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Culture Time (culture + time)

Distribution by Scientific Domains

Life Sciences	50%
Medical Sciences	40%

Selected Abstracts

P28 Interleukin-8 from keratinocytes can be used to test for contact allergy

CONTACT DERMATITIS, Issue 3 2004
Bolli Bjarnason
Objective:, To investigate whether secretion of interleukin-8 (IL-8) proteins by keratinocytes following in vitro exposure to a contact allergen can be used to detect contact allergy. Methods:, Suction blisters were made on skin of allergic and anergic subjects to urushiol, the contact allergen of poison ivy. Keratinocyte cultures were prepared and exposed to the allergen in vitro. Controls were the allergen solvent. Variable allergen concentrations, allergen exposure times and cell culture times were used. At the end of each culture time, IL-8 RNA and protein of the culture supernatants were analyzed by PCR and ELISA. Results:, The concentration of IL-8 in the supernatants proved to be a successful way to distinguish between subjects who patch tested positive with a non-toxic concentration of urushiol and subjects who tested negative. In the allergic subjects, a correlation was established between the dose of the allergen and the IL-8 protein concentration in the supernatants. Conclusions:, In vitro testing of contact allergies in patients makes possible an objective assessment of their allergic status without causing a booster effect or risking active sensitizations. The results indicate that the method may be used as an alternative method to animal models for testing consumer products before their marketing, thus avoiding ethical problems and problems related to interpretation of tests because of biological differences between animals and humans. [source]

Effect of recombinant Rv1009 protein on promoting the growth of Mycobacterium tuberculosis

JOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008
X. Wu
Abstract Aims:, To determine whether resuscitation-promoting factor (RPF) from Mycobacterium tuberculosis can promote mycobacterial growth and shorten culture time. Method and Results:, We cloned, expressed and purified an RPF from M. tuberculosis, Rv1009 protein and subsequently studied the biological activity of the recombinant Rv1009 (rRv1009) in liquid and on solid media. Our results indicate that the molecular weight of rRv1009 protein expressed in Escherichia coli BL21 was approximately 39 kDa. At picomolar and micromolar concentrations, rRv1009 protein could increase the optical density of freeze-dried Mycobacterium bovis BCG three to fivefold in Middlebrook 7H9 medium, stimulate the growth of viable mycobacteria on solid medium, and shorten positive growth detection time of a small number of M. tuberculosis in BACTEC 960 medium. Conclusions:, The rRv1009 could promote proliferation of mycobacteria. It may be useful for culture of mycobacteria presented in clinical samples. Significance and Impact of the Study:, rRv1009 protein can be used as a growth-promoting reagent of mycobacteria in the medium to shorten the time of culture. [source]

Intracellular signaling involved in macrophage adhesion and FBGC formation as mediated by ligand,substrate interaction

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 4 2002
Weiyuan John Kao
Abstract Fibronectin and RGD- and/or PHSRN-containing oligopeptides were preadsorbed onto physicochemically distinct substrata: polyethyleneglycol-based networks or tissue culture polystyrene (TCPS). The role of selected signaling kinases (namely protein tyrosine kinases, protein serine/threonine kinases, PI3-kinase, Src, and MAPK) in the adhesion of human primary blood-derived macrophages and the formation of foreign-body giant cells (FBGC) on these modified substrata was investigated. The involvement of individual intracellular signaling molecules in mediating macrophage adhesion dynamically varied with the culture time, substrate, and ligand. For example, fibronectin on TCPS or networks involved similar signaling events for macrophage adhesion; however, fibronectin and G3RGDG6PHSRNG, but not peptides with other RGD and/or PHSRN orientations, mediated similar signaling events for macrophage adhesion on TCPS but mediated different signaling events on networks. Depending on the substrate, a specific molecule (i.e., Src, protein kinase C) within the protein tyrosine kinase or protein serine/threonine kinase family was either an antagonist or agonist in mediating FBGC formation. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 62: 478,487, 2002 [source]

Subculture affects the phenotypic expression of human periodontal ligament cells and their response to fibroblast growth factor-2 and bone morphogenetic protein-7,in vitro

JOURNAL OF PERIODONTAL RESEARCH, Issue 5 2008
S. Lossdörfer
Background and Objective:, Although periodontal ligament cells display several osteoblastic traits, their phenotypic expression is still not well established. It remains a matter of debate whether they resemble a terminally differentiated cell type or an intermediate maturation state that potentially can be directed towards a fibroblastic or an osteoblastic phenotype. Material and Methods:, To explore the characteristics of periodontal ligament cells in greater detail, fourth-passage, sixth-passage and eighth-passage human periodontal ligament cells were cultured for up to 3 wk. Ki-67, alkaline phosphatase, osteocalcin, osteoprotegerin and receptor activator of nuclear factor-,B ligand (RANKL) mRNA expression was quantified by real-time polymerase chain reaction. Furthermore, the cellular response to fibroblast growth factor-2 and bone morphogenetic protein-7 was examined in first-passage and fourth-passage cells. Dermal fibroblasts (1BR.3.G) and osteoblast-like cells (MG63) served as reference cell lines. Results:, Proliferation decreased over time and was highest in fourth-passage cells. The expression of differentiation parameters, osteoprotegerin and RANKL increased with culture time and was higher in fourth-passage cells than in cells of later passages. The RANKL/osteoprotegerin ratio increased steadily until day 21. Administration of fibroblast growth factor-2 enhanced cell numbers in both passages, whereas alkaline phosphatase and osteocalcin production remained unchanged. By contrast, exposure of periodontal ligament cells to bone morphogenetic protein-7 resulted in a reduction of cell number in the first and fourth passages, whereas the production of alkaline phosphatase and osteocalcin was enhanced. In dermal fibroblasts, differentiation parameters did not respond to both stimuli. MG63 cells behaved similarly to periodontal ligament cells. Conclusion:, These results indicate that subculture affects the phenotypic expression of human periodontal ligament cells with respect to the characteristics that these cells share with osteoblasts. Furthermore, the periodontal ligament cell phenotype can be altered by fibroblastic and osteoblastic growth factors. [source]

PRODUCTION OF PARALYTIC SHELLFISH TOXINS BY APHANIZOMENON SP.

JOURNAL OF PHYCOLOGY, Issue 4 2002
LMECYA 31 (CYANOBACTERIA)
We examined intracellular and extracellular paralytic shellfish toxins (PST) in a strain of Aphanizomenon sp. (LMECYA31) isolated from a Portuguese freshwater reservoir throughout the growth cycle and under different conditions affected by temperature and nitrate and phosphate availability. PST concentrations and compositions were greatly influenced by cell density, growth stage, and temperature and nutrients conditions. On a per-cell basis results showed (1) the enhancement of PST cell quota after the end of exponential growth phase in nutrient replete batch cultures, (2) the absence of a PST increment at late growth stages under phosphate limitation, (3) a rise in PST maximum cell quota under nitrate depletion, and (4) the enhancement of toxin production at higher temperatures. The relative proportion of the four toxins detected, neoSTX, dcSTX, STX and GTX5, also changed within and between culture settings. While growing under phosphate rich media cells produced mainly GTX5 and neoSTX, whereas under phosphate limitation the proportion of STX and dcSTX increased substantially with culture age. Large amounts of extracellular toxins were found in the culture medium, increasing during culture time. Extracellular toxin composition in each culture was fairly constant and always similar to the intracellular composition found at late stages of growth. This further supported other research that indicates that PSTs are released to the water through cell lysis, and a significant concentration of PST may be expected to remain in the water upon the collapse of a toxic bloom or after cells removal by water treatment. [source]

Comparative IFN- , Secretion after Hatching by Bovine Blastocysts Derived Ex Vivo and Completely Produced In Vitro

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2007
JA Neira
Contents The interferon-tau (IFN- ,) secretion levels after hatching by bovine blastocysts derived from in vitro maturated oocytes (Group A) and from in vivo (Group B) were investigated considering embryo quality. Only very homogeneous blastocysts of excellent or good quality were considered from day 7 of culture (Group A) and day 7 after artificial insemination with frozen-thawed from the same bull used for in vitro fertilization (Group B). All embryos were individually cultured into a 50 ,l droplet of synthetic oviduct fluid medium with 10% fetal calf serum. After 24-h culture both Group A (n =44) and B (n = 40) secreted <54 pm IFN- ,. After 48-, 72-, 96- and 120-h culture, Group A daily secreted 143 ± 24 pm IFN- , (n = 19) vs 85 ± 12 pm IFN- , (n = 21) for Group B (p < 0.01), 491 ± 128 pm IFN- , (n = 29) vs 216 ± 37 pm IFN- , (n = 23) (NS), 499 ± 135 pm IFN- , (n = 26) vs 353 ± 93 pm IFN- , (n = 21) (NS), 559 ± 136 pm IFN- , (n = 22) vs 333 ± 75 pm IFN- , (n = 20) (NS), respectively. Taken all together during 5 days, Group A produced per embryo 1690 ± 290 pm IFN- , (n = 22) vs 982 ± 182 pm IFN- , (n = 20) for Group B (p < 0.05). For all culture time there were sizable percentages of embryos that did not produce concentrations of IFN- , above a certain cut-off level, and as such were not used to compute the means. In respect of the embryo quality whatever the groups after days 7,12 of culture, IFN- , secretions were 1815 ± 453 pm (n = 10) for the embryos of excellent quality vs 1356 ± 200 pm (n = 28) for those of good quality (NS) and 360 ± 188 pm (n = 4) (p < 0.05) for embryos of fair quality. A positive relationship between IFN- , production and in vitro development of quality I embryos was observed, whatever the embryos origins and, the embryos completely produced in vitro secreted more IFN- , than the embryos produced in vivo. [source]

Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2001
R. Robert Balcarcel
Abstract Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 1,10, 2001. [source]

Dependence of Apparent Viscosity on Mycelial Morphology of Streptomyces fradiae Culture in Various Nitrogen Sources

BIOTECHNOLOGY PROGRESS, Issue 4 2000
Du Bok Choi
To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa·s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration,which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa·s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 × 104 ,m2. In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 × 104 ,m2 comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology. [source]

Drug metabolic activity of cultured hepatocytes can synchronize with bile acid concentration in the medium

CELL BIOCHEMISTRY AND FUNCTION, Issue 1 2002
Nobuhiro Sugihara
Abstract The regulation of drug metabolic activity of cultured hepatocytes can be applied to the evaluation of pharmacokinetics, analysis of drug delivery and the bioartificial liver system. It is very difficult to maintain the drug metabolic activity mediated by cytochrome P-450 (CYP) 3A. Recently we found that the CYP3A aminopyrine N-demethylase (AMND) activity of hepatocytes cultured on collagen surface oscillated with culture time. This phenomenon was related to the concentration of bile acid in the culture medium. CYP3A, multidrug resistant gene 2 (MDR2) and heat shock protein 84 (HSP84) mRNA appeared in a manner corresponding to this oscillation. When a large quantity of bile acid was taken up into hepatocytes from the medium, low AMND activity was observed, and these proteins did not appear. When bile acid was secreted and the bile acid concentration inside the hepatocytes was low, high AMND activity was obtained, and these proteins appeared. In order to clarify the mechanism of oscillation between AMND activity and bile acid, 8,,M glycocholic acid was added to the culture medium 15,h before the measurement. No oscillation in AMND activity was observed in the presence of 8,,M glycocholic acid. Bile acid controls the AMND activity in the transcription of hepatocytes. Copyright © 2001 John Wiley & Sons, Ltd. [source]