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The reported incidence of colonization of oropharyngeal medical devices with Candida spp. has increased in recent years, although few studies that have systematically examined the adherence of yeast cells to such biomaterials, the primary step in the process of colonization. This study, therefore, examined the effects of oropharyngeal atmospheric conditions (5% v/v carbon dioxide) and the presence of a salivary conditioning film on both the surface properties and adherence of Candida albicans, Candida krusei and Candida tropicalis to PVC and silicone. Furthermore, the effects of the salivary conditioning film on the surface properties of these biomaterials are reported. Growth of the three Candida spp. in an atmosphere containing 5% v/v CO2 significantly increased their cell surface hydrophobicity and reduced the zeta potential of C. albicans and C. krusei yet increased the zeta potential of C. tropicalis (p<0.05). Furthermore, growth in 5% v/v CO2 decreased the adherence of C. tropicalis and C. albicans to both PVC and silicone, however, increased adherence of C. krusei (p<0.05). Pre-treatment of the microorganisms with pooled human saliva significantly decreased their cell surface hydrophobicity and increased their adherence to either biomaterial in comparison to yeast cells that had been pre-treated with PBS (p<0.05). Saliva treatment of the microorganisms had no consistent effect on microbial zeta potential. Interestingly, adherence of the three, saliva-treated Candida spp. to saliva-treated silicone and PVC was significantly lower than whenever the microorganisms and biomaterials had been treated with PBS (p<0.05). Treatment of silicone and PVC with saliva significantly altered the surface properties, notably reducing both the advancing and receding contact angles and, additionally, the microrugosity. These effects may contribute to the decreased adherence of saliva-treated microorganisms to these biomaterials. In conclusion, this study has demonstrated the effects of physiological conditions within the oral cavity on the adherence of selected Candida spp. to biomaterials employed as oropharyngeal medical devices. In particular, this study has ominously shown that these materials act as substrates for yeast colonization, highlighting the need for advancements in biomaterial design. Furthermore, it is important that physiological conditions should be employed whenever biocompatibility of oropharyngeal biomaterials is under investigation.
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PMID:Conditioning film and environmental effects on the adherence of Candida spp. to silicone and poly(vinylchloride) biomaterials. 1534 78

The creation of nonfouling surfaces is one of the major prerequisites for microdevices for biomedical and analytical applications. Poly(ethylene glycol) (PEG), a water soluble, nontoxic, and nonimmunogenic polymer has the unique ability of reducing nonspecific protein adsorption and cell adhesion and, therefore, is generally coupled with a wide variety of surfaces to improve their biocompatibility. The performance of these modified surfaces for long-term biomedical applications largely depends on the stability of these PEG films. To this end, we have investigated the stability of covalently coupled ultrathin PEG films on silicon in aqueous in vivo like conditions for a period of 4 weeks. The PEG-modified silicon substrates were incubated in PBS (37 degrees C, pH 7.4, 5% CO2) for different periods of time and then characterized using the techniques of ellipsometry, contact angle measurement, X-ray photoelectron spectroscopy, and atomic force microscopy. The ability of the PEG-modified surfaces to control protein fouling was examined by protein adsorption studies using fluorescein isothiocyanate labeled bovine serum albumin and ellipsometry. Furthermore, the ability of these films to control fibroblast adhesion was examined. Studies suggest that the PEG-modified surfaces retain their protein and cell repulsive nature even though the PEG film thickness decreases for the period of investigation.
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PMID:Evaluation of the stability of nonfouling ultrathin poly(ethylene glycol) films for silicon-based microdevices. 1574 77

We successfully established a novel cell line (OS-1) derived from human ovarian small cell carcinoma, hypercalcemic type secreted PTH, PTH-rP and ACTH. The OS-1 cell line was established from metastatic focus of uterus. A patient was 25-year-old Japanese woman. The first she received left ovariectomy on April 2002. The histopathological diagnosis was ovarian small cell carcinoma, pT2c, Nx, Mx. Then on June 2003, metastatic focus of uterus was ectomied. A part of the recurrent tumor of uterus was cut into small pieces with razor blades, and dissociated with 0.1% trypsin-0.02% EDTA/ PBS(-) solution at room temperature. The single cells and small cell clusters were seeded into 60mm dishes and cultured in growth medium (GM: DMEM/F12 supplemented with 20% fetal bovine serum and 0.1% non-essential amino acids solution) at 37 degrees C, 4.7% CO2 in humidified air. Medium was exchanged twice a week. The OS-1 cells grew as floating cultures in the dishes. Radioimmunoassay of the conditioned media was revealed that the cultures secreted large amount of PTH, PTHrP and ACTH simultaneously. Susceptibilities of anti-cancer drugs to the OS-1 cells were examined using oxygen electrode meter (Daikin), and the results suggested VLB and TXL were effective, and CDDP, CPT-11, VP-16, VCR, CPA, MMC and CBDCA were not effective. In our knowledge, it is the first report that the cell line secreting PTH, PTHrP and ACTH was successfully established from ovarian small cell carcinoma, hypercalcemic type. We expect that OS-1 cell line contribute to study on the mechanism of ectopic hormone secretion and susceptibility of anti cancer drugs to the small cell carcinoma.
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PMID:Establishment and characterization of a human ovarian small cell carcinoma, hypercalcemic type, cell line (OS-1) secreting PTH, PthrP and ACTH--special reference to the susceptibility of anti-cancer drugs. 1603 5

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

The objective of this study was to investigate the in vitro and in vivo developmental abilities of equine embryos cryopreserved by vitrification. Twenty-eight embryos were recovered from Native pony and Thoroughbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following exposure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). Single embryos were loaded in 0.25-ml straws, cooled for 1 min in liquid nitrogen vapor and immersed in liquid nitrogen. Straws were warmed in water (20 degrees C, 20 sec), and the contents were expelled with 0.5 M sucrose in PBS. Then the sucrose was diluted in 1-step (Groups 1 and 2) or 4-steps (Group 3). Embryos (n=21) were cultured for 120 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO2 in air and evaluated morphologically. Development to the hatching or hatched blastocyst stage was obtained in 0 7 , 4 7 and 4 7 embryos in Groups 1, 2 and 3, respectively. An additional 7 embryos were vitrified-warmed according to the treatment of Group 2 (4 embryos) and Group 3 (3 embryos). Five embryos were selected after in vitro culture for 4 h and were transferred nonsurgically into the uterine horn of Day-4 recipient mares. Transfer of 2 embryos (both Day-6 blastocysts: Group-2 treatment) resulted in pregnancies with a viable fetus at Day-60 of the gestation period.
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PMID:Pregnancies following transfer of equine embryos cryopreserved by vitrification. 1672 55

The objective of the present study was to assess the in vitro viability of ovine embryos at different stages of development after combining cell sampling and vitrification. Precompacted morulae, compacted morulae and blastocysts were obtained from superovulated Sarda ewes at 4, 5 or 6 d following insemination. Embryo cell biopsy was carried out in a 100-microl drop of PBS + 10% fetal calf serum (FCS) with 10 micromol nocodazole and 7.5 microg/ml cytochalasin-b by aspiration (3-5 cells). Embryos were cryopreserved at room temperature after exposure of 2 solutions for 5 min, transferred into a vitrification solution, loaded into the center of 0.25-ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. In Experiment 1, the in vitro viability of manipulated or vitrified embryos after in vitro co-culture in TCM 199 medium with 10% FCS and sheep oviductal epithelial cells (SOEC) in 5% CO2 humidified atmosphere in air at 39 degrees C was significantly lower (P < 0.05 and P < 0.01, respectively) at precompacted morula (60 and 30%) and compacted morula (62 and 39%) stages than intact embryos at the same stages (87 and 88%). No differences were found at the blastocyst stage. In Experiment 2, the in vitro survival rate of precompacted morulae which were manipulated and immediately vitrified was lower (P < 0.05) than in those manipulated and, after a temporary period of culture, vitrified at blastocyst stage (21 vs 48%); while no differences were found at compacted morula and blastocyst stages. The results show that 1) the stage of development influences the subsequent in vitro viability of manipulated and vitrified ovine embryos, 2) temporary culture after manipulation and before vitrification improves the in vitro viability of embryos, and 3) the hole in the zona pellucida resulting from biopsy does not affect blastocyst survival after subsequent vitrification.
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PMID:Effect of biopsy and vitrification on in vitro survival of ovine embryos at different stages of development. 1672 45

The isolation and culturation of SSCs of different stage of Wuzhishan Mini Porcine (WZSP) with different way of enzymatic digestion and culturation were deaded in this study. The results of the experiment described are as the following: The proper time of isolation and culturation of SSCs of WZSP is 1-20 old days. Different old of piglets with different method. Using DMEM medium as a fundmental culture medium add different gradient at 34 degrees C in a water-saturated atmosphere of 95% air, 5% CO2. The mulberry-shaped SSCs clusters appeared as original generation in 7-8 days culture. The SSCs clusters developed half-suspendedly in the culture medium. SSCs alkaline phosphatase (AKP) staining expressed positively. Mouse embryonic fibroblast was used as feeder layer for the SSCs passage cultured, The SSCs show good attached attributes, but the number of SSCs decreased quickly after 4 days culture. By seminiferous cord fragment culturation can also appear SSCs clusters in 5 days, The SSCs clusters developed half-suspendedly in the culture medium. In addition, the testes placed in cold (4 degrees C) PBS banlanced salt solution for 24 h also can be used as a good matierials for preparation of SSCs. These results indicate that the method of solation and culturation of SSCs are very correct and efficient, all these can be utilized as a good reference for future studies.
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PMID:[Studies on spermatogonial stem cells cultured in vitro of Wuzhishan Mini Porcine]. 1689 12

In this study, possibility of the method of immobilization of basic fibroblast growth factor (bFGF) on polylactone-type polymer scaffolds via plasma treatment was investigated. To introduce acid carboxylic functional groups on the surface of the polymer matrix, poly(lactide-co-glycolide) (PLGA) film was treated with carbon dioxide (CO2) plasma and then incubated in a phosphate buffer saline (PBS, pH 7.4) solution of bFGF. The bFGF binding efficiency to the CO2 plasma-treated PLGA (PT-PLGA) films under different treating parameters was investigated and compared. It was found bFGF binding efficiency to PLGA was enhanced by CO2 plasma treatment. The binding efficiency of bFGF to PLGA was variational with CO2 plasma treating time and it reached a maximum after a treating time of 20min under the power of 20W. The changes of surface chemistry and surface topography induced by CO2 plasma treatment played main roles in improving binding efficiency. Bound bFGF was released continuously from the films for up to 7 days in vitro. The stability of bFGF immobilized on PLGA film via CO2 plasma treatment was tested further under dynamic conditions by a Parallel Plate Flow Chamber. Mouse 3T3 fibroblasts were cultured on the bFGF bound PLGA with a prior plasma treatment (20W, 20min) (PT-PLGA/bFGF) film, which showed that bFGF released from PT-PLGA/bFGF film was bioactive. Adhesion and growth of cells on PLGA scaffolds were greatly improved by immobilization of bFGF on them. Therefore, the method of CO2 plasma treatment combining bFGF anchorage not only was usable in delivering bFGF, but also could be applied extensively for surface modification of scaffolds in tissue engineering.
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PMID:The immobilization of basic fibroblast growth factor on plasma-treated poly(lactide-co-glycolide). 1831 47

A ratiometric fluorimetry is proposed for the determination of peroxynitrite (ONOO-) and the evaluation of its scavenger based on synchronous fluorescence spectroscopy. L-tyrosine, an intrinsic fluorescent aminoacid, reacts with peroxynitrite and carbon dioxide, yielding a highly fluorescent dimer of tyrosine in pH 8.5 PBS buffer solution. By synchronous fluorescence scanning, both monomer and dimer emission bands of L-tyrosine appeared at 364 and 406 nm, respectively. The ratio of F406/ F364 is quantitatively related to the concentration of peroxynitrite, where F406 represents the intensity of synchronous emission band of dimer and F364 represents that of monomer. The method has been showing merit of being insensitive to the changes in experimental parameters and offers a higher sensitivity and a broader responding linear range of the analyte concentration, compared to fluorimetric method, giving a LOD of 1.84 X 10(-8) mol x L(-1) and a linear range of 1.60 X 10(-7) mol x L(-1)-6.00 X 10(-6) mol x L(-1) for ONOO-, respectively, with a deviation of 2.4% for 1.00 X 10(-6) mol x L(-1) ONOO- (n = 8). A IC50 of 0.065 microg x mL(-1) for mitoxantrone, an antioxidant and anticancer drug, was also obtained. This method has proved to be simple and speedy. It would be easily used in the determination of ONOO- and its scavenger.
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PMID:[Study on ratiometric determination of peroxynitrite and its scavenger based on synchronous fluorescence spectroscopy]. 1897 23

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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