Establishment of a 3D co-culture model to investigate the role of primary fibroblasts in the development of an invasive phenotype of DCIS lesions

A differentiation-arrested primary cell culture model was used to examine the role of reactive oxygen species in the control of prostacyclin (PGI2) production in the perinatal rat lung. Coincubation of the lung cells with arachidonic acid (AA) and xanthine (X, 0.25 mM) plus xanthine oxidase (XO, 10 mU/ml) or with AA and glucose (25 mM) plus glucose oxidase (25 mU/ml) augmented the AA-induced PGI2 output. Superoxide dismutase (10 U/ml) did not alter the X + XO effect, whereas catalase (10 U/ml) eliminated both X + XO and glucose plus glucose oxidase effects. H2O2 (1–200 microM) showed a dose-related biphasic augmentation with peak stimulation at 20 microM. Catalase again blocked this effect, but dimethylthiourea, a hydroxyl radical scavenger, did not. A 20-min pretreatment of the cells with X + XO, glucose plus glucose oxidase, or H2O2, however, diminished the capacity of the cells to convert exogenous AA to PGI2. This pretreatment effect was also blocked by catalase. The responses were similar in lung cells obtained from day 20 rat fetuses (term = 22 days) and 1-day-old newborn rats. Lactate dehydrogenase release was not detected during treatment periods but increased significantly after exposure to reactive oxygen species.(ABSTRACT TRUNCATED AT 250 WORDS)

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SETD1 and NF-κB Regulate Periodontal Inflammation through H3K4 Trimethylation

Journal of Dental Research ◽

10.1177/0022034520939029 ◽

2020 ◽

Vol 99 (13) ◽

pp. 1486-1493 ◽

Cited By ~ 1

Author(s):

M. Francis ◽

G. Gopinathan ◽

A. Salapatas ◽

S. Nares ◽

M. Gonzalez ◽

...

Keyword(s):

Gene Expression ◽

Histone Methylation ◽

Gene Promoters ◽

Cell Culture Model ◽

Inflammatory Gene Expression ◽

Inflammatory Gene ◽

H3k4 Trimethylation ◽

Periodontal Inflammation ◽

Culture Model

The inflammatory response to periodontal pathogens is dynamically controlled by the chromatin state on inflammatory gene promoters. In the present study, we have focused on the effect of the methyltransferase SETD1B on histone H3 lysine K4 (H3K4) histone trimethylation on inflammatory gene promoters. Experiments were based on 3 model systems: 1) an in vitro periodontal ligament (PDL) cell culture model for the study of SETD1 function as it relates to histone methylation and inflammatory gene expression using Porphyromonas gingivalis lipopolysaccharide (LPS) as a pathogen, 2) a subcutaneous implantation model to determine the relationship between SETD1 and nuclear factor κB (NF-κB) through its activation inhibitor BOT-64, and 3) a mouse periodontitis model to test whether the NF-κB activation inhibitor BOT-64 reverses the inflammatory tissue destruction associated with periodontal disease. In our PDL progenitor cell culture model, P. gingivalis LPS increased H3K4me3 histone methylation on IL-1β, IL-6, and MMP2 gene promoters, while SETD1B inhibition decreased H3K4me3 enrichment and inflammatory gene expression in LPS-treated PDL cells. LPS also increased SETD1 nuclear localization in a p65-dependent fashion and the nuclear translocation of p65 as mediated through SETD1, suggestive of a synergistic effect between SETD1 and p65 in the modulation of inflammation. Confirming the role of SETD1 in p65-mediated periodontal inflammation, BOT-64 reduced the number of SETD1-positive cells in inflamed periodontal tissues, restored periodontal tissue integrity, and enhanced osteogenesis in a periodontal inflammation model in vivo. Together, these results have established the histone lysine methyltransferase SETD1 as a key factor in the opening of the chromatin on inflammatory gene promoters through histone H3K4 trimethylation. Our studies also confirmed the role of BOT-64 as a potent molecular therapeutic for the restoration of periodontal health through the inhibition of NF-κB activity and the amelioration of SETD1-induced chromatin relaxation.

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Human vascular adhesion protein 1 (VAP-1) is a unique sialoglycoprotein that mediates carbohydrate-dependent binding of lymphocytes to endothelial cells.

Journal of Experimental Medicine ◽

10.1084/jem.183.2.569 ◽

1996 ◽

Vol 183 (2) ◽

pp. 569-579 ◽

Cited By ~ 82

Author(s):

M Salmi ◽

S Jalkanen

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Vascular Endothelial Cells ◽

Lymphoid Tissues ◽

Vascular Endothelial ◽

Adhesion Protein ◽

Culture Model ◽

Vascular Adhesion ◽

Vascular Adhesion Protein 1

The regulated interactions of leukocytes with vascular endothelial cells are crucial in controlling leukocyte traffic between blood and tissues. Vascular adhesion protein-1 (VAP-1) is a novel, human endothelial cell molecule that mediates tissue-selective lymphocyte binding. Two species (90 and 170 kD) of VAP-1 exist in lymphoid tissues. Glycosidase digestions revealed that the mature 170-kD form of VAP-1 expressed on the lumenal surfaces of vessels is a heavily sialylated glycoprotein. The sialic acids are indispensable for the function of VAP-1, since the desialylated form of VAP-1 no longer mediates lymphocyte binding. We also show that L-selectin is not required for binding of activated lymphocytes to VAP-1 under conditions of shear stress. The 90-kD form of VAP-1 was only seen in an organ culture model, and may represent a monomeric or proteolytic form of the larger species. These data indicate that L-selectin negative lymphocytes can bind to tonsillar venules via the VAP- 1-mediated pathway. Moreover, our findings extend the role of carbohydrate-mediated binding in lymphocyte-endothelial cell interactions beyond the known selectins. In conclusion, VAP-1 naturally exists as a 170-kD sialoglycoprotein that uses sialic acid residues to interact with its counter-receptors on lymphocytes under nonstatic conditions.

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Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation

In Vitro Cellular & Developmental Biology - Animal ◽

10.1007/s11626-009-9192-7 ◽

2009 ◽

Vol 45 (7) ◽

pp. 378-387 ◽

Cited By ~ 10

Author(s):

Mainak Das ◽

John W. Rumsey ◽

Neelima Bhargava ◽

Cassie Gregory ◽

Lisa Riedel ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Culture ◽

Growth Factors ◽

Free Cell ◽

Skeletal Muscle Differentiation ◽

Cell Culture Model ◽

Serum Free ◽

Myotube Formation ◽

Culture Model

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281. The role of insulin in milk protein synthesis

Reproduction Fertility and Development ◽

10.1071/srb05abs281 ◽

2005 ◽

Vol 17 (9) ◽

pp. 117

Author(s):

K. K. Menzies ◽

K. L. Macmillan ◽

K. R. Nicholas ◽

C. Lefevre ◽

C. Ormandy

Keyword(s):

Gene Expression ◽

Protein Synthesis ◽

Dairy Cows ◽

Milk Protein ◽

Explant Culture ◽

Milk Protein Gene ◽

Culture Model ◽

Milk Protein Genes ◽

Milk Volume

The mammary explant culture model has been frequently used to mimic lactation and to examine the endocrine control of milk protein gene expression. Studies in the mouse show the expression of the milk protein genes in explants requires insulin in the presence of prolactin and cortisol. The role of insulin in milk protein synthesis in the dairy cow is not as clear. The bovine mammary explant culture model has been utilised to show that insulin is essential for alpha-s1-casein gene expression and the synthesis of the casein proteins. In addition, mouse culture experiments were undertaken to provide an insight into the underlying molecular mechanisms of insulin action in hte mammary gland. A global analysis of the genes induced in the cultured explants was done using Affymetrix microarray and showed 132 genes, including the major milk protein genes, required the complement of insulin, cortisol and prolactin for maximal expression. Twenty-seven genes showed a 3-fold change in gene expression in response to insulin. The function of these genes can be largely categorised into maintenance of cell integrity, signal transduction, transport mechanisms, cellular metabolism and a direct effect on gene transcription in the nucleus. The requirement for insulin in milk protein synthesis is highlighted by its role in inducing the STAT5 gene, known to be a key transcription factor for the milk protein genes. Interestingly, dairy cows of high genetic merit have unusually low serum concentrations of insulin. This has occured in association with a high selection pressure for milk volume that has altered the regulation of blood glucose homeostasis. Our study indicates that this intensity of selection for high milk volume could be compromising the dairy cow’s potential for milk protein production: Has selecting for milk volume in many populations of dairy cows been achieved by lowering circulating insulin levels with consequent effects on the efficiency for milk protein yield as well as compromised reproductive performance.

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