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Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension

Nuñez, Eutimio Gustavo Fernández; De Rezende, Alexandre GonÇalves; Puglia, Ana Lia Pradella; Leme, Jaci; Boldorini, Vera Lucia Lopes; Caricati, Celso Pereira; Tonso, Aldo
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 1153-1163
ENG
Relevância na Pesquisa
55.79%
Objective To assess the expression of rabies virus G-glycoprotein (RVGP) expression using Semliki Forest virus as a vector in combination with BHK-21 cells cultured in suspension. Results A multilevel factorial design was used to quantify effects of temperature (33–37 C), fresh medium addition after the viral adsorption step (100–200 % with respect to the initial cell suspension volume before infection) and harvest time (8–40 h) on RVGP production. Experimental runs were performed in 24-well cell culture plates at a multiplicity of infection (MOI) of 16. An additional experiment in spinner-flask was performed at MOI of 9, using the optimal conditions determined in cell culture plates. Values for temperature, fresh medium addition and harvest time of 33 C, 100 % and 16 h, respectively, ensured the optimal RVGP production in culture plates. The volumetric yield (239 ng ml-1 ) in these conditions was higher than that reported previously for adherent cell culture. In spinner-flasks, the volumetric yield was improved (559 ng ml-1 ). Conclusion These results establish the basis for designing bioprocess to produce RVGP.

Avaliação do papel do marcador de superfície CD166 na diferenciação osteoblástica/cementoblástica de células mesenquimais indiferenciadas do ligamento periodontal de humanos; Evaluation of the role of the surface marker CD166 along osteoblastic/cementoblastic differentiation of mesenchymal stem cells from periodontal ligament of humans

Guilherme Henrique Costa Oliveira
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 25/02/2015 PT
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45.96%
Células mesenquimais isoladas do ligamento periodontal (PDLSCs) de dentes humanos mostraram ser multipotentes e possuem a capacidade de se diferenciar em adipócitos osteoblastos in vitro. Além disso, possuem a capacidade de formar tecido semelhante ao cemento e ao ligamento periodontal quando transplantadas para animais imunossuprimidos. Por outro lado foi mostrado na literatura uma grande heterogeneidade destas linhagens de células e muito tem sido feito na tentativa de isolar grupos com fenótipos mais favoráveis a diferenciação em tecido mineralizado. A literatura aponta que existe uma modulação na expressão do marcador de superfície CD166 durante a diferenciação osteoblástica e a partir destes achados levanta-se a hipótese de sua participação neste processo. Em acréscimo, células positivas para este marcador mostraram alta capacidade de diferenciar-se em fenótipo osteoblástico. Deste modo, este estudo tem como objetivo avaliar se um grupo de células enriquecido para o marcador CD166 através de separação magnética (PDL/CD166+) apresenta maior potencial de diferenciação osteoblástica/cementoblástica quando comparado ao pool de células não separadas (PDL) e ao grupo de células que não foram retidas na coluna magnética (PDL/CD166-). Este estudo ainda se propõe a avaliar se o marcador de superfície CD166 é regulado no processo de diferenciação osteoblástica. Após a separação magnética de três populações celulares...

Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three dimensional scaffolds

Gomes, Manuela E.; Sikavitsas, V. I.; Behravesh, E.; Reis, R. L.; Mikos, Antonios G.
Fonte: Wiley Interscience Publicador: Wiley Interscience
Tipo: Artigo de Revista Científica
Publicado em //2003 ENG
Relevância na Pesquisa
46%
This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds...

Influence of the porosity of starch-based fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal cells cultured in a flow perfusion bioreactor

Gomes, Manuela E.; Holtorf, H. L; Reis, R. L.; Mikos, Antonios G.
Fonte: Universidade do Minho Publicador: Universidade do Minho
Tipo: Artigo de Revista Científica
Publicado em //2006 POR
Relevância na Pesquisa
45.99%
This study investigates the influence of the porosity of fiber mesh scaffolds obtained from a blend of starch and poly(!-caprolactone) on the proliferation and osteogenic differentiation of marrow stromal cells cultured under static and flow perfusion conditions. For this purpose, biodegradable scaffolds were fabricated by a fiber bonding method into mesh structures with two different porosities– 50 and 75%. These scaffolds were then seeded with marrow stromal cells harvested from Wistar rats and cultured in a flow perfusion bioreactor or in 6-well plates for up to 15 days. Scaffolds of 75% porosity demonstrated significantly enhanced cell proliferation under both static and flow perfusion culture conditions. The expression of alkaline phosphatase activity was higher in flow cultures, but only for cells cultured onto the higher porosity scaffolds. Calcium deposition patterns were similar for both scaffolds, showing a significant enhancement of calcium deposition on cellscaffold constructs cultured under flow perfusion, as compared to static cultures. Calcium deposition was higher in scaffolds of 75% porosity, but this difference was not statistically significant. Observation by scanning electron microscopy showed the formation of pore-like structures within the extracellular matrix deposited on the higher porosity scaffolds. Fourier transformed infrared spectroscopy with attenuated total reflectance and thin-film X-ray diffraction analysis of the cell-scaffold constructs after 15 days of culture in a flow perfusion bioreactor revealed the presence of a mineralized matrix similar to bone. These findings indicate that starch-based scaffolds...

Exploring neural differentiation of adipose stem cells for therapeutic use

Correia, C.; Martins, Ema G.; Sousa, R. A.; Reis, R. L.
Fonte: Universidade do Minho Publicador: Universidade do Minho
Tipo: Conferência ou Objeto de Conferência
Publicado em /06/2014 ENG
Relevância na Pesquisa
46.02%
Spinal cord injuries (SCI) are one of the most complex injuries to treat in the medical area. Due to the complexity of the spinal cord, the treatments are mostly palliative, used to prevent the progression of the injury, handle spasticity, deafferentation pain syndromes and dysautonomia1. Herein, we propose a cell-based approach, taking benefit of particular characteristics of adipose derived stem/stromal cells (hASC). When compared to other stem cell sources, hASC present key advantages such as their immuno-modulatory properties, low immunogenicity, high cell yield per gram of processed tissue, and, their neural differentiation potential, which have been described2. In this project, we aimed to explore and validate procedures to differentiate hASC into cells of the neural lineage, through the use of xeno-free and therapeutic grade reagents along the entire cell manufacturing process. Human ASC (hASC xeno-free, irisbiosciences, Portugal) were cultured in xeno-free media as control, or in neural induction media (Cell Therapy Systems, Life Technologies). Three culturing phases were tested: i) Expansion, for cell proliferation up to p2-3; ii) Pre-Induction, where cells were stimulated with 20ng/mL bFGF and 20ng/mL EGF and iii) Differentiation...

Experimental model of cultured skin graft

Gragnani,Alfredo; Morgan,Jeffrey R.; Ferreira,Lydia Masako
Fonte: Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia Publicador: Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2004 EN
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45.99%
One of the most used animal models of cultured keratinocytes autografting is based on xenografting of human keratinocytes to the rat or athymic mice, immunological neutral recipient that acts as biological carrier. It could be studied in this model many facts that occur after transplant without the ethical aspect in the clinical study. The proposition of the experimental model is related to the sequence of the total or partial skin transplant, as autografting or xenografting, cultured or not, to the back of athymic mice. The model presents the possibility of study in vivo athymic animal, when the in vivo study in anima nobili is not ethical. It permits the xenografting evaluation of cultured cells graft or of the genetically modified cells and of the association of the cultured cells and the dermal substitutes, the composite grafts, and of the autografting.

Initial Organic Products of Assimilation of [13N]Ammonium and [13N]Nitrate by Tobacco Cells Cultured on Different Sources of Nitrogen 1

Skokut, Thomas A.; Wolk, C. Peter; Thomas, Joseph; Meeks, John C.; Shaffer, Paul W.; Chien, W.-S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1978 EN
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45.95%
Glutamine is the first major organic product of assimilation of 13NH4+ by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of 13NO3− by tobacco cells cultured on nitrate. The percentage of organic 13N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. 13NO3−, used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of 13N in glutamine, and a concomitant increase in the percentage of 13N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of 13N from 13NH4+ into glutamine more extensively than it inhibits the incorporation of 13N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when 13NH4+ is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of 13NH4+ is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of 13N from 13NO3− into glutamate more strongly than it inhibits the incorporation of 13N into glutamine...

Dexamethasone inhibits the development of mast cells from dispersed human fetal liver cells cultured in the presence of recombinant human stem cell factor.

Irani, A A; Nilsson, G; Ashman, L K; Schwartz, L B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1995 EN
Relevância na Pesquisa
45.98%
Human fetal liver cells cultured in the presence of recombinant human stem cell factor (rhuSCF) give rise to highly purified mast cell populations. This study examined the effect of steroid hormones on mast cell differentiation. Dispersed fetal liver cells cultured in the presence of rhuSCF at 50 ng/ml and in the presence or absence of various steroid hormones for 4 weeks, were analysed for the presence of mast cells by metachromatic staining with toluidine blue, by immunohistochemistry with a monoclonal antibody against tryptase, and by immunofluorescent flow cytometry with a monoclonal antibody against Kit. Dexamethasone added to the cultures at day 0 resulted in a dose-dependent inhibition of rhuSCF-induced mast cell differentiation with > 85% inhibition seen at a dose of 10(-6) M. A similar effect was seen with hydrocortisone, but not with oestradiol or progesterone. The addition of dexamethasone resulted in decreased DNA synthesis in 14-day-old cultured cells, as assessed by incorporation of bromodeoxyuridine. Addition of dexamethasone to 3-week-old SCF-dependent fetal liver mast cells had no significant effect on mast cell survival. Removal of dexamethasone after 3 weeks of culture with SCF did not result in mast cell development. Thus...

Regulation of IgM memory expression by spleen cells cultured in diffusion chambers

Borella, L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1971 EN
Relevância na Pesquisa
45.95%
In intact mice IgM memory to sheep erythrocytes (SRBC) has an early peak followed by a declining phase. The aim of this study was to determine whether the decay of IgM memory is regulated by the early appearance of cells producing antibodies of IgG classes. The expression of IgM memory was studied in SRBC-primed mouse spleen cells restimulated with SRBC in Millipore diffusion chambers implanted in irradiated hosts. Restimulation of spleen cells obtained from mice primed with SRBC 2 days before the beginning of the culture (D2 cells) induced a ten-fold increase in IgM PFC above the number expected in cultures of non-primed cells. The number of IgM PFC significantly decreased in cultures of cells obtained 4 days after priming (D4 cells). Inhibition of IgM PFC occurred in cultures of D2 cells co-cultured with D4 cells in single or double compartment diffusion chambers. In primed spleen cells cultured in double compartment chambers there was an inverse relationship between the number of early IgG PFC in one side and appearance of IgM PFC in the other. A 500-fold increase in antigen concentration in chambers containing D4 cells induced a slow, but consistent, rise in IgM PFC. The data indicate the expression of IgM memory in this culture system is inhibited by the appearance of cells producing antibody of IgG classes as early as 4–10 days after primary antigenic stimulation. It is postulated that a similar process may also regulate the kinetics of IgM memory decay in the intact animal.

Suppression of interleukin 1α and interleukin 1β in human limbal epithelial cells cultured on the amniotic membrane stromal matrix

Solomon, A.; Rosenblatt, M.; Monroy, D.; Ji, Z.; Pflugfelder, S.; Tseng, S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/2001 EN
Relevância na Pesquisa
45.98%
AIMS—Amniotic membrane (AM) transplantation reduces inflammation in a variety of ocular surface disorders. The aim of this study was to determine if AM stroma suppresses the expression of the IL-1 gene family in cultured human corneal limbal epithelial cells.
METHODS—Human corneal limbal epithelial cells were cultured from limbocorneal explants of donor eyes on plastic or on the AM stroma. Transcript expression of IL-1α, IL-1β, IL-1 receptor antagonist (RA), and GAPDH was compared with or without addition of lipopolysaccharide to their serum-free media for 24 hours using RNAse protection assay (RPA). Their protein production in the supernatant was analysed by ELISA.
RESULTS—Expression of IL-1α and IL-1β transcripts and proteins was significantly reduced by cells cultured on the AM stromal matrix compared with plastic cultures whether lipopolysaccharide was added or not. Moreover, expression of IL-1 RA by cells cultured in the lipopolysaccharide-free medium was upregulated by AM stromal matrix. The ratio between IL-1 RA and IL-1α protein levels in AM cultures was higher than in plastic cultures.
CONCLUSIONS—AM stromal matrix markedly suppresses lipopolysaccharide induced upregulation of both IL-1α and IL-1β. These data may explain in part the effect of AM transplantation in reducing ocular surface inflammation...

Proteomics-Based Identification of Proteins Secreted in Apical Surface Fluid of Squamous Metaplastic Human Tracheobronchial Epithelial Cells Cultured by Three-Dimensional Organotypic Air-Liquid Interface Method

Kim, Seung-Wook; Cheon, Kyounga; Kim, Chang-Hoon; Yoon, Joo-Heon; Hawke, David H.; Kobayashi, Ryuji; Prudkin, Ludmila; Wistuba, Ignacio I.; Lotan, Reuben; Hong, Waun Ki; Koo, Ja Seok
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/07/2007 EN
Relevância na Pesquisa
45.94%
Squamous cell carcinoma (SCC) in the lung originates from bronchial epithelial cells that acquire increasingly abnormal phenotypes. Currently, no known biomarkers are clinically efficient for the early detection of premalignant lesions and lung cancer. We sought to identify secreted molecules produced from squamous bronchial epithelial cells cultured with organotypic culture methods. We analyzed protein expression patterns in the apical surface fluid (ASF) from aberrantly differentiated squamous metaplastic normal human tracheobronchial epithelial (NHTBE) and mucous NHTBE cells. Comparative two-dimensional PAGE analysis revealed 174 unique proteins in the ASF of squamous NHTBE cells compared to normal mucociliary differentiated NHTBE cells. Among them, 64 well-separated protein spots were identified using liquid chromatography-tandem mass spectrometry, revealing 22 different proteins in the ASF from squamous NHTBE cells. Expression of six of these proteins (SCCA1, SCCA2, S100A8, S100A9, annexin I, and annexin II) in the squamous NHTBE cells was further confirmed with immunoblot analysis. Notably, SCCA1 and SCCA2 were verified as being expressed in squamous metaplastic NHTBE cells but not in normal mucous NHTBE or normal bronchial epithelium. Moreover...

Vascular endothelial cells cultured from patients with cerebral or uncomplicated malaria exhibit differential reactivity to TNF

Wassmer, Samuel Crocodile; Moxon, Christopher Alan; Taylor, Terrie; Grau, Georges Emile; Molyneux, Malcolm Edward; Craig, Alister Gordon
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Publicado em /02/2011 EN
Relevância na Pesquisa
45.95%
Plasmodium falciparum malaria is a major cause of morbidity and mortality in African children, and factors that determine the development of uncomplicated (UM) versus cerebral malaria (CM) are not fully understood. We studied the ex vivo responsiveness of microvascular endothelial cells to pro-inflammatory stimulation and compared the findings between CM and UM patients. In patients with fatal disease we compared the properties of vascular endothelial cells cultured from brain tissue to those cultured from subcutaneous tissue, and found them to be very similar. We then isolated, purified and cultured primary endothelial cells from aspirated subcutaneous tissue of patients with CM (ECCM) or UM (ECUM) and confirmed the identity of the cells before analysis. Upon TNF stimulation in vitro, ECCM displayed a significantly higher capacity to upregulate ICAM-1, VCAM-1 and CD61 and to produce IL-6 and MCP-1 but not RANTES compared with ECUM. The shedding of endothelial microparticles, a recently described parameter of severity in CM, and the cellular level of activated caspase-3 were both significantly greater in ECCM than in ECUM. These data suggest that inter-individual differences in the endothelial inflammatory response to TNF may be an additional factor influencing the clinical course of malaria.

Increased mechanosensitivity of cells cultured on nanotopographies

Salvi, Joshua D.; Lim, Jung Yul; Donahue, Henry J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
46.01%
Enhancing cellular mechanosensitivity is recognized as a novel tool for successful musculoskeletal tissue engineering. We examined the hypothesis that mechanosensitivity of human mesenchymal stem cells (hMSCs) is enhanced on nanotopographic substrates relative to flat surfaces. hMSCs were cultured on polymer-demixed, randomly distributed nanoisland surfaces with varying island heights and changes in intracellular calcium concentration, [Ca2+]i, in response to fluid flow induced shear stress were quantifide. Stem cells cultured on specific scale nanotopographies displayed greater intracellular calcium responses to fluid flow. hMSCs cultured on 10-20 nm high nanoislands displayed a greater percentage of cells responding in calcium relative to cells cultured on flat control, and showed greater average [Ca2+]i increase relative to cells cultured on other nanoislands (45-80 nm high nanoislands). As [Ca2+]i is an important regulator of downstream signaling, as well as proliferation and differentiation of hMSCs, this observation suggests that specific scale nanotopographies provide an optimal milieu for promoting stem cell mechanotransduction activity. That mechanical signals and substrate nanotopography may synergistically regulate cell behavior is of significant interest in the development of regenerative medicine protocols.

Growth and differentiation of neural stem cells in a three-dimensional collagen gel scaffold★

Huang, Fei; Shen, Qiang; Zhao, Jitong
Fonte: Medknow Publications & Media Pvt Ltd Publicador: Medknow Publications & Media Pvt Ltd
Tipo: Artigo de Revista Científica
Publicado em 05/02/2013 EN
Relevância na Pesquisa
46.01%
Collagen protein is an ideal scaffold material for the transplantation of neural stem cells. In this study, rat neural stem cells were seeded into a three-dimensional collagen gel scaffold, with suspension cultured neural stem cells being used as a control group. Neural stem cells, which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor, actively expanded and formed neurospheres in both culture groups. In serum-free medium conditions, the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group. Immunofluorescence staining showed that neurospheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker βIII-tubulin, the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic marker 2’,3’-cyclic nucleotide 3’-phosphodiesterase. Compared with neurospheres cultured in suspension, the differentiation potential of neural stem cells cultured in collagen gels increased, with the formation of neurons at an early stage. Our results show that the three-dimensional collagen gel culture system is superior to suspension culture in the proliferation...

Aβ Oligomers-Induced Toxicity is Attenuated in Cells Cultured with NbActiv4™ Medium

Zhou, Yan; Klein, William L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.96%
Pathogenic Aβ-derived diffusible ligands (ADDLs) bind to post-synaptic targets, induce excessive reactive oxygen species (ROS) and stimulate tau hyperphosphorylation in cultured neurons. Recently, NbAc-tiv4™ medium was reported to increase neuron synapse densities in cultured hippocampal neurons. We aimed to investigate the effect of this novel medium on ADDL-induced toxicity. We found that ADDL-induced ROS was attenuated in cells cultured with NbActiv4™. ADDL binding assay was performed in neurons cultured by different feeding conditions with NbActiv4™. Feeding cells with 30 % medium once a week, ADDL binding sites were abundant at days in vitro (DIV) 18. However, changing 50 % medium once a week decreased ADDL binding about 80 %. NbActiv4™ produced about 40 % more glial fibrillary acidic protein (GFAP) positive astrocytes than the widely used hippocampal culture medium, neurobasal supplemented with B27 (neurobasal/B27). Astrocytes are reported to produce kinds of trophic factors including insulin-like growth factor 1 (IGF-1). Consistently, when cultured with NbActiv4™, neurons were sensitive to inhibitors of insulin/IGF-1 signaling in response to ADDL attack. Overall, this study supports the important role of astrocytes in neuroprotection and indicates that targeting astrocytes dysfunction may lead to new therapeutic strategies for Alzheimer’s disease.

Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells

Kitahama, S.; Gibson, M.; Hatzinikolas, G.; Hay, S.; Kuliwaba, J.; Evdokiou, A.; Atkins, G.; Findlay, D.
Fonte: Elsevier Science Inc Publicador: Elsevier Science Inc
Tipo: Artigo de Revista Científica
Publicado em //2000 EN
Relevância na Pesquisa
55.84%
Fibrillin-containing microfibrils are structural components of extracellular matrices of a diverse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive...

Type 1 collagen influence on gene expression in UMR106-06 osteoblast-like cells is inhibited by genistein

Celic, S.; Katayama, Y.; Chilco, P.; Martin, T.; Findlay, D.
Fonte: Universidade de Adelaide Publicador: Universidade de Adelaide
Tipo: Artigo de Revista Científica
Publicado em //1998 EN
Relevância na Pesquisa
55.97%
We have previously shown that an exogenous type I collagen matrix can regulate expression of mRNA for parathyroid hormone (PTH)-related protein (PTHrP) and its receptor, the PTH/PTHrP receptor, in the UMR106-06 osteogenic sarcoma cell line, which is considered to be representative of a relatively mature osteoblast phenotype. Consistent with those data, we show here that growth of UMR106-06 cells on type I collagen increased PTH/PTHrP receptor-binding capacity. Analysis of the binding data showed that the number of PTH/PTHrP receptors expressed by cells cultured on collagen was at least 2-fold greater than that of cells cultured on plastic. Expression of mRNA encoding alkaline phosphatase (ALP) and osteopontin (OP) was also upregulated in cells cultured on collagen, suggesting that interaction with collagen promotes the osteoblast phenotype in this cell line. Retinoic acid (RA), which has also been shown to promote osteoblastic differentiation, synergized with type I collagen to cause super-induction of OP mRNA. In contrast, RA abolished the collagen-induced increase in ALP mRNA and PTH/PTHrP receptor mRNA. The collagen-mediated increase in the expression of OP and PTH/PTHrP receptor mRNA, but not that of ALP, was perturbed by prior covalent modification of the collagen by non-enzymatic glycation. The collagen effects did not occur via interaction with RGD amino acid domains in type I collagen...

High glucose attenuates protein S-nitrosylation in endothelial cells - Role of oxidative stress

Wadham, C.; Parker, A.; Wang, L.; Xia, P.
Fonte: Amer Diabetes Assoc Publicador: Amer Diabetes Assoc
Tipo: Artigo de Revista Científica
Publicado em //2007 EN
Relevância na Pesquisa
55.82%
OBJECTIVE: Hyperglycemia-induced endothelial dysfunction, via a defect of nitric oxide (NO) bioactivity and overproduction of superoxide, is regarded as one of the most significant events contributing to the vascular lesions associated with diabetes. However, the mechanisms underlying such hyperglycemic injury remain undefined. We hypothesized that alterations in cellular protein S-nitrosylation may contribute to hyperglycemia-induced endothelial dysfunction. RESEARCH DESIGN AND METHODS: We exposed endothelial cells to high glucose in the presence and absence of reactive oxygen species inhibitors and used the biotin switch assay to analyze the alteration in the global pattern of protein S-nitrosylation compared with cells cultured under normal glucose conditions. We identified endogenous S-nitrosylated proteins by mass spectrometry and/or immunoblotting with specific antibodies. RESULTS: High-glucose treatment induced a significant reduction of endogenous S-nitrosylated proteins that include endothelial NO synthase, beta-actin, vinculin, diacylglycerol kinase-alpha, GRP78, extracellular signal-regulated kinase 1, and transcription factor nuclear factor-kappaB (NF-kappaB). Interestingly, these changes were completely reversed by inhibition of superoxide production...

Slow Adaptive Changes in Urease Levels of Tobacco Cells Cultured on Urea and Other Nitrogen Sources 1

Skokut, Thomas A.; Filner, Philip
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1980 EN
Relevância na Pesquisa
45.96%
Tobacco (cv. Xanthi) XD cells cultured for more than a year on urea as the sole source of nitrogen have urease activities about four times higher than cells which have been cultured on nitrate. When cells which had always been grown on nitrate were transferred to urea, the urease activity in these cells remained at a lower level for eight transfers (40 generations), then gradually increased 4-fold during the next seven to 10 transfers. Cells with high urease activity multiplied 19% more rapidly and accumulated less urea than cells with low urease activity. These findings suggest that elevated urease accelerates urea assimilation; therefore, urea limited growth. Clones of cells with low urease activity responded in the same way as uncloned populations when transferred from nitrate to urea, indicating that high urease cells originate from low urease cells, rather than from a preexisting subpopulation of high urease cells. The urease levels in clones of cells from a population with high urease activity were three to seven times the low urease level. The observed dependence of urease activity on generations of growth on urea was matched with a model in which high urease cells originated at mitosis of low urease cells at a frequency of 8 × 10−5...

Co-cultivo de zigotos e embriões de duas células de camundongos (Mus musculus domesticus) em suspensão de células epiteliais de ovidutos de camundongos e bovinos nos meios CZB sem glicose, TCM199 e Ménézo B2; Zygotes and two cells mouse embryos in co-culture of mouse and bovine epithelial cells in CZB glucose free, TCM199 and Ménézo B2 mediuns

Tavares, Liliam Mara Trerrisan; Assumpção, Mayra Elena Ortiz D'’Avila; Ferreira, Fernando; Lima, Alecsandra Sobreira; Mello, Marco Roberto Bourg; Visintin, José Antonio
Fonte: Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia Publicador: Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; Formato: application/pdf
Publicado em 01/01/1999 POR
Relevância na Pesquisa
46.02%
Zigotos e embriões de 2 células de camundongos F1 (C57/DBA) foram cultivados (controle) e co-cultivados em suspensões de células epiteliais de ovidutos de camundongos (MOEC) e de bovinos (BOEC) até o estádio de blastocistos nos meios CZB, TCM199 e Ménézo B2. Os zigotos que atingiram os estádios de blastocistos expandido e eclodido no CZB foram 44 (38,26%) para o controle, 2 (1,89%) para as MOEC e 8 (6,72%) para as BOEC, sendo que o controle apresentou-se estatisticamente diferente (p<=0,05) em relação às MOEC e às BOEC. Os zigotos cultivados e co-cultivados nos meios TCM199 e B2 não clivaram além de 2 células. Os embriões de 2 células que desenvolveram até blastocistos no CZB foram 46 (43,80%) para o controle, 45 (38,79%) para as MOEC e 37 (29,60%) para as BOEC, sendo que o controle apresentou-se estatisticamente diferente (p<=0,05) em relação às BOEC. No TCM199, o total de blastocistos foi de 52 (48,15%) para o controle, 68 (39,08%) para as MOEC e 64 (48,49%) para as BOEC, não havendo diferença estatística (p>;0,05) entre os três grupos. No B2, o total de blastocistos foi de 28 (24,78%) para o controle, 34 (28,10%) para as MOEC e 25 (23,81%) para as BOEC, não havendo diferença estatística (p>;0...