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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.
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PMID:Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei. 1618 75

A hallmark of renal cell carcinoma (RCC) invasion is its ability to degrade ECM by local production of gelatinase enzymes. Although many studies on RCC have demonstrated the importance of MMPs, very little information is currently known regarding the effect of inducers and inhibitors. We therefore investigated the effect of inducers and inhibitors on RCC 786-0 in vitro. Human RCC 786-0 (ATCC) was grown in RPMI medium supplemented with 10% FBS, penicillin, and streptomycin in 24-well tissue plates. At near confluence, the cells were washed with PBS; the serum-free medium was incubated with various inducers: phorbol ester (PMA), tumor necrosis factor alpha (TNF-alpha), interleukin 1-beta (IL-1beta) and lipopolysaccharides (LPS). Cells were also incubated with inhibitors: EGCG, doxycycline, and a nutrient mixture with and without PMA; retinoic acid, dexamethasone, H-7; actinomycin D, or cyclohexamide. After 24 h, the medium was removed and analyzed for MMP-2 and MMP-9 by gelatinase zymography. RCC 786-0 secreted two bands, a major band corresponding to MMP-2 and a faint band corresponding to MMP-9. PMA and TNF-alpha, with increased concentration, increased MMP-9 secretion, while IL-1beta and LPS did not significantly modify MMP-9 activity. MMP-2 secretion was not affected by any of the inducers. All the inhibitors tested without and with PMA showed a dose-dependent decrease in both MMP-2 and -9 expression. Further studies are in progress to confirm the role of MMP-9 on Matrigel invasion using PMA, cytokines, and LPS.
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PMID:Modulation of human renal cell carcinoma 786-0 MMP-2 and MMP-9 activity by inhibitors and inducers in vitro. 1672 Sep 25

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.
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PMID:Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays. 1675 May 70

A tissue culture procedure was utilized to compare tissue cell invasion by Salmonella enteritidis from molted and full feed hens. Three identical trials were performed in which 80-wk-old active laying hens were divided into 2 groups of 6 birds each. The molted hen group was subjected to a 14-d feed withdrawal, and the full-fed hen group was administered a standard layer ration. After feed treatment, crop, ileum, cecum, and ovary (small and large yellow follicles removed) were collected, rinsed in PBS, and placed into 50 mL of RPMI medium. The ends of intestine and crop tissues were tied to allow attachment of Salmonella only to the lumen surface. The RPMI medium containing 10(7) to 10(8) cfu of novobiocin and nalidixic acid-resistant phage type 13 Salmonella enteritidis was injected into the lumen of the intestine and crop tissues. Additionally, ovaries were incubated in 50 mL of RPMI medium containing 10(6) to 10(7) cfu of the Salmonella enteritidis. Tissues were incubated with Salmonella at 37 degrees C for 2 h, after which tissues were placed in 50 mL of fresh RPMI medium containing 500 microg/mL of gentamicin and incubated for 5 h at 37 degrees C to remove any Salmonella that had not penetrated tissues. Tissues were rinsed, stomached in 10 mL of PBS, serially diluted, and plated onto brilliant green agar containing novobiocin and nalidixic acid for Salmonella enumeration. Salmonella invasion of ovaries was reduced in tissues from molted hens in trials 1 and 2 as compared with full-fed controls (> 1.2 log reduction) but not in trial 3. Salmonella invasion of ceca from molted hens was numerically increased in trials 1 and 2 and significantly increased in trial 3 as compared with controls (> 0.8 log increase). No significant differences in Salmonella invasion were detected for crops and ileum. These data suggest that molting may affect invasion of tissues by Salmonella enteritidis.
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PMID:The effect of feed deprivation on tissue invasion by Salmonella enteritidis. 1690 61

In recent years, several investigators have shown that transfer of dendritic cells (DC) prevents diabetes development in non-obese diabetic (NOD) mice. Accumulating evidences showing that DC cultured in medium containing fetal calf serum (FCS) can induce a dominant unspecific immune response in tumor models after i.v. injection prompted us to investigate if the protecting effect of DC on diabetes development in NOD mice might be supported by the induction of an anti-FCS immune response in recipient mice. Five-week-old NOD mice were injected i.v. with FCS-cultured bone marrow-derived DC or PBS as control. Levels of anti-FCS and anti-bovine serum albumin (BSA) antibodies were measured in the serum of recipient mice. Anti-FCS cellular immune responses were also analysed after a single DC injection using in vitro proliferation of splenocytes either in RPMI supplemented with FCS, AIMV-BSA or RPMI containing autologous mouse serum or BSA as a read out. DC injection prevented diabetes development in NOD mice and high titers of anti-FCS and anti-BSA antibodies were detected in serum of all DC-injected mice. Besides, splenocytes isolated from DC-injected mice proliferated vigorously in the presence of bovine proteins in contrast to splenocytes isolated from control mice but removing bovine proteins abrogated the high level of proliferation of those splenocytes suggesting that lymphocytes have been primed against bovine proteins in vivo after DC injection. All together, our data show that DC transfer induced cellular and humoral anti-FCS immune responses in recipient NOD mice suggesting that the protective effect of DC relies on their unspecific immunostimulatory effects.
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PMID:Fetal calf serum-primed dendritic cells induce a strong anti-fetal calf serum immune response and diabetes protection in the non-obese diabetic mouse. 1719 60

The influence of the composition of the polymer coated polyvinyl alcohol (PVA), vinyl alcohol/vinyl amine copolymer (A-PVA) and polyethylenimine (PEI) coated superparamagnetic iron oxide nanoparticles (SPIONs) on the colloidal stability, cytotoxicity and cellular uptake of these particles in different cell media is reported in this paper. Although all examined polymer coated SPIONs were stable in water and PBS buffer these colloidal systems had different stabilities in DMEM or RPMI media without and supplemented with fetal calf serum (FCS). We found that A-PVA coating onto the surface of the SPIONs decreased the cytotoxicity of the polymer compared to the same concentration of A-PVA alone. As well, polyplexes of PEI-SPIONs with DNA in concentration used for transfection experiments showed no cytotoxicity compared to PEI and PEI-SPIONs. Our data show that the choice of medium largely influences the uptake of these particles by HeLa cells. The optimal medium is different for the different examined polymer coated SPIONs and it should be determined in each case, individually.
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PMID:Effect of cell media on polymer coated superparamagnetic iron oxide nanoparticles (SPIONs): colloidal stability, cytotoxicity, and cellular uptake studies. 1788 Dec 3

Tailorable cationic chitosan/PLGA nanoparticles (CPNP) were used for the delivery of an antisense 2'-O-methyl-RNA (2OMR) directed against RNA template of human telomerase. Here, we describe the influence of the chitosan content on binding efficiency, complex stability, uptake in different human lung cell types and finally demonstrate the efficacy of this nanoplex system. CPNPs were prepared by the emulsion-solvent evaporation method using different amounts of chitosan and purified by preparative size exclusion chromatography. The characterization by photon correlation spectroscopy and zeta potential measurements showed a small increase in size and an increase of zeta potential with increasing amounts of chitosan. Binding efficiency and complex stability with 2OMR was high in water and correlated well with the chitosan content of particles but was weak in physiologically relevant media (PBS and RPMI cell culture medium). However, flow cytometry analysis showed that the uptake of 2OMR into A549 lung cancer cells was considerably higher in combination with nanoparticles and dependent on the amount of chitosan when compared to 2OMR alone. Confocal laser scanning microscopy revealed that the uptake into A549 cells is mediated via complexes of 2OMR and chitosan/PLGA nanoparticles despite the weak binding in cell culture medium. The nanoparticles were well tolerated and efficient in inhibiting telomerase activity.
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PMID:The influence of chitosan content in cationic chitosan/PLGA nanoparticles on the delivery efficiency of antisense 2'-O-methyl-RNA directed against telomerase in lung cancer cells. 1870 37

RECIPES: Ammonium chloride lysing solution, 10x. Complete DMEM. Complete RPMI. DTT (DL-dithiothreitol), 0.1 M. EDTA (ethylenediamine tetraacetic acid), 0.5 M, pH 8. Ethidium bromide staining solution. FBS (fetal bovine serum). Formamide, deionized. Gel loading buffer, 6x. L-Glutamine, 0.2 M (100x). HBSS (Hanks' buffered salt solution). PBS (phosphate-buffered saline). RNase A stock solution (DNase-free), 2 mg/ml. SSC, 20x. TAE buffer, 50x. TBE buffer, 10x. TE buffer. TrisCl, 1 M. Trypsin/EDTA solution.
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PMID:Common stock solutions, buffers, and media. 1877 Jun 54

We tested whether granulocytes, which contaminate PBMC isolates after prolonged blood storage at room temperature, are responsible for inhibited T cell function in aged blood. We extend previous observations by characterizing these contaminating granulocytes as CD11b+ CD15+ cells comparable to activated CD11b+ CD15+ granulocytes induced by incubation of blood with FMLP. Granulocyte contamination of PBMC was observed within 6-8 h after venipuncture and room temperature storage (2.3 fold increase), and increased 11.3-fold by 24-26 h in comparison to PBMC from fresh blood. Refrigerated 22-26 hour storage of blood exacerbated granulocyte contamination (84-fold increase). In contrast, granulocyte contamination was markedly reduced if blood was diluted in RPMI-1640 medium (3.9-fold increase) or PBS (1.8-fold increase) prior to 22-26 hour room temperature storage. Granulocyte contamination significantly correlated with reduced CD3zeta chain expression, a marker of T cell dysfunction. Correspondingly, T cell proliferation following PHA stimulation was significantly decreased in PBMC with contaminating granulocytes from aged blood (77% of control) or FMLP treated blood (44% of control). Minimizing granulocyte contamination in PBMC of aged blood by cell sorting, or by reducing granulocyte activation by diluting blood in PBS prior to storage, increased CD3zeta chain expression and increased T cell proliferation following stimulation. These data indicate that granulocytes inhibit T cell function in aged blood. Therefore, preventing granulocyte activation in blood specimens is critical to maintain optimal T cell function. This may be accomplished by limiting the time from venipuncture to PBMC isolation to <8 h and may be extended to 26 h by simply diluting blood in PBS prior to room temperature storage.
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PMID:Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function. 1904 16

In this study, anti proliferative activity of structural proteins and fraction of supernatant from culture of Candida albicans on proliferation responses of lymph node cells in Balb/c mice have been evaluated. For this reason Candida albicans was cultured in RPMI medium supplemented with 10% FBS at 37 in 5% CO2 until reaching a confluent state for 2 weeks. The culture supernatant was obtained by centrifugation and purified by gel filtration chromatography and structural proteins of C. albicans were obtained from breakage of cell wall by vortexing with glass beads in suspension of PBS and 1 mM PMSF and then cultured with lymph node cells and evaluated by MTT assay. The results in this study demonstrated that both of structural proteins and fraction of supernatant from culture of Candida albicans suppress immune responses compared with Control group. Our study provides evidence that proteins of C. albicans have anti proliferative activity.
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PMID:Evaluation the anti proliferative activity of structural proteins and fraction of supernatant from culture of Candida albicans. 1907 35


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