GSTP1 DNA methylation and expression status is indicative of 5-aza-2'-deoxycytidine efficacy in human prostate cancer cells

DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi) have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) either with a single high dose (5-20 μM), every alternate day (0.1-10 μM) or daily (0.005-2.5 μM). A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 μM or greater (p<0.0001) and induction of cell death from 0.5 μM (p<0.0001). In contrast, treatment with a single high dose of 20 μM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥ 0.05 μM). Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥ 0.5 μM) associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥ 50 μM) to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy.     

The mutagenicity of 2-amino-N6-hydroxyadenine to L5178Y tk +/- 3.7.2C mouse lymphoma cells: measurement of mutations to ouabain, 6-thioguanine and trifluorothymidine resistance, and the induction of micronuclei

2-Amino-N6-hydroxyadenine (AHA) was tested in the mouse lymphoma L5178Y tk +/- assay using the microtitre cloning technique over concentrations from 0.005 micrograms/ml-1 (100% viability) to 6 micrograms/ml (10% viability) as measured by cloning efficiency immediately after treatment. At low, non-toxic concentrations (0.005-0.25 micrograms/ml) a dose-related linear increase in the frequency of ouabain-resistant mutants was seen, in addition to an increase in 6-thioguanine- and trifluorothymidine-resistant mutants. No consistent induction of micronucleated cells was observed in this concentration range. Toxic concentrations (20-90% kill) induced a dose-related increase in micronuclei, while the frequency of ouabain-resistant mutants fell (although it was still highly significantly above the control value). These results suggest that the mechanism of action of AHA depends on the concentration, with point mutations being induced at low, non-toxic doses and detectable chromosome breakage occurring only at higher doses. Both large-colony and small-colony trifluorothymidine-resistant mutants were induced at all concentrations. The utility of using multiple genetic end-points in one cell line and the importance of dose range selection for risk assessment and an understanding of the mode of action of test substances is underlined.     

DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size,dose, route of injection and duration of protection-early protection correlates with Mx expression

Rainbow trout of different sizes (10 and 100g) were injected intramuscularly (i.m.) or intraperitoneally (i.p.) with different doses (range 10 ng-10 microg) of a viral haemorrhagic septicaemia (VHS)-DNA vaccine (pcDNA3vhsG). As controls, fish were injected with the pcDNA3 plasmid alone, or with inactivated VHS virus. Fish were challenged at different times post-vaccination (p.v.) to assess protection. At certain times p.v., serum samples were analysed for neutralising antibody and liver tissue was analysed for Mx mRNA expression. A DNA dose of 0.5 microg injected by the i.m. route induced protection in fish of all sizes in challenges performed either 1 or 4 weeks p.v. This dose also conferred effective protection up to 9 months p.v. in fish >100 g. With lower doses of DNA (0.1 and 0.01 microg) and challenge at 4 weeks p.v., 10 g fish were partially protected but protection was not observed in 100 g fish. Vaccination by the i.p. route induced no or lower levels of protection compared with the i.m. route. Fish vaccinated with 0.5 microg DNA i.m. had no detectable serum neutralising antibody (NAb) at 4 weeks p.v. (with the exception of a single 10 g fish) but antibody was detected at 8 weeks and 6 months p.v. but not at 9 months p.v. However, cohorts of these fish showed effective protection at all timepoints. Lack of detectable levels of NAb (at 9 weeks p.v.) despite partial protection in challenge at 4 weeks p.v. was also observed with 0.01 microg doses of DNA i.m. NAb was detected in sera of fish at 8 weeks after vaccination with 0.1 microg i.m. but not in fish vaccinated with doses of 0.01-0.5 microg i.p. Early protection (1 week p.v.) correlated with elevated Mx gene expression.     

Effect of Type of Carbohydrate on Growth and Protein Synthesis by Tetrahymena pyriformis

SYNOPSIS. In chemically defined media at carbohydrate concentrations ≧ 0.5% (w/v) Tetrahymena pyriformis W multiplied more rapidly, developed larger cells, and achieved greater growth as measured by optical density when carbohydrate was provided as dextrin rather than glucose. In media containing 0.3 mg/ml of amino acid nitrogen, growth increased with glucose concentration from 0.1 to 1%, did not change significantly to 3%, and was sharply inhibited at higher glucose levels. With dextrin, maximum growth paralleled carbohydrate concentration from 0.1 to 3%. At higher N levels the inhibitory concentration of glucose was lowered, but growth in dextrin media was not affected except at N concentrations that were inhibitory independent of carbohydrate source. At 1% carbohydrate levels, total cell protein per ml of culture was 60% greater, protein per cell approximately 50% greater, and cells were 1.5 to 2 times larger in media with dextrin than with glucose. Comparable differences in protein synthesis were observed at 2% carbohydrate levels and efficiency of conversion of substrate-N to protein-N was greater in the medium with dextrin than glucose.
Growth as measured by optical density in media with 0.3 mg/ml of N and 1 or 2% (w/v) of dextrin was not significantly reduced by the simultaneous presence of 1 or 2% glucose. This observation appeared to negate osmotic pressure as an explanation of reduced growth in the presence of glucose. At higher osmolar concentrations osmotic pressure appeared to be a major determinant of overall growth but not of cell size.     

石牌广藿香原生质体的分离及培养研究

以石牌广藿香悬浮细胞为材料,对影响其原生质体分离和培养的酶浓度、作用时间、溶液渗透压和材料的生理状态等因素进行了研究。结果表明:以0.5%的果胶酶、0.2%离析酶和0.8%的纤维素酶组合处理继代培养3～11d的悬浮细胞8h,渗透压调节剂为9%甘露醇,原生质体产量达1.65×106 protoplasts·mL-1 PCV,活力超过86%。在原生质体的液体浅层培养中,细胞分裂频率为13.5%。     

碳源对粉核油球藻生长和脂肪酸组成特性的影响

研究了不同碳源类型（CO2、NaHCO3和葡萄糖）及其浓度对粉核油球藻（Pinguiococcus pyrenoidosus CCMP 2078）生长及脂肪酸组成的影响。结果表明：（1）培养液中适量添加碳源促进了粉核油球藻的生长,三种碳源的适宜添加浓度分别是0.5% CO2,5mmol/L NaHCO3和20g/L葡萄糖,对数生长末期的细胞密度分别是对照的3.10倍、1.47倍和2.78倍;（2）除了低浓度葡萄糖外,其他碳源类型和浓度均降低了TPUFA和EPA占总脂肪酸的比例,提高了TSFA的比例,胞内EPA和TSFA含量均下降;（3）低浓度碳源提高了TSFA和EPA产量。通入0.5% CO2培养的EPA和TSFA产量分别是对照的2.30倍和2.69倍,5mmol/L NaHCO3培养的TSFA产量是对照的1.85倍,5g/L和10g/L葡萄糖培养的EPA和TSFA产量最高分别可达对照的2.11倍和1.58倍。因此,通入低浓度CO2最有利于粉核油球藻的生长以及EPA和饱和脂肪酸的生产,EPA和饱和脂肪酸含量的提高主要是通过生物量的增大来实现的。     

Effect of spermatozoal concentration and number on fertility of frozen equine semen

Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy rates. Several ejaculates from each of 5 stallions were frozen in a skim milk-egg yolk based freezing medium at 2 spermatozoal concentrations in 0.5-mL polyvinyl-chloride straws. Half of each ejaculate was frozen at 400 x 10(6) cells/mL and half at 1,600 x 10(6) cells/mL. Insemination doses were based on post-thaw spermatozoal motility and contained approximately 320 x 10(6) (320 to 400) motile spermatozoa or approximately 800 x 10(6) (800 to 900) motile spermatozoa. Sixty-three mares were assigned to 1 of 4 spermatozoal treatments (1--low spermatozoal number, low concentration; 2--low spermatozoal number, high concentration; 3--high spermatozoal number, low concentration; 4--high spermatozoal number, high concentration) and were inseminated daily. Post-thaw spermatozoal motility was similar for cells frozen at both spermatozoal concentrations (P > 0.1). One-cycle pregnancy rates were 15, 40, 28 and 33%, respectively, for Treatments 1, 2, 3 and 4. Packaging spermatozoa at the high concentration tended to increase pregnancy rates vs packaging at the low concentration (37 vs 22%; P = 0.095). Furthermore, when the lower spermatozoal number was used, there tended (P < 0.1) to be a higher pregnancy rate if spermatozoa were packaged at the higher concentration. There was no increase in pregnancy rates when higher numbers of motile spermatozoa were inseminated (27 vs 31%; P > 0.1). Based on these results, a single 0.5-mL straw dose containing 800 x 10(6) spermatozoa should be used and each insemination dose should contain approximately 320 x 10(6) motile spermatozoa. Fertility trials utilizing other freezing extenders are necessary before recommending a single 0.5-mL insemination dose for all freezing extenders.     

Investigation of biomass concentration,lipid production,and cellulose content in Chlorella vulgaris cultures using response surface methodology

The microalgae Chlorella vulgaris produce lipids that after extraction from cells can be converted into biodiesel. However, these lipids cannot be efficiently extracted from cells due to the presence of the microalgae cell wall, which acts as a barrier for lipid removal when traditional extraction methods are employed. Therefore, a microalgae system with high lipid productivity and thinner cell walls could be more suitable for lipid production from microalgae. This study addresses the effect of culture conditions, specifically carbon dioxide and sodium nitrate concentrations, on biomass concentration and the ratio of lipid productivity/cellulose content. Optimization of culture conditions was done by response surface methodology. The empirical model for biomass concentration (R² = 96.0%) led to a predicted maximum of 1123.2 mg dw L^?1 when carbon dioxide and sodium nitrate concentrations were 2.33% (v/v) and 5.77 mM, respectively. For lipid productivity/cellulose content ratio (R² = 95.2%) the maximum predicted value was 0.46 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 when carbon dioxide concentration was 4.02% (v/v) and sodium nitrate concentration was 3.21 mM. A common optimum point for both variables (biomass concentration and lipid productivity/cellulose content ratio) was also found, predicting a biomass concentration of 1119.7 mg dw L^?1 and lipid productivity/cellulose content ratio of 0.44 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 for culture conditions of 3.77% (v/v) carbon dioxide and 4.01 mM sodium nitrate. The models were experimentally validated and results supported their accuracy. This study shows that it is possible to improve lipid productivity/cellulose content by manipulation of culture conditions, which may be applicable to any scale of bioreactors. Biotechnol. Bioeng. 2013; 110: 2114–2122. © 2013 Wiley Periodicals, Inc.     

Cytotoxicity of tin on human peripheral lymphocytes in vitro.

The comparative effects of inorganic and organic tin compounds on chromosomes were assessed in human peripheral blood lymphocytes of healthy donors 20-40 years of age. The endpoints observed were chromosomal abnormalities, sister-chromatid exchanges (SCEs) and cell cycle kinetics. The maximum concentrations which reduced the replicative index by about 50%, of stannic chloride and trimethyltin chloride were 40 micrograms and 2 micrograms per culture respectively. The tested doses were 20 micrograms and 10 micrograms of stannic chloride and 1 microgram and 0.5 microgram of trimethyltin chloride. Both doses of stannic chloride induced a much higher frequency of chromosomal abnormalities (P less than 0.05-P less than 0.001) and a greater reduction of cell cycle kinetics than the corresponding relative doses of trimethyltin chloride. The frequencies of SCEs/cell induced by the latter were, however, slightly higher than those induced by the former.     

Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica

The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.     

Determination of tacrolimus in rabbit aqueous humor by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of tacrolimus (FK506) in rabbit aqueous humor. After a simple protein-precipitation by methanol, the post-treatment samples were separated on a reversed-phase, Thermo-Hypersil-BDS-C18 column with a mobile phase of a mixture of 0.1% formic acid in water, methanol and acetonitrile (5:85:10, v/v/v). Tacrolimus and ritonavir (internal standard, IS) were all detected by the selected reaction-monitoring (SRM) mode. The method developed was validated in rabbit aqueous humor with a daily working range of 0.5-100 ng/ml with correlation coefficient, r>0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision, possible matrix effect and stability. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of tacrolimus in rabbit aqueous humor.     

Effect of low doses of ionizing irradiation on viability of cortical thymocytes

The cortical thymocytes of rats in whole organism, isolated lobes of thymus and cells suspension were exposed to ionizing radiation in a wide range of doses (0.1-200 cGy). In contrast to relatively high dose radiation (50-200 cGy), exposure to doses of 10 cGy resulted in cell death without DNA degradation. The level of doses lower than 10 cGy (0.5-5 cGy) induced thymocyte death which is independent of DNA degradation, RNA and protein synthesis. With decrease in radiation dose, the increase of latent period preceding cell death took place.     

High cell density perfusion culture of mouse-human hybridomas

Summary Five mouse-human hybridomas, H2, H3, V1, V2 and V6 cells secreting anti-virus human monoclonal immunoglobulin G (IgG) were cultured in serum-free media at high density in a settling perfusion culture vessel with an inner cell sedimentation zone. The H2, H3 and V6 cells reached a density of 10⁷ cells/ml in 0.5% (w/v) BSA-ITES-eRDF (see Materials and methods). The H2 cells reached only 6.8 × 10⁶ cells/ml in the absence of bovine serum albumin (BSA), but the addition of 0.2% (w/v) Pluronic F68 increased the maximum cell density to 1.1 × 10⁷ cells/ml, which was the same level as in BSA including medium. On the other hand, Pluronic F68 showed no stimulative effect on the growth of H2 cells in static culture. Pluronic F68 also increased the maximum cell density of V2 cells from 4.6 × 10⁶ cells/ml to 6.9 × 10⁶ cells/ml even in the presence of 0.5% (w/v) BSA.     

Relationship between radiation induced adaptive response in human fibroblasts and changes in chromatin conformation

Chromatin conformation changes in the normal human fibroblasts VH-10 were studied by the method of anomalous viscosity time dependence (AVTD). Gamma-irradiation of cells in a dose range of 0.1–3 Gy caused an increase in maximal viscosity of cell lysates. Conversely, irradiation of cells with low doses of 0.5 or 2 cGy resulted in a decrease in the AVTD peaks with a maximum effect approximately 40 min after irradiation. The same exposure conditions were used to study a possible adaptive effect of low doses, measured by changes in cell survival. A primary dose of 2 cGy caused significant modification of cell response to a challenge dose. Approximately 20% protection to challenge doses of 0.5 Gy (p < 0.003), 2 Gy (p < 0.02) and 2.5 Gy (p < 0.002) was observed. However, the direction of this effect (adaptation or synergism) was found to be dependent on a challenge dose. The combined effect of 2 cGy and 1 Gy was significantly synergistic, while no modification was observed for 1.5 Gy and 3 Gy. A partial correlation was found between the AVTD changes and cell survival when the combined effect of a primary dose of 2 cGy and challenge dose was examined. The dose of 2 cGy alone increased survival by 16% (p < 0.0003). These results suggest that the low-dose induced effects on survival may be related to chromatin reorganization.     

The effect of vasoactive intestinal polypeptide (VIP) on the canine gallbladder motility.

1. VIP at doses of 10(-9) to 10(-8) M was ineffective and at doses of 5 x 10(-8) to 10(-7) M exerted a slight inhibitory effect on the tone of the canine gallbladder muscle strip. However, VIP (0.1-1 micrograms/kg) injected intravenously (i.v.) in conscious dogs dose-dependently decreased the gallbladder pressure. 2. VIP did not influence significantly the acetylcholine (ACh)- or carbachol- induced contractions of canine gallbladder under in vitro or in vivo conditions, but it decreased the electrically-induced, atropine-sensitive contractions of gallbladder muscle strips. 3. VIP (5 x 10(-9) to 5 x 10(-8) M) did not influence significantly the dose-response curve for cholecystokinin octapeptide (CCK OP) of canine and guinea-pig gallbladder muscle strips. VIP injected i.v. (0.1-0.5 micrograms/kg) in conscious dogs greatly decreased the CCK OP-induced gallbladder pressure.     

High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600mg/l while residual methanol level was maintained below 2g/l. Clarification of culture supernatant through a 0.1μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5×10(8)IU/mg. The optimised process has a great potential for large scale production of fully functional hIFNα2b.     

Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris

The methylotrophic yeast Pichia pastoris is a powerful system for production of recombinant proteins, showing high ability to secrete properly folded proteins. A major plus is the strong AOX1 promoter highly induced by methanol. During growth on methanol, however, oxygen readily becomes limiting. In oxygen-limited cultivations of recombinant Pichia pastoris, the methanol concentration had a strong impact on the production of a single-chain antibody fragment (scFv). High methanol concentrations were required to compensate the lack of oxygen and fully induce recombinant protein production, at the same time reducing gratuitous biomass formation due to a lower biomass yield. Product concentrations of 60, 150, and 350 mg/L were obtained with methanol concentrations of 0.3, 1, and 3% (v/v). Moreover, accumulation of a putative product fragment that cannot be removed during affinity purification was prevented at high methanol concentrations. Cell vitality after 100 h was maintained above 98% and 96% of the culture with 0.3% and 3% methanol, respectively. In cultivations supplemented with oxygen, in contrast, methanol concentration between 0.3% and 3% did not influence the product yield of 300-400 mg/L. Thus, efficient recombinant protein production under oxygen-limitation seems to require high methanol concentrations, enabling product concentration as high as otherwise obtained only with expensive supply of pure oxygen.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.