Later Stages Of Culture Research Articles

Death and transfiguration in static Staphylococcus epidermidis cultures.

The overwhelming majority of bacteria live in slime embedded microbial communities termed biofilms, which are typically adherent to a surface. However, when several Staphylococcus epidermidis strains were cultivated in static liquid cultures, macroscopic aggregates were seen floating within the broth and also sedimented at the test tube bottom. Light- and electron microscopy revealed that early-stage aggregates consisted of bacteria and extracellular matrix, organized in sheet-like structures. Perpendicular under the sheets hung a network of periodically arranged, bacteria-associated strands. During the extended cultivation, the strands of a subpopulation of aggregates developed into cross-connected wall-like structures, in which aligned bacteria formed the walls. The resulting architecture had a compartmentalized appearance. In late-stage cultures, the wall-associated bacteria disintegrated so that, henceforth, the walls were made of the coalescing remnants of lysed bacteria, while the compartment-like organization remained intact. At the same time, the majority of strand-containing aggregates with associated culturable bacteria continued to exist. These observations indicate that some strains of Staphylococcus epidermidis are able to build highly sophisticated structures, in which a subpopulation undergoes cell lysis, presumably to provide continued access to nutrients in a nutrient-limited environment, whilst maintaining structural integrity.

In vitro biosafety profile evaluation of multipotent mesenchymal stem cells derived from the bone marrow of sarcoma patients

BackgroundIn osteosarcoma (OS) and most Ewing sarcoma (EWS) patients, the primary tumor originates in the bone. Although tumor resection surgery is commonly used to treat these diseases, it frequently leaves massive bone defects that are particularly difficult to be treated. Due to the therapeutic potential of mesenchymal stem cells (MSCs), OS and EWS patients could benefit from an autologous MSCs-based bone reconstruction. However, safety concerns regarding the in vitro expansion of bone marrow-derived MSCs have been raised. To investigate the possible oncogenic potential of MSCs from OS or EWS patients (MSC-SAR) after expansion, this study focused on a biosafety assessment of MSC-SAR obtained after short- and long-term cultivation compared with MSCs from healthy donors (MSC-CTRL).MethodsWe initially characterized the morphology, immunophenotype, and differentiation multipotency of isolated MSC-SAR. MSC-SAR and MSC-CTRL were subsequently expanded under identical culture conditions. Cells at the early (P3/P4) and late (P10) passages were collected for the in vitro analyses including: sequencing of genes frequently mutated in OS and EWS, evaluation of telomerase activity, assessment of the gene expression profile and activity of major cancer pathways, cytogenetic analysis on synchronous MSCs, and molecular karyotyping using a comparative genomic hybridization (CGH) array.ResultsMSC-SAR displayed comparable morphology, immunophenotype, proliferation rate, differentiation potential, and telomerase activity to MSC-CTRL. Both cell types displayed signs of senescence in the late stages of culture with no relevant changes in cancer gene expression. However, cytogenetic analysis detected chromosomal anomalies in the early and late stages of MSC-SAR and MSC-CTRL after culture.ConclusionsOur results demonstrated that the in vitro expansion of MSCs does not influence or favor malignant transformation since MSC-SAR were not more prone than MSC-CTRL to deleterious changes during culture. However, the presence of chromosomal aberrations supports rigorous phenotypic, functional and genetic evaluation of the biosafety of MSCs, which is important for clinical applications.

Nontypeable Haemophilus influenzae inhibits autolysis and fratricide of Streptococcus pneumoniae in vitro

Streptococcus pneumoniae (SP) and nontypeable Haemophilus influenzae (NTHi) are common commensals of the human airway and major bacterial pathogens of otitis media (OM) and other upper airway infections. The interaction between them may play an important role in the pathogenesis of polymicrobial infections. Although previous studies suggested NTHi could promote pneumococcal survival and biofilm formation, how NTHi affects pneumococcal activities has not been defined. Our data in the present studies indicated that the outcome of the interaction between SP and NTHi was in a cell-density-dependent manner and the enhancement of pneumococcal survival happened at the later stages of culturing. Using quantitative PCR, we found that the expression of pneumococcal genes regulating autolysis and fratricide, lytA and cbpD, were significantly down-regulated in co-culture with NTHi. We further observed that influence of NTHi was not on direct cell-to-cell contact, but that this contact may contribute to the interaction between these two microorganisms. These results suggest that pneumococcal survival and biofilm formation can be enhanced by down-regulating pneumococcal cell wall hydrolase production thereby inhibiting pneumococcal autolysis and fratricide in the presence of NTHi.

Coupled affinity and sizing chromatography: A novel in-process analytical tool to measure titer and trend Fc-protein aggregation

The expanding use of monoclonal antibodies in the biopharmaceuticals industry has brought the need for new analytical tools. We have developed a coupled affinity and gel-filtration high-performance liquid chromatography method to simultaneously analyze titer and quality of monoclonal antibodies. Before this assay, available analytical methods for protein aggregation required highly purified proteins. This assay can qualitatively describe a protein from a clarified cell culture solution by trending protein aggregation over time while measuring protein titer. It can be used to assess proteins in both early- and late-stage culture due to its dynamic range and sensitivity. This assay is a sensitive technique that overcomes the time limitations of previous approaches. It provides an essential tool to accomplish process optimization.

The Occurrence of Soft Rot (Janthinobacterium Agaricidamnosum) In Mushroom (Agaricus Bisporus) Crops

Abstract This study evaluated the development of soft rot disease (Janthinobacterium agaricidamnosum) in Agaricus bisporus crops. Symptoms of the disease appeared at the end of the first break of the mushroom’s harvest and mostly occurred in the later stages of cultivation (in the second break and between the 1st and 2nd crop). Distribution of lesions in growing rooms indicated that employees served as a vector of disease infection, which was spread when harvesting the mushrooms. An evaluation of the post-harvest mushrooms showed that fruiting bodies, especially those stored in closed containers, showed disease symptoms when infected at harvest.

Molecular characterization of AfuFleA, an l-fucose-specific lectin from Aspergillus fumigatus

Aspergillus fumigatus causes life-threatening infections in immunocompromised patients. We have found that extracts of mycelial mats of A. fumigatus contain a potent hemagglutinin. To clarify the characteristics of this factor, the hemagglutinin was purified from late-stage cultures and characterized at the molecular level. The hemagglutinin is a 32-kilodalton protein that shows activity as an L-fucose lectin. The gene encoding this protein, AfufleA, was identified from a genomic DNA library utilizing consensus primers designed for amino acid sequences obtained from peptides following limited trypsin proteolysis. An open reading frame was found that consists of 942 nucleotides encoding 314 amino acids with a deduced molecular mass of 34,498 and contains all seven of trypsin-digested peptide sequences; four short introns, 49-63bp, were also identified. AfufleA shares homology with a fucose-specific lectin produced by the orange peel mushroom, Aleuria aurantia. The role of AfufleA fucose-specific lectin is not clear, but this lectin may enhance attachment of fungal spores to mammalian cell membranes and contribute to the pathogenicity of A. fumigatus.

A Possible Role for Wnt3a in Enhancing StemRegenin1-Mediated Expansion of Hematopoietic Stem Cells

AbstractAbstract 4130Insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT) may be improved by the administration of ex vivo expanded UCB-derived hematopoietic stem cells (HSC). Although culturing HSC with cytokines such as SCF, Flt3L and TPO results in robust proliferation, it is accompanied with extensive differentiation and loss of self renewal capacity. Inhibition of the Aryl hydrocarbon Receptor (AhR) leads to potent expansion of hematopoietic progenitor cells (HPC) (Boitano et al. Science 2010). Reportedly, Wnt3a inhibits differentiation in different types of stem cells including hematopoietic stem cells(Reya et al. Nature 2003, Ten Berge et al. Nature Cell Biology 2011). Here, we evaluated possible additive or synergistic effects of combining the AhR antagonist StemRegenin1 (SR1) with recombinant purified Wnt3a to expand hematopoietic stem cells ex vivo.UCB-derived CD34-selected cells were cultured in serum-free Glycostem Basal Growth Medium (GBGM) supplemented with the early-acting growth factors SCF, Flt3L and TPO (SFT medium) with or without the addition of SR1 and Wnt3a. Cell number, viability and subset composition within the CD34+ cells were measured using flowcytometry. The multilineage differentiation potential and self renewal capacity of expanded CD34+HPC were evaluated in stroma-supported long-term culture-initiating cell (LTC-IC) assays.Culturing CD34+ cells in SFT medium resulted in a mean 10.2-fold increase in CD34+ cells after 1 week of culture (n=3). Addition of SR1 to the SFT-medium resulted in a 16-fold increase of CD34+ input cells within 7 days, while on the other hand, addition of Wnt3a alone to the SFT-medium resulted in a 7-fold increase in CD34+ cells. However, combining SR1 and Wnt3a in the SFT medium resulted in a 20-fold expansion of CD34+ cells compared to input. The early additive effect of Wnt3a on SFT+SR1-induced expansion of CD34+ cells, however, disappeared upon prolonged culture up to 2–3 weeks.Approximately 3–10% of UCB-derived CD34+ cells could be characterized as phenotypic HSC, as was defined by CD34+CD38lowCD45RAlowCD90+ cells. After culture, we sorted different CD34+-populations to evaluate their functional capacity. Evaluation of LTC-IC frequencies yielded a frequency of 1/23 LTC-IC in the phenotypic HSC-subset (CD34+CD38lowCD45RAlowCD90+) after 28 days of culture in SFT medium with SR1. However, no LTC-IC appeared to be present in the multipotent progenitor subset (MPP, CD34+CD38lowCD45RAlowCD90low). These results indicate that phenotypic HSC maintain their functional LTC-IC capacity after expansion culture.Collectively, our results confirm that SR1 expands HSC with preservation of self-renewal capacity and ability to differentiate into various hematopoietic lineages. In addition, we show that Wnt3a initially enhances SFT+SR1-driven expansion of CD34+ HPC, but reduces the increase in number of CD34+ cells at later stages of culture. These data may suggest that the period of expansion needed for clinical application may be shortened by combining SR1 and Wnt3a. Disclosures:Spanholtz:Glycostem Therapeutics: Employment. Groenewegen:Glycostem Therapeutics: Equity Ownership.

On metabolic shift to lactate consumption in fed-batch culture of mammalian cells

Fedbatch culture is the prevalent cell cultivation method in producing protein therapeutics. A metabolic shift to lactate consumption in late stage of cultivation has been shown to extend the culture viability and increase product concentrations. To better understand the factors, which trigger metabolic shift we performed transcriptome and metabolic flux analysis on a fedbatch culture of mouse myeloma cell line (NS0) and developed a mechanistic kinetic model for energy metabolism. Experimental observation indicates that the shift to lactate consumption occurs upon the cessation of rapid growth and under conditions of low glycolysis flux and high extracellular lactate concentrations. Although the transition is accompanied by a general down regulation of enzymes in energy metabolism, that alone was insufficient to elicit a metabolic shift. High lactate level has been reported to exert an inhibitory effect on glycolysis enzyme phosphofructokinase; model simulation suggests that a high lactate level can contribute to reduced glycolytic flux as well as providing a driving force for its conversion to pyruvate. The transcriptome data indicate that moderate alteration in the transcript levels of AKT1 and P53 signaling pathways genes occurs in the late stage of culture. These signaling pathways are known to regulate glycolytic activity. Model simulations further suggest that AKT1 signaling plays a key role in facilitating lactate consumption. Collectively, our results strongly suggest that lactate consumption in fedbatch culture is an outcome of reduced glycolysis flux, which is a product of lactate inhibition and regulatory action of signaling pathway caused by reduced growth rate.

Analysis of biosynthetic fluctuations of cultured Taxus seedlings using a metabolomic approach

Fluctuations in the biosynthesis of taxoids in 1–5 year old cultured seedlings of Taxus chinensis var. mairei were investigated using LC–IT-TOF-MS and a metabolomics approach. In the total ion chromatogram (TIC) of the extracts, 16 prominent peaks were observed. Ten compounds were identified by comparison of retention times and MS/MS spectra with those of reference compounds. An additional 6 taxoids were isolated by preparative HPLC and identified by comparison of their spectroscopic data with those reported in the literature. It was clarified that the relative concentrations of taxoids with 4(20) double bonds are high at early stages of cultivation. On the other hand, relatively higher amounts of 5-acetoxy taxoids oxidized at the 4- and 10- positions and taxoids having 5(20)-oxetane rings were found at later stages of cultivation. This approach provides practical information on the biosynthetic flow of taxoids in cultured yew seedlings.

A novel biocatalytic approach to acetylation of 1-β-d-arabinofuranosylcytosine by Aspergillus oryzae whole cell in organic solvents

Biocatalytic acylation of 1-β-D-arabinofuranosylcytosine (ara-C) was developed using whole cell of Aspergillus oryzae as a novel catalyst. (13)C nuclear magnetic resonance (NMR) analysis indicated that the whole-cell biocatalyst had more specific activity toward the 3'-hydroxyl group than 5'-hydroxyl group among the available hydroxyl groups in sugar moiety of ara-C. Except for glucose and maltose, 11 carbon sources supplemented to basal media, including Spans, Tweens, olive oil and oleic acid, exhibited notable enhancement effects on both the cell growth and the acylation reactions. It was suggested that the carbon sources containing controlled-release oleic acid were the important substrates for the production of fungal cell-bound lipase with specific activity, partially due to a gradual induction effect of their released oleic acid on the cell-bound lipase production. Despite the low initial reaction rate and substrate conversion, the addition of 2.0 g/l Span 80 resulted in a higher 3'-regioselectivity of the cells than 81%. By using Tween 85 at its optimum concentration of 5.0 g/l, however, the highest initial rates (3.2 mmol/l h) and substrate conversion (76%) of the whole-cell catalyzed acylation of ara-C can be achieved. It was also found that the 3'-regioselectivity of the cells showed observable increase by extending the culture time. And the activity of cell-bound lipase drastically increased in the early stage of cell growth and then declined in the late culture stage, whatever the culture media used. Our results thus indicated that A. oryzae whole cell was a promising green tool for biosynthesis of nucleoside esters with potential bioactivities.

Interleukin-4 enhances trafficking and functional activities of GM-CSF-stimulated mouse myeloid-derived dendritic cells at late differentiation stage

Mouse bone marrow-derived dendritic cells (BMDCs) are being employed as an important model for translational research into the development of DC-based therapeutics. For such use, the localization and specialized mobility of injected BMDCs within specific immune tissues are known to define their immunity and usefulness in vivo. In this study, we demonstrate that IL-4, a key driving factor for in vitro propagation and differentiation of BMDCs, when added during a late culture stage can enhance the in vivo trafficking activity of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced BMDCs. It suggests that the temporal control of IL-4 stimulation during the in vitro generation of DCs drastically affects the DC trafficking efficiency in vivo. With this modification of IL-4 stimulation, we also show that much less cytokine was needed to generate BMDCs with high purity and yield that secrete a high level of cytokines and possess a good capacity to induce proliferation of allogeneic CD4+T cells, as compared to the conventional method that uses a continuous supplement of GM-CSF and IL-4 throughout cultivation. These results provide us with an important know-how for differentiation of BMDCs from myeloid stem cells, and for use of other immune cells in related medical or stem cell applications.

Changes in expression of VE-cadherin and MMPs in endothelial cells: Implications for angiogenesis

The mechanism of cell-cell contact dependent regulation of pericellular proteolysis in angiogenesis was examined by studying the expression of MMPs using isolated HUVECs in culture. Zymography, Immunoblot and RT-PCR analysis showed that the production and secretion of matrixmetalloproteinase-2 and matrixmetalloproteinase-9 by HUVECs in culture were high when they remain as individual cells and significantly decreased during later stages of culture when cells developed cell-cell contact and tubular network-like structure. As MMPs decreased there was significant upregulation of VE-cadherin in cells undergoing angiogenic transition. Investigations to understand the signaling pathways downstream of VE-cadherin showed a relatively high level of β-catenin in the nucleus of endothelial cells in culture during initial stages and decrease in its levels in the nucleus, associated with an increase in the cytosol during later stages of culture. The distribution of β-catenin was found to be regulated by Tyr/Ser phosphorylation status of this protein. Cell-cell contact dependent downregulation of MMPs during angiogenesis was also observed in experiments using proangiogenic substances which caused a rapid rate of downregulation of MMP-2 and MMP-9 and absence of downregulation of MMPs when treated with anti-angiogenic agents.

Raman spectroscopic monitoring of the osteogenic differentiation of human mesenchymal stem cells

The differentiation of stem cells into multi-lineages is essential to aid the development of tissue engineered materials that replicate the functionality of their tissue of origin. For this study, Raman spectroscopy was used to monitor the formation of a bone-like apatite mineral during the differentiation of human mesenchymal stem cells (hMSCs) towards an osteogenic lineage. Raman spectroscopy observed dramatic changes in the region dominated by the stretching of phosphate groups (950-970 cm(-1)) during the period of 7-28 days. Changes were also seen at 1030 cm(-1) and 1070 cm(-1), which are associated with the P-O symmetric stretch of PO(4)(3-) and the C-O vibration in the plane stretch of CO(3)(2-). Multivariate factor analysis revealed the presence of various mineral species throughout the 28 day culture period. Bone mineral formation was observed first at day 14 and was identified as a crystalline, non-substituted apatite. During the later stages of culture, different mineral species were observed, namely an amorphous apatite and a carbonate, substituted apatite, all of which are known to be Raman markers for a bone-like material. Band area ratios revealed that both the carbonate-to-phosphate and mineral-to-matrix ratios increased with age. When taken together, these findings suggest that the osteogenic differentiation of hMSCs at early stages resembles endochondral ossification. Due to the various mineral species observed, namely a disordered amorphous apatite, a B-type carbonate-substituted apatite and a crystalline non-substituted hydroxyapatite, it is suggested that the bone-like mineral observed here can be compared to native bone. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analyses for monitoring the various mineral species, degree of mineralisation and the crystallinity of hMSCs as they differentiate into osteoblasts.

Effects of vascular endothelial growth factor on porcine preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer

This study examined the effects of vascular endothelial growth factor (VEGF) on porcine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) at different developmental stages. Four sets of experiments were performed. In the first, supplementation of the in vitro culture medium with 5 ng/mL VEGF was suitable for porcine IVF embryo development, and the blastocyst formation rate was significantly higher than the control and other groups (57.73 ± 6.78% (5 ng/mL VEGF) vs. 43.21 ± 10.22% (control), 42.16 ± 10.24% (50 ng/mL VEGF) and 41.91 ± 11.74% (500 ng/mL VEGF); P < 0.05). The total cell number after supplementation with 5 ng/mL VEGF was significantly higher than the control and other groups (151.85 ± 39.77 (5 ng/mL VEGF) vs. 100.00 ± 34.43 (control), 91.2 ± 31.51 (50 ng/mL VEGF), and 112.53 ± 47.66 (500 ng/mL VEGF); P < 0.05). In the second experiment, when VEGF was added at different developmental stages of IVF derived embryos (early stage, days 1–3, late stage, days 4–7), the blastocyst formation rate and total cell number were significantly higher at the late stage (47.71 ± 9.13% and 131.5 ± 20.70, respectively) than in the control (34.32 ± 7.44% and 85.50 ± 20.41, respectively) and at the early stage (33.60 ± 5.78% and 86.75 ± 25.10, respectively; P < 0.05). There was no significant difference in the blastocyst development rate or total cell number between the whole culture period (days 1–7) and the late stage culture period after supplementation with 5 ng/mL VEGF (P > 0.05). In the third experiment, the cleavage rate was significantly higher when SCNT embryos were cultured with VEGF during the whole culture period than in the late stage (63.56 ± 15.52% vs. 39.72 ± 4.94%; P < 0.05), but there was no significant difference between the control and the early stage culture period (P > 0.05). The blastocyst formation rate was significantly higher at the late stage culture period with VEGF than at the early stage culture period (34.40 ± 15.06% vs. (16.07 ± 5.01%; P < 0.05). There was no significant difference in the total cell number between the groups (P > 0.05). In experiment 4, using real-time PCR, VEGF mRNA expression was detected in all the developmental stages of IVF and SCNT embryos, but the expression level varied according to the developmental stage. VEGF receptor, KDR mRNA was detected in all stages IVF and SCNT embryos. However, flt-1 mRNA was not expressed in all embryonic stages of IVF and SCNT embryos. These data suggest that VEGF supplementation at the late embryonic developmental stage might improve the developmental potential of both IVF and SCNT preimplantation porcine embryos through its receptors.

Ex vivo live imaging of melanoblast migration in embryonic mouse skin

Ex vivo live imaging of melanoblast migration in embryonic mouse skin

Variations in Laboratory-Scale Actinomycete Communities Exposed to Cadmium as Assessed by Denaturing Gradient Gel Electrophoresis Profiles

The actinomycete populations and functions in cadmium (Cd) contaminated soil were investigated by the cultivation-independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with various concentrations of Cd in the laboratory. The partial 16S rDNA genes were amplified by polymerase chain reaction (PCR) using specific primers bound to evolutionarily conserved regions within these actinomycete genes. The diversity in PCR-amplified products, as measured by denaturing gradient gel electrophoresis (EGGE), was used as a genetic fingerprint of the population. Principle component analysis and Shannon-Weaver diversity index ( H) analyses were used to analyze the DGGE results. Results showed that the two principal components accounted for only a low level of the total variance. The value H in contaminated soil was lower than that in the control at later stages of cultivation, whereas at earlier stages it was higher. Among the six sampling time points, the first, fifth and sixth weeks had the highest values of H. Significantly negative correlations between bioavailable Cd concentration and H values existed in the samples from weeks 2 ( R =0.929, P< 0.05) and 4 ( R =0.909, P< 0.05). These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.

Functional difference of receptor-type protein tyrosine phosphatase ζ/β isoforms in neurogenesis of hippocampal neurons

Receptor-type protein tyrosine phosphatase ζ/β (RPTPζ) is a transmembrane chondroitin sulfate proteoglycan (CSPG) and has been shown to play crucial roles in controlling axonal growth and neuronal migration. The RPTPζ has two transmembranous isoforms, shorter receptor form of RPTPζ (sRPTPζ) and full-length receptor form of RPTPζ (fRPTPζ), but no studies have been reported about functional difference of these two isoforms. In the present study, therefore, we examined whether or not two RPTPζ isoforms have different role in controlling dendritic morphology and synaptic number in cultured hippocampal neurons using the quantitative morphometrical analysis. Confocal microscopic observation showed that the immunoreactivity of RPTPζ was observed throughout cells such as axons, growth cones, and dendrites at the early stages of neuronal culture, while it was seen predominantly on dendrites at the late stages. Western blotting analysis revealed that fRPTPζ was mainly expressed at the early stages of culture and both RPTPζ isoforms were expressed at late stages of culture. The overexpression of sRPTPζ in hippocampal neurons increased the dendritic arborization without altering the average length of dendritic branches, whereas that of fRPTPζ decreased the dendritic arborization and increased the average length of dendritic branches. The RNA interference of fRPTPζ expression increased the dendritic arborization without altering the average length of dendritic branches. The overexpression of fRPTPζ decreased the density of hippocampal dendritic synapses, but that of sRPTPζ had no effects. Pleiotrophin, a ligand for RPTPζ to interfere the phosphatase activity, increased the density of hippocampal dendritic synapses. Thus, the present study demonstrates that two transmembranous RPTPζ isoforms have different functions for regulating dendritogenesis and synaptogenesis.

Lipid production in Porphyridium cruentum grown under different culture conditions

Autotrophic growth of Porphyridium cruentum under 18:12 h and 12:12 h light:dark cycles showed the maximum cell concentration of 2.1 g-dry wt./L, whereas the specific growth rate, 0.042 (1/h), at 18:6 h is faster than that of 12:12 h, 0.031 (1/h), respectively. The highest lipid accumulation level, 19.3 (%, w/w), was achieved at 12:12 h cycle. Under dark cultivation condition with 10 g/L of glucose, the lipid accumulation in the cell was 10.9 (%, w/w), whereas the heterotrophic growth with glycerol as the carbon resource showed low level of cell concentration and lipid production, compared to that of glucose. The glucose was decided to be a suitable carbon resource for the heterotrophic growth of P. cruentum. The lipids from P. cruentum seemed be feasible for biodiesel production, because over 30% of the lipid was C16-C(18:1). The cultivation time and temperature were important factors to increase the maximum cell concentration. Extending the cultivation time helps maintain the maximum cell concentration, and higher lipid accumulation was achieved at 25 degrees C, compared to 35 degrees C. The fed-batch cultures showed that, under the light condition, the specific production rate was slightly decreased to 0.4% lipid/g-dry wt./day at the later stage, whereas, under the dark condition, the specific production rate was maintained to be a maximum value of 1.1% lipid/g-dry wt./day, even in the later stage of cultivation. The results indicate that the heterotrophic or 12:12 h cyclic mixotrophic growth of P. cruentum could be used for the production of biodiesel in long-term fed-batch cultivation of P. cruentum.

Transition of Cleavage Divisions During In Vitro Development of Bovine Embryos

Abstract: Bovine in vitro fertilized (IVF) embryos were cultured in either CR1aa (CR1) or TCM-199 (TCM) medium and compared for their daily development based on the number of cleavage divisions (CDN), as calculated from the total cell numbers of the embryos. Embryos with advanced developmental stage and higher morphological quality were selected for use in the experiment. The relation between the CDN and the number of embryonic days after IVF in both groups showed a linear correlation; no significant difference (P > 0.05) was found between the two groups. However, CDN on days 3 and 8 after IVF in the CR1 group did not increase, which suggests that transient developmental arrests occurred at these stages. In contrast, embryos in the TCM group showed a transient evelopmental arrest 3 d after IVF, but CDN increased regularly with age in days at later stages of culture with 100 µM βmercaptoethanol. A significant difference was found between the regression lines of the two groups during 5–9 d after IVF (P < 0....

The G9A Histone Methyltransferase BIX-01294 Reactivates ε-Globin Expression and Blocks Erythroid Differentiation in Primary Baboon Erythroid Progenitor Cell Cultures

The development of new therapies to increase fetal hemoglobin (HbF) levels in patients with sickle cell disease and β-thalassemia depends on an increased understanding of the mechanism responsible for the developmental regulation of globin gene expression. A role for epigenetic modifications in the mechanism of of globin gene regulation is suggested by the presence of high levels of DNA methylation near the 5' regions of developmentally silenced ε- and γ-globin genes and the ability of pharmacological inhibitors of DNA methyltransferase (DNMTase) to reactivate ε- and γ-globin expression in adults. Whether additional epigenetic modifications associated with gene silencing and DNA methylation, such as histone H3 (lys9) dimethylation, are also involved is unknown. To investigate the hypothesis that histone H3 (lys9) dimethylation may function in the mechanism of developmental globin gene silencing, chromatin immunopreciptation assays were performed to determine the distribution of histone H3 (lys9) dimethyl and histone H3 (lys9) acetyl throughout the β-globin gene complex in purified primary baboon bone marrow (BM) erythroid cells from phlebotomized baboons expressing low levels (5–10%) of HbF and purified erythroid cells from erythroid progenitor cell cultures expressing high levels of HbF (30–50%). In BM erythroid cells, the level of histone H3 (lys9) acetyl associated with the β-globin gene was 10–20 fold higher than with the ε- and γ-globin genes, while the level of histone H3 (lys9) dimethyl associated with the ε- and γ-globin genes was 2–4 fold higher than with the β-globin gene. In erythroid cells from day 12 erythroid progenitor cell cultures, the level of histone H3 (lys9) acetyl associated with the highly expressed γ- and β-globin genes was 10–20 fold higher than with the silent ε-globin gene, while the level of histone H3 (lys9) dimethyl associated with the ε-globin gene was 2–4 fold higher than with the γ- and β-globin genes. Therefore a reciprocal relationship was observed between levels of histone H3 (lys9) acetylation and dimethylation associated with active and inactive globin genes. Experiments were performed to further investigate the role of histone H3 (lys9) dimethyl in ε-globin gene silencing by determining the effect of the G9A histone methyltransferase inhibitor BIX-01294 on ε-globin expression. Erythroid progenitor cell cultures derived from CD34+ BM cells of three individual baboons were treated with the varying doses of the DNMTase inhibitor decitabine (0.125–1.0μM), and BIX-01294 (1.25–5μM), alone and in combination. Changes in ε- globin were assessed by real time PCR using the ΔΔCT method with α-globin as the standard. Decitabine (0.5μM) increased ε-globin 25.8±7.7 fold while BIX-01294 (2.5μM) increased ε-globin 3.09±1.16 fold. Decitabine (1μM) and BIX-01294 (2.5μM) in combination increased ε-globin 55.7±24.9 fold. BIX-01294 enhanced ε-globin expression approximately twofold at all decitabine doses ranging from 0.125–1.0μM (mean increase=103± 44.7%). BIX-01294 also blocked terminal erythroid differentiation and allowed expansion of more primitive cells as evidenced by the presence of a large population of basophilic erythroblasts at late stages of culture (day 14). These results demonstrate that BIX-01294 reactivates expression of the silenced ε-globin gene and that synergistic reactivation can be achieved using combinations of BIX-01294 and decitabine. While these results are consistent with the hypothesis that epigenetic modifications are important in the mechanism of developmental globin gene silencing, the observation that BIX-01294 blocks erythroid differentiation suggests the possible involvement of a reprogramming mechanism.