationally active females.2max and lactate threshold, and multiple Wingate tests for anaerobic exercise performance.Thirty-two females were randomized in a double-blind placebo controlled manner into one of four supplementation groups including β-ALA only (BA), creatine only (CRE), β-ALA and creatine combined (BAC) and placebo (PLA). Participants supplemented for four weeks using an individualized daily dosing strategy that included a loading phase for the creatine for week 1 of 0.3g/kg of body weight and a maintenance phase for weeks 2-4 of 0.1g/kg of body weight, with or without a continuous dose of β-ALA of 0.1g/kg of body weight. They reported for testing at baseline, day 7 and day 28. Testing sessions consisted of a resting muscle biopsy of the vastus lateralis, body composition measurements (DEXA), a graded exercise test on the cycle ergometer for VO2max (p=0.27), peak lactate (p=0.05) lactate threshold (p=0.67), ventilatory threshold (p=0.35), peak power (p=0.42), mean power (0.28), total work (p=0.28) or rate of fatigue (0.20). There were some trends for anaerobic exercise indicating groups supplementing with creatine may have greater improvements, however, these findings were not statistically significant.Results showed all supplementation strategies increasing muscle carnosine levels over placebo after four weeks, but not between groups. The percent change for each group after four weeks were 35.3±44.8% (p=0.02) for BA, 42.5±99.3% (p=0.01) for BAC, 0.7±27.1% (p=0.04) for CRE versus 13.9±44.0% for PLA. Muscle total creatine showed trends of increasing for all active supplement groups after four weeks, but not between groups. The percent change in muscle creatine after four weeks was 4.6±71.4% for BA, 154.0±375.0% for BAC, 1.7±41.6% for CRE and -4.1±10.9% for PLA (p=0.72). There were improvements for all groups with percent body fat after four weeks (p=0.01), despite the present study not including a specific training protocol. The delta values were -2.3±2.6% BAC, -1.4±4.5% CRE, 0.2±1.8% BA and -1.3±2.2% PLA. There were no group differences observed for VOThe present study failed to show any additive effects of β-ALA and creatine supplementation for body composition, aerobic exercise, lactate threshold or anaerobic exercise measures. This could be due to the small sample size resulting in low power and effect sizes. Previous research has demonstrated that four weeks of β-ALA and creatine supplementation was enough time to increase muscle carnosine and phosphagen levels. However, perhaps more time is needed for performance adaptations to occur, especially without the addition of an exercise training component.


Retinopathy of prematurity is becoming obvious with the improvement of neonatal ambulance. However there is still not a good treatment. The present study is to observe the effect of 17 beta-estradiol (E2) on oxygen-induced retinopathy (OIR), and explore the relationship between the changes of avascular area and malondialdehyde (MDA) in retina.Newborn oxygen-exposed mice underwent subcutaneous injections of different dose of E2 , tamoxifen or phosphate buffered saline everyday from post-natal day (p)7 to p17. At p17, retinal flat mounts were scored for the percentage of avascular/total retinal area, and pathological changes during revascularization. The MDA concentration in the retina was determined also. In the most efficacious E2 group (10.0 µg), 100.0 µg tamoxifen was also administered, and the percentage of capillary-free/total retinal area determined, and the retinal malondialdehyde concentration assayed.F = 2778.759, P < 0.01), and the difference between any two groups were also significant . The predilection of tufts and clusters during revascularization was mainly aggregated in zones 2 and 3, but the difference of retinal neovascular clusters and tufts in fourth zone among different groups were significant [clusters  vs tufts ]. The mean MDA concentration were 0.711±0.037, 2.084±0.066 , 1.829±0.091(0.1 µg E2), 1.152±0.067(1.0 µg E2), 0.796±0.027(10.0 µg E2), 1.988±0.049(10.0 µg E2 +100.0 µg tamoxifen) . The difference between any two groups were also significant . The close relation between the percentage of avascular/total retinal area and MDA concentration was also verified .The mean percentage of capillary-free area over total retinal area was 0, 34.197±1.301, 23.685±0.407 (0.1 µg E2), 14.648±0.355 (1.0 µg E2), 4.693±0.450 (10.0 µg E2) and 32.240±0.654 (10.0 µg E2 +100.0 µg tamoxifen). The difference was significant (Oxidative stress responses play a pivotal role in OIR, by means of receptor pathway. E2 can alleviate oxidative stress reaction, and thus ameliorate the severity of oxygen induced retinopathy. Retinopathy of prematurity (ROP), a kind of vasoproliferative retinal disease associated with premature and low body weight infants, is becoming more and more serious with the improvement of neonatal ambulanceAll experimental mice were neonatal C57BL/6J mice, purchased from Animal Experimental Center of Xian Jiaotong University. All animals were cared for in accordance with the Xian Jiaotong University Institute for Laboratory Animal Research  and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.E2, tamoxifen and fluorescein labeled high-molecular-weight dextran  were obtained from Sigma Corp., USA. The Mini malondialdehyde (MDA) assay kit was purchased from Biological and Engineering Institute of Nanjing Jiancheng, China.2 from p7 to p12, along with nursing mothers. At p12, they were returned to room air and kept on feeding. The pups were killed on p17.Neonatal C57BL/6J mice was divided into six groups according to the treatment: room air with subcutaneous vehicle injection (control 1), hyperoxia with vehicle injection (control 2), hyperoxia with 0.1 µg E2 injection group, hyperoxia with 1.0 µg E2 injection group, hyperoxia with 10.0 µg E2 injection group, and hyperoxia with 10.0 µg E2 and 100.0 µg tamoxifen injection group. The pups received daily subcutaneous injections of either E2 in vehicle (dissolved in ethanol and diluted in 0.05 ml PBS) or vehicle alone from post-natal (p)7 to p17.The pups were exposed to 75% OTo study the retinal vascular pattern, retinal flat mounts were obtained by using perfusion of a high-molecular-weight dextran  conjugated with fluorescein (Sigma), as described previouslyDigitized images of the total retinal area and the central capillary-free area was measured using Optimas software (Version 5.2).The central capillary-free area was expressed as a percentage of total retinal area.et alDuring the phase of revascularisation, pathological vessel, neovascular tufts and clusters in retina appeared as described previouslyThe lipid peroxidation concentration was determined by a method that measures the amount of thioibarbituric acid reactivity, which is expressed as the amount of MDA formed during acid hydrolysis of the lipid peroxide compound. Retinal tissue homogenates were prepared and reacted with sodium dodecyl sulfate, acetic acid, and thiobarbituric acid as described previouslyP < 0.05 was considered statistically significant. All analyses were performed using the SPSS software All data were expressed as mean ±SD. Differences among groups were analyzed by one-way analysis of variance (ANOVA), and the Student-Newman- Keuls (SNK) method was used for multiple comparison. The P-value reported was two-sided and value of p < 0.01 vs control 2, n = 6), 14.648±0.355  and 4.693±0.450%  respectively (n = 6). 100.0 µg tamoxifen, a selective antagonist of estrogen receptor, significantly decreased the effect of 10.0 µg E2 on the ratio of NCFA/TA (n = 6). These results  , 1.152±0.067  and 0.796±0.027  respectively. 100 µg tamoxifen, a selective antagonist of estrogen receptor, significantly reversed the effect of 10.0 µg E2 on the concentration of MDA 1.988±0.049 . These results suggested that reduced free radicals formation may be involved in the protective effect of activation of estrogen receptor against hyperoxia-induced retina injury.Compared with control 1 group (0.711±0.037), the concentration of MDA in retina was significantly increased in hyperoxia-exposed mice in control 2 (2.084±0.066). Treatment with E2, at the concen- tration of 0.1, 1.0, and 10.0 µg, decreased the concentration of MDA to 1.829±0.091 .In order to more easily understand the relation among retinal capillary- free area as a percentage of total area and MDA, the results were plotted and the trends between them was clearly manifested . With administration of E2, the increase of MDA was reduced in a dose-dependent manner, and an estrogen receptor antagonist reversed this action. Thus our data suggest that a certain amount of oxidative stress reaction may contribute to retinal angiogenesis, while excessive oxidative stress has the opposite effect. Through its receptor pathway, E2 can alleviate the excessive oxidative stress response, and finally ameliorate retinal angiogenesis. In hyperoxia, the percentage of retinal avascular area was not further improved with an increase in the E2 dose above 10.0 µg (results not shown here), indicating that the OIR can not be completely eliminated by E2. Therefore other elements must be involved in this pathological process, which need to be further explored.By determining the differences in the percentage of avascular area/ total retinal area in different groups, we can understand the effect of E2 on retinal angiogenesis of OIR mice. The retina, extremely rich in membranes with polyunsaturated lipidsAnother study demonstrated that retinal angiogenesis could be modulated by E2 through HIF-1 alpha and VEGF pathway, and thus improve OIRAltogether, oxidative stress response may play a pivotal role in OIR through receptor pathways, E2 can mitigate the injury of excessive oxidative stress response in oxygen exposed retina, and thus ameliorate OIR. Further more, the results of this study suggest that fully studying the change of endocrine hormone in pregnancy and fully understanding their function in fetal tissue development, may provide better prophylaxis for ROP. Recently, it has been reported that the fragment of prolactin (17-PRL) exerts more powerful anti-angiogenesisin vivo. The pathways related to estrogen include P13K/Akt-Nrf2 signaling pathwayThe mechanism of antioxidative stress is complicated


Cardiovascular morbidity is a well known late complication of chemotherapy, for example for treatment of testicular cancer. In addition, development of a combination of overweight, hypertension and abnormal lipid profiles has been observed in these patients suggesting increased risk of developing the metabolic syndrome and diabetes. The early effects of chemotherapy on myocardial function and metabolic profile are largely unknown. Therefore, the purpose of this study was to assess short-term effects of chemotherapy in testicular cancer on myocardial function in relationship with alterations in metabolic profile.Fourteen patients with testicular cancer were treated with bleomycin, etoposide and cisplatin.Before and after chemotherapy, magnetic resonance imaging techniques were used to assess cardiac systolic and diastolic function, and abdominal fat volume (summation of 3 slices at the level of the L5 vertebra). In addition, hepatic and myocardial triglyceride content were assessed using MR spectroscopy. Blood samples were taken at both occasions to obtain plasma lipid profile and to estimate insulin sensitivity.After chemotherapy, an unfavorable shift in metabolic profile was observed: Visceral abdominal fat volume was increased significantly (from186 ± 125 ml to 227 ± 162 ml. P< 0.05) without significant changes in BMI. Hepatic triglyceride content increased, although non-significant (from 3.23 ± 2.72 % to 4.65 ± 4.85% P> 0.05). In addition proxy-measures of insulin sensitivity (Quicki) decreased from 0.39 ± 0.05 to 0.36 ± 0.05 P < 0.05 and serum LDL-cholesterol increased significantly after chemotherapy treatment from 3.12 ± 1.15 mmol/l to 3.74 ± 1.41 mmol/l.These metabolic derangements were paralleled by subclinical changes in myocardial function. Diastolic function, as assessed as the E/A ratio, decreased (from 1.87 ± 0.43 to ± 1.64 ± 0.45 P< 0.05.) Furthermore, left ventricular end-diastolic volume was decreased (from192 ± 27 ml. to 175 ± 26 ml. P< 0.05), indicating disturbed ventricular relaxation. Myocardial systolic function and myocardial triglyceride content were unaltered.Chemotherapy for testicular cancer induces unfavorable metabolic changes, which are paralleled by impairment in diastolic heart function.None.


Rhynchophorus palmarum L. (Coleoptera: Curculionidae) by chromatography on anion-exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gels, β-glucosidase is a monomeric protein with a molecular weight of 58 kDa based on its mobility in SDS-PAGE and 60 kDa based on gel filtration. Maximal β-glucosidase activity occurred at 55°C and pH 5.0. The purified β-glucosidase was stable at 37°C and its pH stability was in the range of 5.0–6.0. The enzyme readily hydrolyzed p-nitrophenyl-β-D-glucoside, cellobiose, cellodextrins and required strictly β-gluco configuration for activity. It cleaved glucose-glucose beta-(1–4) linkages better than β-(1–2), β-(1–3) and β-(1–6) linkages. The catalytic efficiency (Kcat/KM) values for p-nitrophenyl-β-D-glucoside and cellobiose were respectively 240.48 mM-1s-1 and 134.80 mM-1s-1. Beta-glucosidase was capable of catalysing transglucosylation reactions. The yield of glucosylation of 2-phenylethanol (20 %), catalysed by the beta-glucosidase in the presence of cellobiose as glucosyl donor, is lower than those reported previously with conventional sources of beta-glucosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transglucosylation reactions (pH 6.6).A β-glucosidase was purified from the digestive fluid of the palm weevil  Rhynchosciara americana larvae . This was done in order to find new β-glucosidase for use in glycobiotechnology and propose a biological role for the characterized enzyme in the degradation of cellulose.In this study, we attempted to purify and characterize the β-glucosidase from the digestive fluid of larvae of the palm weevil p-nitrophenyl-glycopyranosides were purchased from Sigma Aldrich, www.sigmaaldrich.com. DEAE—Sepharose CL-6B, Sephacryl-S100 HR, Phenyl Sepharose CL-6B gels were obtained from Pharmacia-LKB Biotech, www.Pharmacia.com. The chemicals used for polyacrylamide gel electrophoresis (PAGE) were from Bio-Rad, www.bio-rad.com. All other chemicals and reagents were of analytical grade.Substrates: cellobiose, sucrose, sophorose, laminaribiose, gentiobiose, xylobiose, lactose, maltose, arabino-galactan, carboxymethylcellulose, inulin, laminarin, xylan, lichenan, starch, glucose, xylose, cellodextrins and R. palmarum larvae were obtained from the commercial oil palm, Elaeis guineensis Jacq. , plantation near the Université d'Abobo-Adjamé . They were collected directly from the palm weevil and dissected in the laboratory.R. palmarum larvae were rinsed in cold water and blotted with filter paper. Guts were dissected in cold 0.9% KCl (w/v) solution and digestive content was removed and stirred in the presence of 0.9% KCl (w/v) solution before centrifugation at 6000 × g for 30 min. The supernatant was then stirred with 100 mM acetate buffer pH 5.0 for 30 min. The homogenate was centrifuged at 10,000 × g for 30 min. The collected supernatant constitued the crude extract. After freezing at -180°C in liquid nitrogen, the crude extract was stored at -20°C.p-nitrophenol from the substrate p-nitrophenyl-β-D-glucopyranoside. An assay mixture (275 µl) consisting of a 100 mM acetate buffer (pH 5.0), 1.25 mM p-nitrophenyl-β-D-glucopyranoside and enzyme solution, was incubated at 37°C for 10 min. The control contained all reactants except the enzyme. Determination of other p-nitrophenylglycosidase activities was carried out under the same experimental conditions. The reaction was stopped by the addition of 1M sodium carbonate (2 ml), and absorbance of the reaction mixture was measured at 410 nm.Under the standard test conditions, β-glucosidase activity was measured by the release of Oligo-saccharidase activity was determined by measuring the amount of glucose or xylose liberated from oligosaccharide by incubation at 37°C for 10 min in a 100 mM acetate (pH 5.0), containing 10 mM oligosaccharide. The amount of glucose was determined by the glucose oxidase-peroxidase method  after hePolysaccharidase activity was assayed by the dinitrosalicylic acid procedure , using 1p-nitrophenol or glucose per min under the defined reaction conditions. Specific activity was expressed as units per mg of protein (U/mg of protein).One unit of enzyme activity was defined as the amount of enzyme capable of releasing one µmol of Protein concentrations were determined by method of Lowry et al.  using boFive ml of crude extract was loaded onto a DEAE-Sepharose CL-6B (2.4 × 6.5 cm) that had been equilibrated previously with 100 mM acetate buffer pH 5.0. The unbound proteins were removed from the column by washing with four column volumes of the same buffer pH 5.0. Proteins were eluted using a stepwise gradient with 0.1; 0.2; 0.3; 0.4; 0.5 and 1 M KCl in 100 mM acetate buffer pH 5.0. Fractions (4 ml each) were collected at a flow rate of 80 ml/h and assayed for enzyme activity. The active fractions were pooled and saturated overnight by 80 % ammonium sulfate in a cold room. The precipitated pellet was then separated by centrifugation at 6000 × g for 30 min and dissolved in 1 ml of 100 mM acetate buffer pH 5.0. The enzyme solution was loaded directly into a Sephacryl S-100 HR column (1.6 × 64 cm) that was pre-equilibrated with the same buffer pH 5.0. Proteins were eluted at a flow rate of 15 ml/h using 100 mM acetate buffer pH 5.0. Fractions of 1 ml were collected and active fractions were pooled together. The pooled fraction from the previous step was saturated to a final concentration of 1.7 M ammonium sulfate and applied on a Phenyl-Sepharose CL-6B column (1.4 × 5 cm) previously equilibrated with 100 mM acetate buffer pH 5.0 containing 1.7 M ammonium sulfate. The column was washed with equilibration buffer and the proteins retained were then eluted using a stepwise gradient of 1, 2, 0.8, and 0.4, 0 M ammonium sulfate in 100 mM acetate buffer pH 5.0. Fractions of 1 ml were collected at a flow rate of 14 ml/h and active fractions were pooled together. The pooled fraction was dialysed against 100 mM acetate buffer pH 5.0 overnight in a cold room.www.hoeferinc.com), according to the procedure of Laemmli  column  was equilibrated with 20 mM acetate buffer (pH 5.0) containing sodium azide 0.5 % (w/v) and calibrated with beta-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (48.8 kDa) and cytochrome C (12.4 kDa). Fractions of 0.5 ml were collected at a flow rate of 0.5 ml/min.p-nitrophenyl-β-D-glucopyranoside in a series of buffers at various pH values ranging from pH 3.6 to 8.0. The buffers used were acetate buffer (100 mM) from pH 3.6 to 5.6 and phosphate buffer (100 mM) from pH 5.6 to 8.0. The pH values of each buffer were determined at 37°C. β-glucosidase activity was measured at 37°C under the standard test conditions. The effect of temperature on β-glucosidase activity was followed in 100 mM acetate buffer pH 5.0 over a temperature range of 30 to 80°C using 1.25 mM p-nitrophenyl-β-D-glucopyranoside under the standard test conditions.The effect of pH on β-glucosidase activity was determined by measuring the hydrolysis of The stability of β-glucosidase was followed over the pH range of 3.6 to 8.0 in 100 mM buffers. The buffers were the same as those used in the study of the pH and temperature optima. After 2 h incubation at 37°C, aliquots were taken and immediately assayed for residual beta-glucosidase activity. The thermal stability of the enzyme was determined at 37 and 55°C after exposure to each temperature for a period from 10 to 140 min. The enzyme was incubated in 100 mM acetate buffer pH 5.0. Aliquots were drawn at intervals and immediately cooled in ice-cold water. Residual activities, determined in both cases at 37°C under the standard test conditions, are expressed as percentage activity of zero-time control of untreated enzyme.M, Vmax and kcat/KM) were determined in 100 mM acetate buffer (pH 5.0) at 37°C. Hydrolysis of P-nitrophenyl-β-D-glucopyranoside was quantified on the basis of released p-nitrophenol as in the standard enzyme assay. Cellobiose hydrolysis was quantified by determination of released glucose, determined with oxidase-peroxidase method (M and Vmax were determined from Lineweaver-Burk plot using different concentrations of p-nitrophenyl-beta-D-glucopyranoside (1–10 mM) and cellobiose (1–20 mM).The kinetic parameters  of different chemical agents for 20 min at 37°C (various cations in the form of chlorides). After incubation, the residual activity was determined by the standard enzyme assay using R. palmarum larvae to catalyse transglucosylation reactions was tested with cellobiose as glucosyl donor and 2-phenylethanol as the glucosyl acceptor. In a typical experiment, the transglucosylation reaction was carried out at 37°C in 2 ml 100 mM acetate buffer or 100 mM phosphate buffer containing an appropriate amount of β-glucosidase corresponding to 15 units of the considered enzymatic activity and various concentrations of glucosyl donor (cellobiose) and of glucosyl acceptor (2-phenylethanol) (www.waters.com), the reaction mixture (20 µl) was analysed quantitatively by HPLC at room temperature. Chromatographic separation of sugars (cellobiose) were performed on a Supelcosyl LC-NH2 (5 µm) column (0.46 × 25 cm) from Supelco  using acetonitrile/water  as the eluent, and monitored by refractometric detection. The flow rate was maintained at 0.75 ml min-1. 2-phenylethanol (PE) and phenylethylglucoside (PEGlc) were analysed on a Thermo Hypersil (www.thermo.com) (5 µm) column (0.46 × 25 cm) using methanol/water  as the eluent. Chromatographic separations were monitored at 257 nm using a constant flow rate of 0.45 mlmin-1  , 2005b.  mlmin-1 , 2005b.The transglucosylation percentage (T) may be expressed as:ectively , 2005b.R. palmarum. p-nitrophenyl-β-D-glucopyranoside was used as the substrate to monitor enzymatic activity. A β-glucosidase was purified from the digestive juice of larvae of the palm weevil From the migration pattern of standards on SDS-PAGE, the molecular weight was calculated to be 58 kDa . The molp-nitrophenyl-β-D-glucopyranoside hydrolysis was found to be 5.0. The enzyme retained more than 70% of its activity in the range pH 4.6 to 6.0 (p-nitrophenyl-β-D-glucopyranoside was 55°C (10) calculated between 40°C and 50°C was 1.90   showed an inhibitory effect on β-glucosidase activity. Others had no effect on enzyme activity , ZnCl2 , FeCl3 , DTNB )  and activity .R. palmarum β-glucosidase f to catalyse transglucosylation reactions was tested with cellobiose as glucosyl donor and with 2-phenylethanol as glucosyl acceptor. Maximum transglucosylation was obtained in the range pH 5.6–7.0  on beta-1,4 linkage.The hinensis , LeuconoR. palmarum β-glucosidase was inactive on high molecular mass polymer such as lichenan, xylan, carboxymethylcellulose, inulin, starch, arabinogalactan and laminarin. The purified enzyme had no contaminating glycosidase activities such as β-glucosidase, β-fucosidase, β-mannosidase, β-arabinosidase and β-xylosidase. The only substrates that were hydrolyzed by the enzyme were cellobiose, cellodextrins, laminaribiose, sophorose, gentiobiose and p-nitrophenyl-β-D-glucopyranoside. These results suggest that this β-glucosidase is an exo-glycosidase with a high specificity for the beta-anomeric configuration of the glucosidic linkage. This pattern is similar to the activity of the β-glucosidases from Sclerotium rolfsii . To explain these differences, Huber et al. .Transglycosylation is a type of hydrolysis in which the glycosyl moiety of the substrate, instead of water, is transferred to other hydroxy compounds . Beta-gls oryzae , Achatinachatina , Thermusmophilus  and M. shyalinus , 2005b or et al.  suggester et al. . In the  1 and 3 . The gluR. palmarum larvae appears to be distinct from other β-glucosidases so far reported in terms of substrate specificity and high affinity towards cellobiose. This enzyme could be used as a tool in the structural analysis of D-glucose containing oligosaccharide chains of glycoproteins, glycolipids and cellulose.The β-glucosidase from


Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated. In this report, we have investigated the molecular mechanism which elucidates the inhibitory effects of dexamethasone on murine osteoblastic MC3T3-E1 cells. It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest. Both the cell cycle arrest and apoptosis were dependent on glucocorticoid receptor (GR), as they were abolished by GR blocker RU486 pre-treatment and GR interference. G1 phase arrest and apoptosis were accompanied with a p53-dependent up-regulation of p21 and pro-apoptotic genes NOXA and PUMA. We also proved that dexamethasone can’t induce apoptosis and cell cycle arrest when p53 was inhibited by p53 RNA interference. These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation. Glucocorticoids (GCs) are the most frequently used anti-inammatory and immunosuppressive drugs in clinic to treat a variety of diseases including inflammation, cancer, and autoimmune disorders The effects of GCs are primarily considered to be mediated by cytosolic glucocorticoid receptor (GR) activation We postulate that GC activates GR, and then leads to activation of P53, granzyme A or GILZ, thereby inducing osteoblasts apoptosis and cell cycle arrest. The results of this study indicate that GR activation indeed up-regulates the expression of P53 and its downstream molecule, which results in growth inhibition.N/A.Dexamethasone, and RU486 (mifepristone) were obtained from Sigma . Dexamethasone was used in a concentration gradient from 0.001 µM to 10 µM. Final concentration of RU486 was 10 µM. Control in all experiments was vehicle (ethanol) unless otherwise indicated. Cell counting kit (CCK-8) were obtained from Dojindo . Antibody of β-action, caspase-3, p53, PUMAand p21were purchased from cell signaling technology , NOXA, granzyme A and GILZ were purchased from Abcam . Annexin V-FITC apoptosis determining kit were purchased from BD PharMingen . TUNEL assay was purchased from Roche Applied Science . Lipofectamine RNAi MAX was purchased from Invitrogen .2 and 5% CO2) and maintained in a subconuent state unless otherwise indicated.The murine osteoblastic cell line MC3T3-E1 was obtained from American Type Culture Collection . Cells were cultured in a-MEM  supplemented with 10% FBS, 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml ascorbic acid Cells were incubated in a humid incubator at 37°C (95% OFor the experiments with RNA interference (RNAi), a mouse P53 and GRα specific double-stranded, small interfering (si) RNA was synthesized . Two of p53 siRNA molecules,sip53-1 and sip53-2, were selected: Sip53-1: (forward) 5- CCACUUGAUGGAGAGUAUUTT -3 and (reverse) 5- AAUACUCUCCAUCAAGUGGTT-3, and sip53-2: (forward) 5- GACCUAUCCUUACCAUCAUTT -3 and (reverse) 5- AUGAUGGUAAGGAUAGGUCTT -3. Two of GRα siRNA molecules, siGR-1 and siGR-2, were selected: siGR-1(forward) 5-GGAGAGGACAACCUGACUUTT-3 and(reverse) 5-AAGUCAGGUUGUCCUCUCCTT-3, siGR-2(forward) 5- CUGCAUGUAUGACCAAUGUTT -3 and(reverse) 5- ACAUUGGUCAUACAUGCAGTT -3.In addition, siRNA molecules that exhibited no homology to the mouse genome sequence were selected as negative controls (forward) 5- UUCUCCGAACGUGUCACGUTT -3 and (reverse) 5- ACGUGACACGUUCGGAGAATT -3 . A further untreated group of MC3T3-E1 cell cultures served as untreated control group (FBS group). Lipofectamine RNAi MAX was used to introduce the siRNA into the MC3T3-E1 cells according to the manufacturer’s instruction.3 cells per well in 96-well plates. To assess the effects of dexamethasone on cell proliferation, the cells were incubated in growth medium or conditioned medium for 24 h at the concentration gradient from 0.001 µM to 10 µM. The sample cells were quantified using WST-8 assay, according to the instructions. Briefly, 10 µl of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium (WST-8) solution reagent was added to 100 µl of culture medium in each well. After incubation for 2.5 h at 37°C, The absorbance of each well was read at a wavelength of 450 nm on a microplate reader. The measurements were represented by the means of at least three independent experiments, with each data point based upon three replicates.The MC3T3-E1 cells were inoculated at 2×10The MC3T3-E1Cells were seeded subconfluently into 12-well plates and treated with conditioned medium for 24 h at the concentration gradient from 0.001 µM to 10 µM. Cells were trypsinized and pelleted with cellular supernatant for 5 min at 400 g. After pellet was resuspended in 60 µL media, cell death rates were determined by counting cells using a hemocytometer after addition of Trypan blue, which stained the cytoplasm of dead cells but not live cells. Cell death rate (%) = number of dead cells/number of total cells (×100%).Cells were analyzed for their cell cycle distribution by flow cytometry. After being treated with dexamethasone for 48 h, the adherent cells were washed once with PBS, then trypsinized, and collected by centrifugation at 400 g for 5 min. The cells (106 cells per sample) were fixed in 4 ml of cold 70% ethanol at −20°C overnight. After centrifugation at 400×g for 5 min, cell pellets were incubated with 0.5 ml of PBS containing 100 g/µml RNase and 5 µg/ml propidium iodide (PI) at 37°C for 30 min. Cell cycle distribution was analyzed by measuring DNA content using a flow cytometer .FITC Annexin V apoptosis determining kit was used to quantitatively determine the percentage of cells within a population that are actively undergoing early apoptosis per manufacture’s instruction. Briefly, the cells were collected, washed twice with Annexin binding buffer and incubated with Annexin V-FITC and propidium iodide under the manufacturer’s recommended conditions. Cells that stain positive for FITC Annexin V and negative for PI were considered undergoing early apoptosis.TUNEL assay was performed using the in situ cell death detection kit according to the manufacturer’s instructions. Briefly, MC3T3-E1 cells were plated onto coverslips and cultured overnight. After treated with various stress stimuli for 24 hours cells were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized in 0.1% Triton X-100 for 2 min at 4°C. Cells were then stained with fluorescein isothiocyanate (FITC)-conjugated terminal deoxynucleotidyl TUNEL reaction mixture for 1 h at room temperature and incubated with DAB for 2 min. All dilutions were performed in PBS, and samples were analyzed and recorded by an OLYMPUS FLUOVIEW microscope.TGTAATAGCTCCTGCATGGGG -3 (forward) and 5- TGCTATGCTTTGCTCCTGATCTG -3 (reverse) primers for P53, 5- TGCTATGCTTTGCTCCTGATCTG (forward) and 5- TGTCAGTTGATAAAACCGCTGCC -3 reverse primers for GRα, 5- ATCATGTTTGAGACCTTCAACA (forward) and 5- CATCTCTTGCTCGAAGTCCA -3 reverse primers forβ-actin. Quantification of the P53 or GRα mRNA expression was calculated using the standard curve method.The total RNA was extracted from the cells using TRIzol reagent  and then reverse-transcribed  following manufacture’s protocol. The detection of P53 or GRα mRNA levels was performed by real-time RT-PCR . The primer were as follows: 5- Whole-cell extracts were prepared using Triton lysis buffer . After the lysates were centrifuged for 12 min at 3,000 g to remove debris, protein concentrations were determined using the Bradford protein assay. 20–40 micrograms of protein from each cell layer homogenate was loaded onto a 10% or 12% polyacrylamide gel and transferred to a PVDF membrane. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies. After extensive washing, the membranes were re-probed with peroxidase conjugated secondary antibody. Blots were processed using an ECL kit and exposed to film Controls for protein loading were made by β-actins as the internal standard.All data were expressed as means ± SD. The differences between groups was analyzed by one-way analysis of variance followed by Bonferroni’s multiple comparisons test using SPSS 18.0 for windows. P-value (P) <0.05 was considered statistically significant.The 24-hour exposure of murine osteoblast MC3T3-E1 to 0.001, 0.01, 0.1,1.10 µmol/L DEX respectively decreased cell proliferation in a concentration-dependent manner  ComparedTo determine whether the inhibitory effect can be attributed to apoptosis and cell cycle arrest, and whether the effect results from GR activation, we treated MC3T3-E1 cells with PBS (control group), 1 µmol/L dexamethasone (DEX group), RU486 (RU486 group), and 1 µmol/L dexamethasone plus 10 µmol/L RU486 (DEX+RU group) respectively. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group (12.1±0.04%) as compared to 3.01±0.04% in the control group, 3.54±0.05% in the RU486 group, and 5.36±0.03% in the DEX+RU group.  A The leTo assess the change of cell cycle induced by dexamethasone in MC3T3-E1 cells, the cell nuclei were stained with propidium iodide and the cell cycle was analyzed through flow cytometry. The percentage of cells in the G0–G1 phase increased significantly (P<0.05) to 84.67±1.61% in the DEX group, as compared to 64.67±2.65% in the control group, 68.5.±2.65% in the RU486 group, and 68.00±0.76% in the DEX+RU group, respectively. The percentage of cells in the S phase was decreased significantly (P<0.05) to 8.90±1.00% in the DEX group as compared to 27.67±0.58% in the control group, 24.00±1.00% in the RU group, and 23.83±1.26% in the DEX+RU group, respectively.  C.To investigate GRαgene function, we tried to silence this gene in MC3T3-E1 cells using siRNA targeting GRαmRNA. The elimination of the GRαmRNA and protein in MC3T3-E1 cell cultures was achieved as determined by real-time PCR  and WestTo further determine the role of GR in DEX-induced apoptosis and cell cycle, we treated MC3T3-E1 cells with PBS (control group), 1 µmol/L DEX (DEX group), siGR-1 (siGR-1 group), 1 µmol/L DEX plus siGR-1 (DEX+siGR-1 group) respectively. Cleaved Capase-3 was used to characterize apoptosis of these groups. Western blot show DEX can significantly activate caspase-3 and then up-regulate cleaved Capase-3 expression, this effect can be reversed by gene silencing of GRα . TUNEL sThe percentage of cells in the G0–G1 phase increased significantly (P<0.05) to 81.14±4.77% in the DEX group as compared to 67.26±3.65% in the control group, 68.4±2.65% in the siGR-1 group, and 69.34±0.76% in the DEX+siGR-1 group. The percentage of cells in the S phase was decreased significantly (P<0.05) to 11.09±0.86% in the DEX group as compared to 24.74±1.58% in the control group, 21.75±2.00% in the siGR-1 group, and 20.83±1.13% in the DEX+siGR-1 group. .To identify the downstream signaling pathways of GR activation, we performed Western blot analysis of proteins affected by GR activation such as granzyme A, GILZ and p53,respectively. This was done when MC3T3-E1 cells was treated with 0, 0.001, 0.01, 0.1, 1.0 and 10.0 µmol/L DEX, respectively. The levels of granzyme A and GILZ did not increased, but the expression of P53 was dose-dependently up-regulated . These rTo further confirm that and identify downstream signaling pathways of P53, we performed western blot analysis of apoptosis and cell cycle associated proteins NOXA, PUMA and p21. In the DEX group, the level of the p53 protein was up-regulated, when compared with that of the control group, siGR-1 group and siGR-1+DEX group, which was consistent with the previous results. The level of the NOXA, PUMA and P21 protein was also up-regulated in the DEX group which suggested that P53 is a key molecular between GR activation and NOXA, PUMA and P21 up-regulation. For comparison, β-actin protein was used as an internal reference, which did not vary remarkably. .To investigate the p53 gene function, we tried to silence this gene in MC3T3-E1 cells with siRNA targeting p53 mRNA. The knockdown of the p53 mRNA in MC3T3-E1 cell cultures was achieved as verified by RT-PCR and western blot . The proWe then treated MC3T3-E1 cells with PBS(control group ), 1 µmol/L dexamethasone(DEX group ), MC3T3-E1 cell silencing the p53 Gene (sip53-1 group), 1 µmol/L dexamethasone plus siRNA (DEX+sip53-1 group) respectively. Cleaved Capase-3 was used to characterize apoptosis of these groups. Western blot show DEX can significantly up-regulate cleaved Capase-3 expression, this effect can be reversed by gene silencing of p53 . TUNEL sThe percentage of cells in the G0–G1 phase increased significantly (P<0.05) to 81.44±3.56% in the DEX group as compared to 66.36±2.44% in the control group, 67.51±3.54% in the siGR-1 group,and 70.68±1.67% in the DEX+siGR-1 group. The percentage of cells in the S phase was also decreased significantly (P<0.05) to 9.54±1.07% in the DEX group as compared to 23.73±0.97% in the control group, 26.57±1.56% in the siGR-1 group, and 22.34±1.34% in the DEX+ siGR-1 group. .In this study, we have illustrated for the first time that GR activation by dexamethasone up-regulates P53 in MC3T3-E1 osteoblast cell line, thereby enhancing the transcriptional activatity of P53. And it ultimately induces MC3T3-E1 cells apoptosis and cell cycle arrest.It has been reported that GCs have potent inhibitory effects on osteoblast proliferation The effect of GR activation by glucocorticoids has long been a research focus. It is well accepted that the effect and molecular mechanism of GR activation is cell context specific. For example, it has been reported that GR activation by dexamethasone promotes survival and inhibits apoptosis of mammary epithelial cells through transcriptional induction of serum and GC-inducible protein kinase-1 (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP-1) GZMA, one of the apoptotic effectors and direct transcriptional targets of glucocorticoid receptor The crosstalk between GR and P53 has long been a dubious topic for quite some time. Many studies verified that GR activation strengthens the transcriptional activity of p53. In a neural cell line HT-22, GR activation by DEX enhances the transcriptional activity of p53, and thus induces apoptosis and cell cycle arrest On the contrary, numerous evidence argue that there is negative cross-talk between GR and p53. Sengupta reported that GR activation resulted in p53 cytoplasmic sequestration, thus inhibiting p53’s transcriptional activity And some other studies have also propounded that there is no relationship between p53 and GR. Dex efficiently suppressed TNF alpha-induced apoptosis through abrogated Bcl-2 reduction, which is independent of p53’s status In this study, we have illustrated that GR activation by dexamethasone up-regulates the expression level of P53, enhances its transcriptional activity, thus resulting in p21, PUMA and NOXA up-regulation. And this subsequently induces osteoblast cell cycle arrest and apoptosis. Furthermore, we verified that disruption of p53 activation by p53 siRNA leads to the decrease in the DEX-induced cytotoxic and apoptotic activities in osteoblast cells.The tumor suppressor p53 provides exquisite decision between cell growth arrest and apoptosis in response to various cellular stress. Sustained stress or irreparable damage trigger p53′s killer functions to initiate transcription of pro-apoptotic genes such as Puma, Noxa, Bax, and Bid. To prevent the unnecessary loss of cells which could cause premature aging, the killer functions of p53 are tightly regulated and balanced against protector functions that arrest cell cycle and support survival in response to low stress or mild damage In conclusion, our findings suggested that GR activation by DEX can up-regulate P53, which then enhances its transcriptional activatity. This mechanism contribute to apoptosis and cell cycle arrest of MC3T3-E1 cells. P53 could be used as a new target for the treatment of GC-induced osteoporosis and osteonecrosis.


Saccharina japonica was fractionated by anion exchange chromatography. The most complex fraction F0.5 was degraded by dilute sulphuric acid and then separated by use of an activated carbon column. Fraction Y1 was fractionated by anion exchange and gel filtration chromatography while Fraction Y2 was fractionated by gel filtration chromatography. The fractions were determined by ESI-MS and analyzed by ESI-CID-MS/MS. It was concluded that F0.5 had a backbone of alternating 4-linked GlcA and 2-linked Man with the first Man residue from the nonreducing end accidentally sulfated at C6. In addition, F0.5 had a 3-linked glucuronan, in accordance with a previous report by NMR. Some other structural characteristics included GlcA 1→3 Man 1→4 GlcA, Man 1→3 GlcA 1→4 GlcA, Fuc 1→4 GlcA and Fuc 1→3 Fuc. Finally, it was shown that fucose was sulfated at C2 or C4 while galactose was sulfated at C2, C4 or C6. A fucoidan extracted from  Fucoidan, which is a family of sulfated heteropolysaccharides extracted from brown algae and invertebrates, has been increasingly studied because of its biological activity, including antitumor activity ,2, proteKjellmaniella crassifolia and investigated by Sakai et al. −  and 474.072 (−2), assigned as B5 and C5, respectively. A series of B-type ions at m/z 175.024 (B1), 417.022 (B2), 377.045 (−2) (B4), 931.131 (B5) along with C-type ions at m/z 193.034 (C1), 773.111 (C4) indicated that the linear sequence was GlcA-ManSO3H-GlcA-Man-GlcA-Man. The ion at m/z 259.011 (ManSO3H) was also confirmed in the above analysis. The ions at m/z 513.102 (B’3), 693.161 (C’4) and 851.173 (B’5) suggested that the sulfate group was lost whereas the ions at m/z 407.055 [2−] and 597.077 (C4-GlcA) suggested that the GlcA residue from the nonreducing end was sensitive in the negative mode. The characteristic ion at m/z 495.079 (−2) was detected, suggesting that the glycosidic bond between the GlcA and Man residues was 1→2 linkage. However, the ion at m/z 326 (−2) did not appear due to its low intensity. Although the linkage of the Man residue was identified as a 2-linked Man, the linkage of the GlcA residue was not determined by ESI-CID-MS/MS. With the assistances of NMR , D-galactose , D-mannose (Man/M), D-glucuronic acid (GlcA), L-rhamnose monohydrate (Rha), D-xylose (Xyl) and D-glucose (Glc)) were purchased from Sigma-Aldrich. 3-Methyl-1-phenyl-2-pyrazolin-5-one (99%) was purchased from Aldrich chemistry. The brown algae et al. [Crude fucoidan was extracted following Wang et al.  and degr−1) for 5 h and then neutralized with barium hydroxide after cooling to room temperature. The solution was centrifugated and the supernate was concentrated. The concentrated solution was fractionated using the activated carbon column (2.6 cm × 30 cm) with a gradient elution from 50% ethanol to 95% ethanol. Then, eluent (Y1) was concentrated and precipitated in 95% ethanol. The activated carbon column was washed with a gradient elution from 50% ethanol to 95% ethanol. The elution (Y2) was combined, concentrated and freeze-dried. Y1 (1.0 g) was separated by anion exchange chromatography on a DEAE-Bio Gel Agarose FF gel (2.6 cm × 30 cm) with elution by water (YF), 0.05 M NaCl (YD) and 0.1 M NaCl (YT). The concentrated solutions of fractions YD and YT were desalted and separated on a Sephadex G-10 column (2.6 cm × 100 cm) with an elution of water. Y2 (0.5 g) was separated on a Bio-Gel P-4 Gel  column (2.6 cm × 100 cm) (4HCO3 at a flow rate 0.14 mL min−1. It was collected every 14 min per pipe after 27 h. Six fractions were collected and lyophilized.Polysaccharide F0.5 was dissolved with reflux in 4% sulphuric acid (60 mg mL 100 cm)  eluted w2CO3 at a flow rate of 0.8 mL min−1 at 45 °C. The molar ratio of monosaccharide composition and the content of fucose were determined following Zhang et al. [−1) was hydrolyzed in 2 M trifluoroacetic acid in a 10 mL ampoule. The ampoule was sealed in a nitrogen atmosphere and hydrolyzed for 4 h at 110 °C. Then the hydrolyzed mixture was neutralized to pH 7 with sodium hydroxide. Later the mixture was converted into its 1-phenyl-3-methyl-5-pyrazolone derivatives and separated by HPLC chromatography. Uronic acid was analyzed by a modified carbazole method [2SO4 at a flow rate of 0.5 mL min−1 at 30 °C.The sulfated content was determined by ion chromatography on Shodex IC SI-52 4E column 4.0 × 250 mm) eluted with 3.6 mM Nag et al. . Briefly0 mm elutESI-MS was performed on a Micromass Q-Tof Ultima instrument .3CN-H2O . Mass spectra were registered in the negative ion mode at a flow rate of 5 μL min−1. The capillary voltage was set to −3000 V, and the cone voltage was set at −50 V. The source temperature was 80 °C, and the desolvation temperature was 150 °C. The collision energy was optimized between 10 and 50 eV. All spectra were analyzed by MassLynx software.Samples were dissolved in CH−1 in a gradient solution. Gradient 1: 0–30 min, water-acetonitrile-ammonium formate buffer : 10:80:10 (v/v/v)→40:50:10(v/v/v). Gradient 2: 30–35 min, water-acetonitrile-ammonium formate buffer : 40:50:10 (v/v/v)→75:15:10(v/v/v).The fractions were analyzed by HPLC with an ELSD detector, performed on a “click” maltose column  at 1 mL minTo further determine the precise structure of F0.5, it was degraded with partial acid hydrolysis (due to the absence of available enzymes). The depolymerized mixture was then separated by use of an activated carbon column. Y1 was purified using anion exchange and gel filtration chromatography while Y2 was purified using gel filtration chromatography. The results suggest that F0.5 consisted of two types of polysaccharides: (1) a glucuronomannan and a sulfated glucuronomannan with the same backbone of repeating 4-linked GlcA and 2-linked Man, and the latter with the first mannopyranose residue from the nonreducing terminus sulfated at C-6 and (2) a glucuronan with a backbone of 3-linked GlcA. There were also some other structural fragments, including GlcA 1→3 Man 1→4 GlcA, Man 1→3 GlcA 1→4 GlcA, Fuc 1→4 GlcA and Fuc 1→3 Fuc. Combined with the results obtained in this study and a previous study , it was


Regional citrate anticoagulation has emerged in critically ill patients to safely treat patients with risk of bleeding. However, liver dysfunction may lead to citrate accumulation and change patients' acid-base status. In 10 years, 100 septic shock patients were treated with CPFA, an extracorporeal therapy that combines unselective plasma adsorption resin (MediaSorb) with continuous hemofiltration. Some patients suffered from liver dysfunction  and we treated them with our citrate protocol. The aim of this study is to evaluated the safety of citrate-like anticoagulation on patients with liver dysfunction.+ 136 mmol/l, citrate 10 mmol/l, citric acid 2 mmol/l) infused to keep inlet citratemia at 3 mmol/l, postdilution solution  and postdilution CaCl2 at a rate restoring the plasma Ca2+ to 1.1 mmol/l  with septic shock and liver dysfunction were treated. Prescribed CPFA parameters were: Qb 150 ml/minute, plasma flow rate (Qp) 30 ml/minute, predilution solution .Twenty-two treatments were performed accounting for 163 hours, mean duration 10.2 ± 2.1 hours, mean plasma volume of 16.4 ± 5.4 l, Qb 150 ml/minute, Qp 28.4 ± 1.8 ml/minute, and a treated plasma dose/kg body weight of 0.8 ± 0.4 l/kg. Mean CaCl2+/iCa2+. We also observed a decrease of bilirubin.In these four patients treated with CPFA and citrate in liver dysfunction, we have observed the absence of alteration: pH, bicarbonate, lactate and Ca


Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM.A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom  Although it is a main storage form of phosphate, phytate cannot be utilized by monogastric animals such as pig, poultry, and humans as they lack phytate-degrading enzymes [Phytic acid . It can catalyze the sequential release of phosphate from phytate [Aspergillus niger and was released to the market in 1991 [The enzyme phytase, chemically known as  phytate , 8. Exte phytate , 10. The in 1991 . Moreove in 1991 , 13.Lentinus edodes, commonly referred to as shiitake mushroom, is a popular edible and medicinal mushroom, which is cultivated and consumed in many Asian countries. Compounds isolated from its fruiting bodies or mycelia, such as lectin, laccase, and polysaccharide, have been demonstrated to show a variety of therapeutic properties, especially antitumor, antivirus, and immunomodulatory activities [L. edodes. The study would supplement the nutritional literature pertaining to this mushroom. tivities –16. The L. edodes were purchased from a local market in Beijing, China. DEAE-cellulose, CM-cellulose, Tris-base, sodium phytate, AMP, ADP, ATP, fructose-6-phosphate (F-6-P), glucose-6-phosphate (G-6-P), and β-glycerophosphate were obtained from Sigma, St. Louis, MO, USA. Affi-gel Blue gel was purchased from Bio-Rad, Richmond, CA, USA. Q-Sepharose, Superdex 75 HR 10/30 column, and molecular mass standards were obtained from GE Healthcare, USA. All other reagents used were of reagent grade from China unless otherwise mentioned.Fresh fruiting bodies of shiitake mushroom μL enzyme solution was incubated with 475 μL of 5 mM sodium phytate in 50 mM Tris-HCl buffer (pH 7.0) at 37°C for 15 min. The enzyme reaction was subsequently terminated by the addition of 500 μL 10% (w/v) trichloroacetic acid. The released phosphate was measured at 700 nm after adding 1000 μL of freshly prepared color reagent, which was composed of 1% (w/v) ammonium molybdate, 3.2% (v/v) sulfuric acid solution, and 7.2% (w/v) ferrous sulfate solution. One unit of phytase activity was defined as the amount of enzyme needed to liberate 1 μmol phosphate per min under the assay conditions. Protein was determined according to Bradford using a protein assay kit  with bovine serum albumin as the standard [Phytase activity was measured using a modified ferrous sulfate-molybdenum blue assay . In briestandard . All det4HCO3 buffer  was added until the molarity of NH4HCO3 attained 10 mM. Ion exchange chromatography on a DEAE-cellulose column (2.5 cm × 30 cm) was conducted in 10 mM NH4HCO3 buffer (pH 9.5). After the removal of unadsorbed materials (fractions D1 and D2), the column was washed with 1 M NaCl in the 10 mM NH4HCO3 buffer (pH 9.5) to remove adsorbed materials. Fraction D2 enriched in phytase activity was dialyzed against 10 mM NH4OAc buffer and then subjected to ion exchange chromatography on a 2.5 cm × 20 cm column of CM cellulose in 10 mM NH4OAc buffer (pH 4.5). The unadsorbed fraction CM1 containing phytase activity was collected before the desorption of inactive adsorbed materials (collected as fraction CM2) with 10 mM NH4OAc buffer (pH 4.5) containing 1 M NaCl. Fraction CM1 was then applied on a 2.5 cm × 20 cm column of Affi-gel Blue gel in 10 mM Tris-HCl buffer (pH 7.5). Unadsorbed fraction with phytase activity (fraction B1) was eluted with the same buffer while adsorbed proteins devoid of phytase activity (fraction B2) were desorbed with 1 M NaCl added to the 10 mM Tris-HCl buffer. Fraction B1 was subsequently chromatographed on a 1.0 cm × 30 cm column of Q-Sepharose in 10 mM NH4HCO3 buffer (pH 9.5). Unadsorbed proteins were eluted into two fractions, Q1 and Q2, while adsorbed proteins were eluted into fraction Q3 with 1 M NaCl in the starting buffer 10 mM NH4HCO3 (pH 9.5). Fraction Q2 with phytase activity was next subjected to gel filtration by fast protein liquid chromatography (FPLC) on a Superdex 75 HR 10/30 column in 0.2 M NH4HCO3 buffer (pH 8.5) using an AKTA Purifier system . The second peak (SU2) represented purified phytase.Fresh fruiting bodies of shiitake mushroom (2000 g) were homogenized and extracted with cold distilled water (4 mL/g) at 4°C for 4 h. Following the centrifugation at 12000 rpm for 15 min, the supernatant obtained was subjected to ultrafiltration until the volume was reduced to 100 mL. NHMr migrate rate curve based on the molecular mass standards used in SDS-PAGE. In FPLC-gel filtration, another curve of elution volume-lgMr was obtained. The molecular mass of purified phytase was calculated using the curve and its elution volume.The molecular mass of the purified phytase was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and FPLC-gel filtration. In SDS-PAGE, a 12% resolving gel and a 5% stacking gel were used, with a procedure as described by Laemmli and Favre . At the The N-terminal sequence of the phytase was determined and carried out using an HP G1000A Edman degradation unit and an HP 1000 HPLC system.A series of sodium phytate solution in buffers with different pH values including 50 mM NaOAc (pH 3.0–5.0), 50 mM Mes (pH 5.0–7.0), and 50 mM Hepes (pH 7.0–9.0) were used to determine the optimal pH value. For determining the optimal temperature, the reaction mixture was incubated at 20°C, 30°C, 37°C, 45°C, 50°C, 60°C, 70°C, 80°C, and 100°C in 50 mM NaOAc (pH 5.0) for 15 min, respectively. In the thermostability assay, enzyme solutions were previously incubated at various temperatures  for various durations , respectively. The residual activity was measured using the standard assay after the enzyme solutions had been cooled down to room temperature. All determinations were performed in triplicate.β-glycerophosphate. The buffer used was 50 mM NaOAc (pH 5.0). The release of Pi was determined as mentioned above.In order to determine the substrate specificity of the purified phytase, several phosphorylated substrates instead of sodium phytate, all at 5 mM concentrations, were added to the assay solution. They included AMP, ADP, ATP, fructose-6-phosphate, glucose-6-phosphate, and μL) of metal ions or EDTA  were mixed with the purified phytase solution in 50 mM NaOAc buffer (pH 5.0) for 2 h at 4°C before the standard phytase assay was performed. The activity assayed in the absence of metal ions was defined as the control. The metal ions tested include K+, Ca2+, Mg2+, Mn2+, Zn2+, Cu2+, Fe3+, and Al3+.Equal volumes  [F. velutipes (14.8 kDa) [Aspergillus fell within the range of 60–120 kDa. Phytases from bacteria such as Bacillus subtilis and Escherichia coli demonstrated molecular masses in the proximity of 40 kDa. On the other hand, plant phytases showed a wide molecular masses range from 60 kDa  to 3699 kDa (characteristic of Lilium longiflorum) [http://blast.ncbi.nlm.nih.gov/Blast.cgi), L. edodes phytase demonstrated no sequence similarity to other phytases. At the same time, it manifested high sequence homology with a nucleoside-diphosphate-sugar epimerase from Synechococcus sp. (YP_001227174.1) and a signal transduction histidine kinase from Kytococcus sedentarius (YP_003150103.1) both of which are phosphorus transportation enzymes.(14 kDa)  and F. v4.8 kDa) . Moleculiflorum) . Based oA. niger. A. niger PhyA exhibited two pH optima at pH 2.5 and pH 5.0, while A. niger PhyB demonstrated only a single pH optimum at pH 2.5 [A. ficcum manifested a pH optimum at pH 1.3, the lowest among all the phytases reported [A. ficcum phytase lost virtually its whole activity when the ambient pH value reached 5.0 [ V. volvacea [F. velutipes [L. edodes, V. volvacea, and F. velutipes) manifested a better pH tolerance than commercial Aspergillus phytases. Although shared similar characteristics in molecular mass and optimal pH value, the three mushroom phytases showed very low N-terminal amino acid sequence similarity and different chromatography behavior.The optimal pH for shiitake mushroom phytase was pH 5.0, with less than 20% variation in activity detected in the pH range of 3.0–9.0. The phytase demonstrates considerable pH adaptability compared with other phytases reported. Two kinds of phytases (PhyA and PhyB) were isolated from t pH 2.5 . Anothervolvacea  and F. velutipes  possesseV. volvacea (45°C) [F. velutipes (45°C) [A. niger PhyA (58°C) [A. niger PhyB (60°C) [A. ficcum PhyB (65°C) [Schwanniomyces castellii (77°C) [L. edodes phytase also demonstrated considerable thermostability with about 60% residual activity at 70°C and 40% residual activity at 95°C. A. ficcum PhyB was relatively stable at 60°C with 14% loss in activity after exposure to the temperature for 60 min, while only about 50% of the highest enzyme activity was retained at 37°C [The purified phytase required a low temperature optimum of 37°C, which was the same as the body temperature in humans, but lower than those of other phytases reported . Most ofa (45°C) , F. velus (45°C) , A. nigeA (58°C) , A. nigeB (60°C) , A. ficcB (65°C) , and Schi (77°C) . L. edod at 37°C . The purA. niger PhyB has been reported to have a wider substrate specificity than A. niger phyA. A. niger phyA showed the highest activity towards sodium phytate, but very low activity towards other substrates such as F-6-P, G-6-P, and ADP. A. niger phyB had an activity toward F-1, 6-P which was 20 times higher than that towards sodium phytate [L. edodes phytase was the highest towards ATP, like phytases from A. ficcum PhyB [V. volvacea [F. velutipes [ρ-nitrophenyl phosphate and F-1, 6-P formed the optimal substrates of A. fumigatus phytase, followed by ATP. On the other hand, alkaline phytases from B. subtilis, B. subtilis natto, B. amyloliquefaciens, and Typa latifolia had narrow substrate specificity and can use sodium phytate as their sole substrate [Phytase from shiitake mushroom exhibited broad substrate specificity on a range of phosphorylated compounds.  phytate . The enzvolvacea , F. veluelutipes , and Speelutipes . ρ-nitroubstrate . 2+ at a concentration of 5 mM, and not significantly affected by K+, Ca2+, Mg2+, Mn2+, Cu2+, Fe3+, and EDTA at a low concentration of 1 mM, but moderately inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a higher concentration of 5 mM. It was previously reported that Ca2+ had a moderately inhibitory effect on phytases from Cladosporium sp. [A. niger [Klebsiella pneumoniae [2+ showed no significant effect on the phytase from A. ficcum [Candida krusei [K. pneumoniae [Bacillus sp. which is strongly inhibited by EDTA at a low concentration of 1 mM [A. ficcum [A. fumigatus [The phytase activity was moderately stimulated by Carium sp. , A. nigeA. niger , and Kleeumoniae . On the a krusei  and K. peumoniae , but dif of 1 mM . On the umigatus .L. edodes phytase displayed some characteristics distinct from those of animal, plant, bacterial, fungal, and mushroom phytases previously reported in the literature. It manifested the advantageous properties of pH tolerance and thermostability. It signifies that the shiitake phytase has a great potential for commercial interest as an animal feed additive or dietary adjuvant.To recapitulate,


The objectives of this study were to synthesize and characterize two types of cytarabine (Ara-C) lipid produgs and evaluate the prodrugs for sustained intraocular delivery after administration by intravitreal injection.Hexadecyloxypropyl cytarabine 5′-monophosphate (HDP-P-Ara-C) and hexadecyloxypropyl cytarabine 3′,5′-cyclic monophosphate (HDP-cP-Ara-C) were synthesized starting from cytarabine (1-β-D-arabinofuranosylcytosine). Their vitreal clearance profile was simulated using a custom dissolution chamber, in vitro cytotoxicity was evaluated using cell proliferation assays, and in vivo ocular properties in rat and rabbit eyes were assessed using biomicroscopy, indirect ophthalmoscopy, tonometry, electroretinography, and histology.HDP-P-Ara-C was cleared from the dissolution chamber (flow rate 2 µL/min) within 7 days. In contrast, HDP-cP-Ara-C, a much more insoluble prodrug, was still detectable 36 days after the dissolution process was started. HDP-P-Ara-C had a 50% cytotoxicity concentration of 52±2.6 μM in human retinal pigment epithelium (ARPE-19) and 32±2.2 µM in a rat Müller cell line, rMC-1. The 50% cytotoxicity concentration values for HDP-cP-Ara-C in ARPE-19 and rMC-1 cells were 50 µM and 25 µM, respectively. HDP-P-Ara-C was not detectable 2 weeks after the highest intravitreal dose (228 µg/rat eye) was injected, and no ocular toxicity was found. With HDP-cP-Ara-C, the drug depot was visible for 26 weeks following a single intravitreal injection (800 µg/rabbit eye). For both compounds, the electroretinogram, intraocular pressure, and other toxicity studies were negative except for the highest dose of HDP-cP-Ara-C (800 µg/eye), which had focal toxicity from the direct touch of the retina and decreased dark adapted a-waves and decreased flicker electroretinogram amplitudes .The cyclic monophosphate prodrug, HDP-cP-Ara-C, was found to have physiochemical properties better suited for sustained delivery of cytarabine to posterior segments of the eye. These properties included limited aqueous solubility, in vitro antiproliferative activity, and good tolerability after injection into rabbit eyes. Proliferative vitreoretinopathy (PVR) is a pathological, exuberant scarring process that is the most common cause of failure after rhegmatogenous retinal detachment surgery -3. The tThe underlying causes of PVR are complex; however, a major component appears to be cell proliferation on the surface and undersurface of the retina after damage to the blood–retinal barrier exposes retinal cells to vitreal growth factors and cytokines . Due to Our efforts to develop long-lasting agents for preventing PVR have focused on synthesizing and evaluating sparingly soluble prodrugs of antiproliferative nucleoside analogs that could be administered by intravitreal injection. For example, we recently described hexadecyloxypropyl 5-fluoro-2'-deoxyuridine cyclic 3′,5′-monophosphate, a lipid prodrug of 5-fluoro-2′-deoxyuridine that stays in the vitreous cavity for months after intravitreal injection and inhibited experimental PVR, presumably by continually releasing minute amounts of the prodrug from its crystalline form into the vitreous .Cytarabine  is another pyrimidine analog that impairs cellular proliferation by inhibiting DNA synthesis. Cytarabine demonstrated better in vitro antiproliferative efficacy on retinal pigment epithelial and fibroblast cells than 5-fluorouracil (5-FU) . However1H NMR) spectra were recorded on Varian HG spectrophotometers operating at 400 MHz and are reported in units of parts per million (ppm) relative to internal tetramethylsilane at 0.00 ppm. Electrospray ionization mass spectra (ESI-MS) were recorded on a Finnigan LCQDECA spectrometer in positive or negative mode. Purity of the compounds was also assessed with thin layer chromatography (TLC) using Analtech silica gel-GF (250 μm) plates and the solvent system: CHCl3/MeOH/con NH4OH/H2O (70:30:3:3 v/v). TLC results were visualized with ultraviolet light, phospray , and charring at 400 °C. Cytarabine  was purchased from Aldrich Chemical Co .All reagents were of commercial quality and used without further purification unless indicated otherwise. Chromatographic purification was done using the flash method with silica gel 60 . Proton nuclear magnetic resonance . The mixture was stirred for 3 days at room temperature. Then the mixture was filtered, and the solvent evaporated. The residue was adsorbed on silica gel and purified with flash column chromatography. Gradient elution CH2Cl2 to 20% EtOH/CH2Cl2 gave HDP-P-Ara-C as a white powder. Yield: 325 mg (38%). 1H NMR (CDCl3/methanol-4d): δ 7.78 ; 6.10 ; 5.85 ; 3.99–3.87 ; 3.50–3.25 ; 3.95–4.05 ; 3.46 ; 3.37 ; 1.80 ; 1.50–1.47 ; 1.27–1.24 ; 0.88 . ESI-MS (m/z) 604 [M-H]-.To a solution of hexadecyloxypropyl-3-phosphate -1-propanol with phosphorous oxychloride as described by Ruiz et al. ) and 1-β1H NMR (CDCl3/methanol-4d): δ 8.22 ; 8.07 ; 7.99 ; 7.25–7.84 ; 6.46 ; 6.03 ; 4.45–4.65 ; 3.90–4.23 ; 0.8–1.20 . ESI-MS (m/z) 694.14 [M+H]+, 716.21 [M+Na]+.Benzoyl chloride  was added dropwise at 0 °C to a solution of 3′,5′-O- cytosine arabinoside  and 4-d4, 2´-O-dibenzoyl-3′,5′-tetraisopropyl disiloxane-1,3-diyl)cytosine arabinoside  in dry methanol (250 ml). The mixture was stirred at room temperature overnight before the solvents were evaporated under vacuum. The residue was purified by column chromatography on silica gel. Eluent: dichloromethane: from 5% to 15% of methanol. Yield: 6.3 g (92%). 1H NMR (CDCl3/methanol-4d): δ 8.49 ; 7.97 ; 7.82 ; 7.58–7.68 ; 7.43–7.58 ; 7.33–7.43 ; 6.45 ; 5.70 ; 4.40–4.42 ; 4.12–4.16 ; 3.85–3.96 ; 3.34–3.36 . ESI-MS (m/z) 452.01 [M+H]+, 474.09 [M+Na]+.Acetic acid (2.9 g) and ammonium fluoride (8.1 g) were added to a mixture of N4, 2’-O-dibenzoyl-cytosine arabinoside  in anhydrous pyridine (100 ml) at 0 °C. The reaction mixture was stirred for 3 h at 0 °C and then quenched by adding water (50 ml). The mixture was concentrated under vacuum, the residue was dissolved in CHCl3 (200 ml) and washed with water (2×20 ml), and the CHCl3 fraction was evaporated. The residue was purified by column chromatography on silica gel. Eluent: ethyl acetate/hexanes (80:20), then ethyl acetate. Yield: 5.4 g (69%). 1H NMR (CDCl3/methanol-4d): δ 8.27 ; 8.05 ; 7.96 ; 7.81; 7.37–7.66 ; 6.49 ; 5.68 ; 4.76–4.85 ; 4.63–4.70 ; 4.45–4.50 ; 4.08–4.15 . ESI-MS (m/z) 555.93 [M+H]+, 578.06 [M+Na]+.A solution of benzoyl chloride  in anhydrous pyridine (25 ml) was added dropwise to a suspension of N4, 2′,5′-tribenzoyl cytosine arabinoside  and hexadecyloxypropyl phosphate  in dry N,N-dimethylformamide (50 ml), N,N-dicyclohexylcarbodiimide  was added. The mixture was stirred for 50 h at 90 °C. The solvent was evaporated, and then the residue was purified by column chromatography on silica gel. Eluent: CH2Cl2 with from 5% to 15% MeOH. Yield: 2.2 g (67%). 1H NMR (CDCl3/methanol-4d): δ 8.27 ; 8.07 ; 7.93 ; 7.83 ; 7.48–7.61 ; 7.35–7.42 ; 6.48 ; 5.90 ; 4.83–4.95 ; 4.72–4.78 ; 4.02–4.08 ; 3.91–3.97 ; 3.49–3.55 ; 3.33–3.44 ; 1.66–1.75 ; 1.46–1.63 ; 1.21–1.39 ; 0.89 . ESI-MS (m/z) 916.32 [M-H]-.To a solution of N4, 2´,5′-O-tribenzoyl-cytosine arabinoside, 3′-monophosphate  was dissolved in dry pyridine (10 ml), and then MeOH/NH3 (7 N solution) (25 ml) was added. The mixture was stirred overnight at room temperature before the solvent was evaporated. The residue was purified by column chromatography on silica gel. Eluent: CH2Cl2: from 5% to 15% of methanol. Yield: 0.66 g (46%). 1H NMR (CDCl3/methanol-4d): δ 7.90 ; 6.19 ; 5.88 ; 4.49–4.51 ; 4.40–4.42 ; 4.13–4.18 ; 3.95–4.05 ; 3.85–3.95 ; 5.56 ; 3.43 ; 1.89–1.95 ; 1.54–1.59 ; 1.21–1.35 ; 0.89 . ESI-MS (m/z) 606.11 [M+H]+, 628.22 [M+Na]+, 644.16 [M+K]+.Hexadecyloxypropyl N4OH:water 80:20:1:1] (100/75%). Yield: 80 mg of equatorial isomer, 80 mg of axial isomer, and 80 mg of mixture these two isomers; total: 240 mg (39%). 1H NMR (CDCl3/methanol-4d): δ 7.36 ; 6.36 ; 5.74 ; 4.69–4.75 ; 4.50–4.55 ; 4.20–4.26 ; 3.95–4.05 ; 3.85–3.95 ; 3.35–3.60 ; 1.69–1.72 ; 1.54–1.59 ; 1.21–1.35 ; 0.89 . ESI-MS (m/z) 588.22 [M+H]+, 620.04 [M+Na]+.A solution of hexadecyloxypropyl cytosine arabinoside 3′-monophosphate  in dry pyridine (250 ml) was added dropwise to a solution of triisopropylbenzenesulfonyl chloride  and 5-methyl-imidazole  in dry pyridine (300 ml). The mixture was stirred for 2 days at room temperature. Solvents were evaporated under vacuum, and the residue was purified by column chromatography on silica gel. Eluent: ethyl acetate/[chloroform:methanol:NHDissolution starting from an intravitreal drug depot was simulated using a custom chamber and a syringe pump. The drug powder (600 µg) was weighed into the chamber, which had 1.5 ml of effective circulation volume to mimic the rabbit vitreous. The chamber was filled with 1.5 ml of Hank’s Balanced Salt Solution (HBSS). The chamber was then connected by tubing to a NE-1000 syringe pump  on one end and open tubing on the other end so that the solution could be collected into a vial . The syr2. All culture media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cell lines were chosen due to their dominant presence in the pathology of proliferative vitreoretinopathy [Human retinal pigment epithelium (ARPE-19) cells were obtained from the American Type Culture Collection . A rat Müller cell line, rMC-1, was provided by Dr. Gabe Silva at UC San Diego . ARPE-19 cells were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 and rMC-1 in DMEM. DMEM/F12 medium and DMEM were maintained at 37 °C in a humidified chamber of 5% COinopathy ,14.−4 cells per well. Serial dilutions of the compounds were added to the wells in a total volume of 100 µl. The plates were maintained in a 37 °C incubator. After 5 days, 50 µl of a solution of XTT  was added to each well, and the plates returned to the incubator for 30 min, after which the ocular density (OD) of each well was measured at a wavelength of 450 nanometers. Percent inhibition was determined by comparing the OD of wells containing compound to the OD of control wells.rMC-1 cells were plated in 96-well plates at 1×10−4 cells per well. Serial dilutions of the compounds were added to the wells in a total volume of 100 µl. The plates were maintained in a 37 °C incubator. After 5 days, 10 µl of a solution of WST-1  was added to each well, and the plates returned to the incubator for 0.5 h, after which the OD450 of each well was measured. Percent inhibition data were obtained from at least three experiments with at least six wells at each concentration in separate 96-well plates. The mean optical density values corresponding to the untreated controls were taken as 100%. The results were expressed as the percentage of the optical density of treated cells relative to that of untreated controls.ARPE-19 cells were plated in 96-well plates at 1×10All animal studies were conducted in adherence with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.Twelve Brown Norway rats were used to evaluate the effect of intravitreal HDP-P-Ara-C versus control injection. HDP-P-Ara-C was intravitreally injected resulting in concentrations of 0.06 mM (2 µg/eye), 0.112 mM (4 µg/eye), 1.12 mM (40 µg/eye), 2.0 mM (73 µg/eye), and 6.32 mM (228 µg/eye) in rat vitreous. Only one eye of each rat was injected with the drug, and the fellow eye was injected with saline as control. Electroretinograms (ERGs) were obtained before drug injection and 1 day before scheduled sacrifice. Following injection, toxicity and safety were evaluated with indirect ophthalmoscopy, biomicroscopy, and fundus photography. At week 2 following drug injections, all of the rats were euthanized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) followed by intraperitoneal injection of 120 mg/kg sodium pentobarbital. Six eyes were used for pathologic evaluation and six eyes used for evaluation of vitreous drug level. Vitreous drug levels were determined at the end of the experiment using high-performance liquid chromatography (HPLC).Sixteen New Zealand Red rabbits were used for this study. Only one eye of each animal was used for the drug testing, and the fellow eye was used as a control by injection of the diluent. Four doses were tested, and each dose was tested in four rabbit eyes after a 50 µl intravitreal injection of the HDP-cP-Ara-C suspension with a resulting dose of 25 µg/eye, 80 µg/eye, 250 µg/eye, and 800 µg/eye. After the intravitreal injection, the rabbits were examined at week 1, 2, 4, 8, and 12 and then once a month for 7 months. The eyes were monitored with slit-lamp exam, tonometry, indirect ophthalmoscope, and fundus camera. The drug depot size was estimated and recorded using the optic disc area and the drug depot contour in the vitreous. The number of disc areas for estimating the two dimensions of the drug depot and the contour  provides an estimate of the density of the drug depot [Intraocular pressure (IOP) measurements and ERG recordings were repeated measurement data. Therefore, generalized estimating equations were used to compare the mean IOP and ERG amplitudes between each treatment group and the control group ,16. All The two lipid prodrugs of cytarabine were successfully synthesized starting from cytarabine. The synthesis procedure is summarized in 50 values were 52 µM in ARPE-19 and 32 µM in rMC-1 for HDP-P-Ara-C, and 50 µM and 25 µM for HDP-cP-Ara-C, respectively . HPLC analysis revealed that no drug was detectable at week 2 following intravitreal injection (including the eyes with the highest dose).In the in vitro study, we noted that HDP-cP-Ara-C was minimally water soluble. We reasoned that the drug depot formed in the vitreous would interfere with fundus exams if we used rat eyes that have a very large lens and very limited vitreous cavity. Rabbit eyes were a better choice for evaluating this formulation. During the 7-month study, two eyes out of the four eyes injected with the 800-µg dose showed direct contact between the drug depot and the optic nerve or inferior retina causing medullary ray gliosis and discoloration of the inferior retina. No other toxicity was noted with the other eyes injected with the 800-µg dose or lower doses. The vitreous drug depot experienced a very slow decrease in size . The depCytarabine is a Food and Drug Administration–approved nucleoside analog used as a chemotherapy agent for hematologic malignancies such as lymphoma, including intraocular lymphoma -19. In aOur approach to sustained PVR therapy involves the intravitreal application of sparingly soluble alkoxyalkyl esters of antiproliferative nucleoside phosphates (or phosphonates) . After i50 value of 25 µM in rMC-1 cells, which was slightly more potent than that previously reported for a similar prodrug, HDP-P-Ara-G , that produced significant inhibition of a laser-induced rat choroidal neovascularization model in a prophylactic study design [In the present study, we synthesized two prodrugs of cytarabine, HDP-P-Ara-C and HDP-cP-Ara-C. Cytotoxicity studies in vitro indicated that both compounds possess more potent antiproliferative activity than unmodified Ara-C, consistent with several earlier studies that show alkoxyalkyl ester prodrugs increase cell membrane permeability and lead to higher intracellular concentrations of active antiviral or antiproliferative metabolites ,28. Notay design .Our simulation of intravitreal clearance using the non-cyclic prodrug HDP-P-Ara-C showed that it was cleared rapidly from the release chamber with all of the drug released by day 6. Compounds of this type are analogs of phospholipids and readily form micelles in water, which probably accounts for the rapid clearance. This behavior was also confirmed in the in vivo study in rat eyes in which no HDP-P-Ara-C was detected in the vitreous 2 weeks after the injection.Since micellization of HDP-P-Ara-C led to rapid clearance, we also evaluated the corresponding cyclic analog HDP-cP-Ara-C. Cyclic monophosphates lack a negatively charged head group and are therefore non-micelle forming and sparingly soluble in water. After HDP-cP-Ara-C was introduced into the release chamber, daily HPLC analysis of the eluate showed a gradual increase in the free drug level. This peaked around 10 days and then decreased thereafter. Low-level sustained release was detected from day 25 to the last time point (day 36) with the drug concentration at about 16 ng/mL in the eluate. This release profile was similar to our previous in vivo rabbit vitreous aspirate study in which a similar formulation of hexadecyloxypropyl ganciclovir monophosphate was studied [Interestingly, in the simulated intravitreal clearance study of HDP-cP-Ara-C, HDP-cP-Ara-C and HDP-P-Ara-C were detected in the eluate with HPLC analysis, indicating that slow hydrolysis of HDP-cP-Ara-C to the more soluble form HDP-P-Ara-C was occurring in PBS during the experiment. These clearance studies are meant to simulate the turnover of water in living rabbit eyes. Thus, the observed release kinetics are relevant to the behavior of drug in a living eye. Both forms of the drug accumulate in retinal tissue  and are In the in vivo study, the HDP-cP-Ara-C drug depots at various doses were observed at the time of euthanasia, and estimation of the vitreal clearance of the drug indicated that the drug depot would be visible for 3 months after 80 or 250 µg/eye doses and even longer with the higher doses.Hexadecyloxypropyl cytarabine 5’-monophosphate was synthesized from cytarabine and found to be unsuitable for providing slow release after intravitreal injection. However, the corresponding 3’, 5’-cyclic monophosphate analog was sparingly water soluble, active against proliferating ocular cells, and thus appeared suitable for evaluation as a long-lasting intravitreal treatment. The in vivo safety of HDP-cP-Ara-C was evaluated, and doses up to 250 µg/eye were well tolerated. In addition to HDP-cP-Ara-C, which possesses a hexadecyloxy propyl ester, we hypothesize that varying the length of the alkoxyalkyl moiety to optimize the water solubility/clearance rate of this compound might improve its efficacy. We recognize that cataract is a possible complication of systemic use of cytarabine and its derivatives. Therefore, in future studies, lens toxicity needs to be closely monitored upon adjusting the length of the alkoxyalkyl moiety to optimize water solubility of the compound. In addition, a pharmacokinetic study of cytarabine delivery to the retina is imperative for optimizing this compound for preventing and treating PVR.


Several studies have shown a high frequency of vitamin D deficiency among HIV patients. Several studies have ascribed these low levels of vitamin D to antiretroviral treatment, especially efavirenz. Tenofovir has been related to changes of bone mineralization in SIV-infected rhesus monkeys and with loss of bone mineral density in children. Adequate vitamin D stores have the potential of an improvement of immune status, lessening of cardiovascular risk and beneficial effects on certain neoplastic disorders.Cross-sectional study of 94 adult HIV outpatients in Leganés  performed in 2008. Risk factors for vitamin D deficiency (< 20 µg/L) were examined using logistic regression.Median age was 44 years (IQR 40 to 48); 69.1% were males, 93,6% whites, 6.4% black race. Mean CD4+ cell count was 446 cells/µL (IQR 312 to 586). Viral load was below 50 copies/mL in 78.7%. Median 25(OH)D level was 17.7 µg/L (IQR 11.9 TO 24.3). 87.2% of patients had 25(OH)D < 30 µg/L , 57.4% had 25(OH)D < 20 µg/L (deficient ) and 19.1% < 10 µg/L (severely deficient). Factors associated with low levels of 25(OH)D were heterosexual vs. IVDU HIV-risk group , season , age >45 vs < 45 years , CD4+ cells nadir <200 vs >200 cells/µL , and tenofovir vs. abacavir therapy . Black race is underrepresented to draw conclusions. In this sample, no association of low 25(OH)D with efavirenz was found.Despite low latitude, low levels of vitamin D are almost universal in our sample of HIV outpatients with satisfactory immunologic and virologic response to ART. Increasing age, less insolation season, heterosexual risk group, and CD4+ nadir were associated with lower levels. Tenofovir use was associated with lower levels of 25(OH)D. Further studies on causality of this association and the need of control bone-mineral density in tenofovir-treated patients seems warranted.