bromochloroacetic-acid and thiazolyl-blue

Studies have reported that epithelial cell proliferation may be involved in the pathogenesis of nasal polyps (NPs). Estrogen receptor (ER)-α, one type of ER, is related to anti-inflammatory action and cell survival in certain tissues. In this study, we examined the presence or absence of ER-α in NPs and healthy inferior turbinate mucosae. We also investigated the effect of dexamethasone on ER-α expression, cell viability, and apoptosis in RPMI 2650 cells.. Immunohistochemical staining and Western blot analysis were conducted to determine the expression of ER-α in 15 NPs and 15 healthy inferior turbinate mucosae. After treating RPMI 2650 cells with dexamethasone, ER-α expression was analyzed using Western blot analysis and cell viability was determined using the MTT assay. Western blot analysis and annexin V-phycoerythrin (PE) staining were used to examine apoptotic cell death.. Western blot analysis showed that ER-α expression was upregulated in 13 of the 15 NP tissues. Immunohistochemical staining for ER-α confirmed the results of the Western blot analysis. When RPMI 2650 cells were treated with dexamethasone, both ER-α expression and cell viability were decreased. Furthermore, the treatment of RPMI 2650 cells with dexamethasone increased apoptotic cell death, as shown by increased levels of BAX and cleaved caspase-3, decreased levels of Bcl-2, and an increased percentage of positive annexin V-PE stained cells.. ER-α expression was higher in NPs than in healthy inferior turbinate mucosae. When RPMI 2650 cells were treated with dexamethasone, ER-α expression was downregulated, cell viability decreased, and apoptosis increased. The decreased cell viability may be related, at least in part, to the decreased ER-α protein levels, which likely contributed to the induction of apoptotic cell death in RPMI 2650 cells.

Topics: Anti-Inflammatory Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line; Cell Survival; Dexamethasone; Endoscopy; Estrogen Receptor alpha; Fulvestrant; Humans; Immunohistochemistry; Keratins; Nasal Polyps; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Thiazoles; Turbinates

The gingiva is the first oral tissue directly exposed to cigarette smoke (CS). Exposure to CS compromises the structure and function of gingival tissue. Damaging or altering the gingival epithelium leads to a compromised protective barrier of the periodontium, resulting in several diseases. The aim of this study was to assess the effect of repeated exposure to CS on gingival epithelial cell growth and on expression of apoptotic protein and keratin.. Primary human gingival epithelial cells were seeded on a collagen scaffold for 5 d to allow growth and stratification. The cells were then exposed for 5 min to whole CS for 3, 6 and 9 d. At the end of each exposure period, cell proliferation [using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays], gene expression [by quantitative reverse transcription polymerase chain reaction (qRT-PCR)] and protein production (by western blot analysis) were investigated.. Higher metabolic activity was found in the CS-exposed cells than in the nonexposed cells, specifically after 3 and 6 d of exposure to CS. At 9 d there was no significant difference between CS-exposed and nonexposed cells. Metabolic activity was supported by the BrdU cell-proliferation analyses, which showed increased cell growth at 3 d compared with the control. However, at 6 and 9 d, cell proliferation in the CS-exposed culture was comparable to that in the nonexposed culture. Interestingly, the Bax/Bcl-2 protein ratios decreased with increased CS exposure, suggesting cell resistance. Moreover, protein analyses showed that CS decreased expression of keratin(K) 5 at 3, 6 and 9 d, and increased expression of K14 at 6 and 9 d. Finally, mRNA analyses showed significant decreases of K1, K6, K10 and K16 in CS-exposed cultures, correlating, at times, with a decrease of protein production.. CS was shown to increase epithelial cell proliferation, which may involve cell resistance to apoptosis. This is supported by the modulation of expression of different keratin genes and proteins. Altogether, these data may explain the hyperplasia reported in gingival tissue, as well as periodontal disease, in smokers.

Topics: Adolescent; Adult; Apoptosis; bcl-2-Associated X Protein; Bromodeoxyuridine; Cell Proliferation; Cell Survival; Epithelial Cells; Epithelium; Gene Expression Regulation; Gingiva; Humans; Keratins; Nicotiana; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Smoke; Tetrazolium Salts; Thiazoles; Time Factors; Young Adult

Keratin is an important protein used for wound healing and tissue recovery. In this study, keratin was first extracted from raw materials and chemically modified to obtain stable keratin (m-keratin). The raw and m-keratin were examined by Raman spectroscopy. The molecular weight of the m-keratin was analysed by SDS-PAGE. The m-keratin was then blended with poly(hydroxybutylate-co-hydroxyvalerate) (PHBV) and electrospun to afford nanofibrous mats. These mats were characterized by field emission scanning electron microscopy (FE-SEM), electron spectroscopy for chemical analysis (ESCA) and atomic force microscopy (AFM). From the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) data, it was found that introduction of keratin enhanced cell proliferation. From wound-healing test and histological examination results, it was shown that the composite mats accelerated wound recovery remarkably as compared to the PHBV control. It was concluded that PHBV-keratin may be a good candidate as a wound dressing.

Topics: Animals; Bandages; Electrophoresis, Polyacrylamide Gel; Keratins; Mice; Mice, Nude; Microscopy, Atomic Force; Nanofibers; NIH 3T3 Cells; Polyesters; Spectrum Analysis, Raman; Tetrazolium Salts; Thiazoles; Wound Healing

Human periodontal ligament cells (hPDLCs) may play an important role in osteoclastogenesis in alveolar bone by expressing the receptor activator of NF-KappaB ligand (RANKL) and osteoprotegerin (OPG). The present study aimed to investigate the differences between the effects of osteogenic induction and 1,25-dihydroxyvitamin D(3) (VD(3)) on hPDLC proliferation and the expression of RANKL, osteoprotegerin, and the vitamin D receptor (VDR) in hPDLCs.. Primary cultures of 11 hPDLC populations from 11 donors were obtained. Three samples of each hPDLC population from passage 3 were, respectively, treated with osteogenic induction medium, 10(-8)M VD(3), or vehicle as a control. Cell proliferation at days 0, 1, 3, 5, and 7 was estimated with the MTT method. At day 6, the mRNA levels of RANKL, OPG and VDR were determined with real-time RT-PCR.. Osteogenic induction significantly promoted hPDLC proliferation, while VD(3) inhibited proliferation. Osteogenic induction significantly down-regulated the mRNA level of RANKL by 1.61-fold (P = 0.033) and decreased the level of VDR by 2.13-fold (P = 0.003), while there was no change in the level of OPG and OPG/RANKL ratio with osteogenic induction. On the contrary, VD(3) significantly up-regulated the level of RANKL by 9.58-fold (P = 0.001) and increased the level of VDR by 3.15-fold (P = 0.004), while down-regulating the OPG/RANKL ratio by 7.14-fold (P=0.004).. Osteogenic induction and VD(3) exert opposite effects in regulating hPDLC proliferation and mRNA expression of RANKL and VDR. This may induce hPDLCs to play different roles in alveolar bone metabolism.

Topics: Adolescent; Calcification, Physiologic; Calcitriol; Calcium Channel Agonists; Cell Proliferation; Cells, Cultured; Child; Coloring Agents; Down-Regulation; Humans; Keratins; Osteoclasts; Osteogenesis; Osteoprotegerin; Periodontal Ligament; RANK Ligand; Receptors, Calcitriol; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles; Time Factors; Transcription, Genetic; Up-Regulation; Vimentin; Young Adult

To determine if the gastroprotective drug OPC-12759 increased proliferation of rat conjunctival goblet cells in culture.. Cultured goblet cells were incubated with 10(-12) to 10(-8) M OPC-12759 for 1 to 7 days. Fetal bovine serum (FBS) was used as a positive control. Cell proliferation was determined by a MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and by immunohistochemical staining with anti-Ki-67, a marker of cell division. Goblet cells were identified by double-labeling with anti-Ki-67, a marker of cell division, and Ulex europaeus agglutinin I lectin, anti-MUC5AC and anticytokeratin 7. Stratified squamous cells were identified by using Griffonia (Bandeiraea) simplicifolia lectin and anticytokeratin 4 antibody.. As determined by MTT conversion to formazan, OPC-12579 at 10(-11) M induced an almost 2-fold increase in goblet cell proliferation on Days 1 and 3 of incubation but not on Days 5 and 7. The FBS at 10% increased cell proliferation by 2- to 3-fold at each time point. Daily replenishment of OPC-12579 for 3 consecutive days induced cell proliferation at all concentrations. Proliferation as determined by the number of Ki-67 positive cells increased by 4- and 3-fold at Days 1 and 3, respectively with addition of 10(-11) M OPC-12579. The FBS at 10% induced a 10-fold increase in goblet cell proliferation on Days 1, 3, and 5. Colocalization of Ulex europaeus agglutinin I, MUC5AC and anticytokeratin 7 with Ki-67 indicated that proliferating cells were goblet cells. Proliferating cells were negative for the nongoblet cell markers Bandeiraea lectin and anticytokeratin 4.. The OPC-12759 stimulates proliferation of conjunctival goblet cells in primary culture.

Topics: Alanine; Animals; Anti-Ulcer Agents; Antioxidants; Cell Proliferation; Cells, Cultured; Coloring Agents; Conjunctiva; Goblet Cells; Histocytochemistry; Keratin-7; Keratins; Ki-67 Antigen; Male; Mucin 5AC; Mucins; Quinolones; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Thiazoles

Improper use of mitomycin-C in ocular medication may result in damage to corneal cells. In this study, the toxic effects of mitomycin-C on cultured porcine keratocytes and endothelial cells were estimated by MTT, 3H-thymidine uptake and cellular counting assay methods. It was found that mitomycin-C caused a dose-dependent toxic effect to keratocytes and endothelial cells. Both cells were treated with mitomycin-C at the concentration ranging from 100, 10, 1, 0.1 to 0.01 microg/ml for 3 min, 5 min or 100 min. The 50% inhibitory dose (ID50) of mitomycin-C to keratocytes and endothelial cells as measured by MTT assay was 0.40, 0.18, 0.16 mg/ml and 0.27, 0.15, 0.14 mg/ml, respectively, after 3, 5 and 100 minutes drug treatment. The ID50 for keratocytes and endothelial cells as measured by 3H-thymidine uptake immediately, 1 day and 7 days after 100 minutes mitomycin-C treatment was 0.3, 0.0002, 143.2 microg/ml and 45.1, 101.1, 450.2 microg/ml, respectively. The ID50 for keratocytes and endothelial cells as measured by cellular counting 1 day and 7 days after mitomycin-C treatment was 232.5, 109.7 microg/ml and 239.9, 367.5 microg/ml, respectively. It is concluded that mitomycin-C is more toxic to cellular proliferation in cultured corneal keratocytes than in endothelial cells.

Topics: Animals; Cell Survival; Cells, Cultured; Coloring Agents; Cornea; Dose-Response Relationship, Drug; Endothelium, Corneal; In Vitro Techniques; Keratins; Mitomycin; Swine; Tetrazolium Salts; Thiazoles; Thymidine; Time Factors

This investigation was undertaken to develop cytotoxicity assay systems using primary cultures of rabbit corneal epithelial cells as an experimental model to evaluate oculotoxic agents and the ability of these in vitro assay systems to predict irritancy potential and delayed toxicity. We have characterized the epithelial nature of the cultures by identifying keratins with antikeratin antibodies (AE1/AE3) and by demonstrating metabolic enzymes important to the integrity of the cells: lactate dehydrogenase, glucose 6-phosphate dehydrogenase and aldolase. Eight surfactants were compared and ranked according to their cytotoxic potential. We evaluated cytotoxicity by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium, by making morphological observations and by assessing lysosomal neutral red uptake and mitochondrial 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. The cells were treated for 1 h with the surfactants and the possibility of delayed toxicity was evaluated 24 h after removal of the surfactant. The cytotoxicity of the different types of surfactants as shown by all the tests was cationic > anionic = amphoteric > non-ionic. Triton X-100, a non-ionic surfactant but a severe irritant, had a ranking similar to anionic surfactants. The in vitro rankings corresponded well to reported in vivo Draize rabbit eye test data. The 24-h test for lactate dehydrogenase leakage showed that mild and non-irritating surfactants did not demonstrate any subsequent damage after a 1-h exposure, but the extreme and severe surfactants continued to show further damage after the 1-h exposure. These in vitro findings were similar to reported in vivo results. The neutral red and MTT tests did not adequately predict the prolonged toxicity of the more irritating surfactants, as was demonstrated by the lactate dehydrogenase leakage test. We conclude that in vitro cytotoxicity assays using primary cultures of rabbit corneal epithelial cells may be used to rank the cytotoxic potential of surfactants, but only the lactate dehydrogenase leakage test was able to assess prolonged cell injury.

Topics: Animal Testing Alternatives; Animals; Cells, Cultured; Coloring Agents; Cornea; Epithelial Cells; Epithelium; Fluorescent Antibody Technique; Fructose-Bisphosphate Aldolase; Glucosephosphate Dehydrogenase; Keratins; L-Lactate Dehydrogenase; Male; Neutral Red; Rabbits; Surface-Active Agents; Tetrazolium Salts; Thiazoles