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Standard Culture Conditions (standard + culture_condition)
Selected AbstractsEffect of an essential oil-containing antiseptic mouthrinse on induction of platelet aggregation by oral bacteria in vitroJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 5 2000E. J. Whitaker Abstract Background: With an increasing body of data suggesting an association between periodontitis and cardiovascular disease, studies have been conducted to elucidate potential mechanisms by which oral bacteria might exert systemic effects. 2 oral bacteria, Streptococcus sanguis and Porphyromonas gingivalis, have been shown to induce platelet aggregation in vitro. This study was conducted to determine the effect of treatment with an essential oil mouthrinse (Listerine® Antiseptic) on the platelet-aggregating activity of these organisms. Method: Bacteria were grown under standard culture conditions. S. sanguis ATCC strain 10556 was exposed for 3 min to the essential oil mouthrinse at either full strength or a 1:1 dilution, while P. gingivalis FDC strain 381 was exposed to the essential oil mouthrinse at a 1:10 dilution. Positive control cells were treated with Hanks balanced salt solution (HBSS). Aggregation was measured using a recording platelet aggregometer. The assay of each organism in its respective mouthrinse dilution(s) or HBSS was repeated 5 times. Results: In all cases, the HBSS-treated organisms induced platelet aggregation, with mean(±S.E.) lag times of 12.30 (±1.36) min and 11.36 (±0.58) min for P. gingivalis and S. sanguis, respectively. In contrast, treatment with the essential oil mouthrinse completely inhibited the platelet aggregating activity of P. gingivalis and of S. sanguis exposed to the 1:1 mouthrinse dilution in all assays; the aggregating activity of S. sanguis treated with full-strength mouthrinse was completely inhibited in 4 of 5 assays, and inhibited by 75% in the 5th, for a mean inhibition of 95±1.5%. Conclusion: This study provides additional evidence that the essential oil mouthrinse can interfere with bacterial cell surface-associated activities which may have clinical relevance. [source] Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in cultureAGING CELL, Issue 6 2003Rita A. Busuttil Summary Oxidative damage is a causal factor in aging and cancer, but it is still not clear how DNA damage, the cellular responses to such damage and its conversion to mutations by misrepair or misreplication contribute to these processes. Using transgenic mice carrying a lacZ mutation reporter, we have previously shown that mutations increase with age in most organs and tissues in vivo. It has also been previously shown that mouse cells respond to oxidative stress, typical of standard culture conditions, by undergoing cellular senescence. To understand better the consequences of oxidative stress, we cultured mouse embryo fibroblasts (MEFs) from lacZ mice under physiological oxygen tension (3%) or the high oxygen tension (20%) associated with standard culture, and determined the frequency and spectrum of mutations. Upon primary culture, the mutation frequency was found to increase approximately three-fold relative to the embryo. The majority of mutations were genome rearrangements. Subsequent culture in 20% oxygen resulted in senescence, followed by spontaneous immortalization. Immortalization was accompanied by an additional three-fold increase in mutations, most of which were G:C to T:A transversions, a signature mutation of oxidative DNA damage. In 3% oxygen, by contrast, MEFs did not senesce and the mutation frequency and spectrum remained similar to primary cultures. These findings demonstrate for the first time the impact of oxidative stress on the genomic integrity of murine cells during senescence and immortalization. [source] Growth of malignant oral epithelial stem cells after seeding into organotypical cultures of normal mucosaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 2 2004Ian C. Mackenzie Background:, Oral squamous cell carcinoma (OSCC) is associated both with the local expansion of clones of malignant cells and with their further migration to regional and distant sites. The interactions that occur between normal and malignant cells during these events are not well modelled by standard culture conditions, but organotypical cultures, in which epithelial cells are grown on a matrix containing fibroblasts, provide a suitable environment for such investigations. Methods:, Cells from five cell lines, each derived from OSCC and marked by retroviral transduction with alkaline phosphatase, were incorporated as small subpopulations (0.1,5%) in uniformly differentiating organotypical cultures constructed from normal oral mucosal cells. The patterns of growth of the malignant cells within the normal epithelium were examined for 3 weeks. Results:, There was variation between the different cell lines in their rates and patterns of growth, but all cell lines produced clusters of malignant cells that had expanded within 3 weeks to replace the normal epithelium. The appearance and spacing of these clusters suggested that each was derived from a single progenitor cell. The number of malignant cells initially present within a given area of organotypical epithelium was much greater than the number of expanding cell clusters subsequently formed. Cluster-forming cells thus represented only a subpopulation of the tumour cells. Conclusions:, The organotypical model allows examination of interactions occurring between cells derived from OSCC and normal epithelia. The three-dimensional nature of organotypical cultures, together with their more normal patterns of differentiation, provides an environment that more closely mimics the in vivo environment in which tumours develop. The finding that only a subpopulation of tumour cells forms expanding tumour colonies suggests a range of growth potentials within a tumour population and may provide preliminary evidence for some form of stem and amplifying cell pattern. [source] Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 5 2009Martha Guerrero-Olazarán Abstract Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 ,g/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Are mesenchymal stromal cells from children resistant to apoptosis?CELL PROLIFERATION, Issue 3 2009H. Dimitriou Objectives:, Mesenchymal stromal cells (MSC) represent a novel cellular candidate in the field of transplantation and tissue regeneration. Their clinical application requires their in vitro expansion. The aim of this study was to assess the effect of conditions that would favour apoptosis, and of long-term expansion, on the characteristics of MSC from children. Materials and methods:, Bone marrow mononuclear cells were cultured for 10 passages (P1,P10). Expression of CD105, CD146, CD95 and apoptosis by 7-amino-actinomycin D staining were evaluated. CFU-F and cell doubling time (DT) were assessed in every passage. Cell-cycle study was performed at P2 and P6. Results:, CFU-F decreased from 38 ± 3.7 at P2 to 9.6 ± 3.2 per 10 MSC/cm2 at P10 and DT increased from 1.93 ± 0.1 (P2) to 6.1 ± 2.45 days (P10). A low percentage of apoptotic (dead) cells was detected at P2 and this did not change until P10. Cells at P2 were at G0/G1 phase, but in advanced passages more cells were in an active state. Induction of apoptosis (addition of anti-Fas agonist antibody) using standard culture conditions, showed a minor effect on MSC survival. Serum deprivation of MSC (up to 72 h) revealed no substantial apoptotic effect while cells retained their tri-lineage differentiation capacity. Conclusions:, We conclude that MSC from children retain their functional characteristics throughout serial passages and remain stable under conditions that usually cause apoptosis. These features render MSC, especially those of early passages, optimal candidates for use in clinical applications. [source] | |||||||||||||