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J W Harbell - One of the best experts on this subject based on the ideXlab platform.

  • measuring depth of injury doi in an isolated rabbit eye irritation test ire using biomarkers of cell death and viability
    Toxicology in Vitro, 2010
    Co-Authors: James V Jester, Joseph Ling, J W Harbell
    Abstract:

    Abstract While DOI is a mechanistic correlate to the ocular irritation response, attempts to measure DOI in alternative tests have been limited to qualitative histopathologic assessment by veterinarian pathologists. The purpose of this study was to determine whether DOI could be measured objectively by fluorescent staining for biomarkers of cell death and viability using an ex vivo isolated rabbit eye (IRE) test. A panel of nine materials characterized by in vivo DOI were selected that caused slight (3% acetic acid and 5% SDS), mild (acetone, sodium hypochlorite and 10% acetic acid), moderate (cyclohexanol and parafluoroanaline) and severe (8% sodium hydroxide and 10% benzalkonium chloride) irritation. Materials were then tested using a modified IRE test with 3 h recovery and then processed for cyrosectioning and staining using a TUNEL assay to detect cell death, Phalloidin to detect intracellular f-actin and DAPI staining to detect nuclei. Control eyes treated with water showed intense Phalloidin staining of the corneal epithelium and stromal keratocytes but no TUNEL labeling. In general, eyes treated with mild, moderate and severe irritants showed regions of TUNEL labeled epithelial and keratocyte nuclei with no Phalloidin stain overlying Phalloidin stained, undamaged cells. DOI measurements showed that slight irritants damaged  50% of the epithelium, extending at times into the anterior stroma ( 50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant ( p r  = 0.785). These data suggest that fluorescent staining of fixed and sectioned tissue using biomarkers can be used to objectively identify the depth of injury caused by ocular irritants.

  • measuring depth of injury doi in an isolated rabbit eye irritation test ire using biomarkers of cell death and viability
    Toxicology in Vitro, 2010
    Co-Authors: James V Jester, Joseph Ling, J W Harbell
    Abstract:

    While DOI is a mechanistic correlate to the ocular irritation response, attempts to measure DOI in alternative tests have been limited to qualitative histopathologic assessment by veterinarian pathologists. The purpose of this study was to determine whether DOI could be measured objectively by fluorescent staining for biomarkers of cell death and viability using an ex vivo isolated rabbit eye (IRE) test. A panel of nine materials characterized by in vivo DOI were selected that caused slight (3% acetic acid and 5% SDS), mild (acetone, sodium hypochlorite and 10% acetic acid), moderate (cyclohexanol and parafluoroanaline) and severe (8% sodium hydroxide and 10% benzalkonium chloride) irritation. Materials were then tested using a modified IRE test with 3h recovery and then processed for cyrosectioning and staining using a TUNEL assay to detect cell death, Phalloidin to detect intracellular f-actin and DAPI staining to detect nuclei. Control eyes treated with water showed intense Phalloidin staining of the corneal epithelium and stromal keratocytes but no TUNEL labeling. In general, eyes treated with mild, moderate and severe irritants showed regions of TUNEL labeled epithelial and keratocyte nuclei with no Phalloidin stain overlying Phalloidin stained, undamaged cells. DOI measurements showed that slight irritants damaged 50% of the epithelium, extending at times into the anterior stroma ( 50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant (p<0.007) correlation (r=0.785). These data suggest that fluorescent staining of fixed and sectioned tissue using biomarkers can be used to objectively identify the depth of injury caused by ocular irritants.

James V Jester - One of the best experts on this subject based on the ideXlab platform.

  • measuring depth of injury doi in an isolated rabbit eye irritation test ire using biomarkers of cell death and viability
    Toxicology in Vitro, 2010
    Co-Authors: James V Jester, Joseph Ling, J W Harbell
    Abstract:

    Abstract While DOI is a mechanistic correlate to the ocular irritation response, attempts to measure DOI in alternative tests have been limited to qualitative histopathologic assessment by veterinarian pathologists. The purpose of this study was to determine whether DOI could be measured objectively by fluorescent staining for biomarkers of cell death and viability using an ex vivo isolated rabbit eye (IRE) test. A panel of nine materials characterized by in vivo DOI were selected that caused slight (3% acetic acid and 5% SDS), mild (acetone, sodium hypochlorite and 10% acetic acid), moderate (cyclohexanol and parafluoroanaline) and severe (8% sodium hydroxide and 10% benzalkonium chloride) irritation. Materials were then tested using a modified IRE test with 3 h recovery and then processed for cyrosectioning and staining using a TUNEL assay to detect cell death, Phalloidin to detect intracellular f-actin and DAPI staining to detect nuclei. Control eyes treated with water showed intense Phalloidin staining of the corneal epithelium and stromal keratocytes but no TUNEL labeling. In general, eyes treated with mild, moderate and severe irritants showed regions of TUNEL labeled epithelial and keratocyte nuclei with no Phalloidin stain overlying Phalloidin stained, undamaged cells. DOI measurements showed that slight irritants damaged  50% of the epithelium, extending at times into the anterior stroma ( 50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant ( p r  = 0.785). These data suggest that fluorescent staining of fixed and sectioned tissue using biomarkers can be used to objectively identify the depth of injury caused by ocular irritants.

  • measuring depth of injury doi in an isolated rabbit eye irritation test ire using biomarkers of cell death and viability
    Toxicology in Vitro, 2010
    Co-Authors: James V Jester, Joseph Ling, J W Harbell
    Abstract:

    While DOI is a mechanistic correlate to the ocular irritation response, attempts to measure DOI in alternative tests have been limited to qualitative histopathologic assessment by veterinarian pathologists. The purpose of this study was to determine whether DOI could be measured objectively by fluorescent staining for biomarkers of cell death and viability using an ex vivo isolated rabbit eye (IRE) test. A panel of nine materials characterized by in vivo DOI were selected that caused slight (3% acetic acid and 5% SDS), mild (acetone, sodium hypochlorite and 10% acetic acid), moderate (cyclohexanol and parafluoroanaline) and severe (8% sodium hydroxide and 10% benzalkonium chloride) irritation. Materials were then tested using a modified IRE test with 3h recovery and then processed for cyrosectioning and staining using a TUNEL assay to detect cell death, Phalloidin to detect intracellular f-actin and DAPI staining to detect nuclei. Control eyes treated with water showed intense Phalloidin staining of the corneal epithelium and stromal keratocytes but no TUNEL labeling. In general, eyes treated with mild, moderate and severe irritants showed regions of TUNEL labeled epithelial and keratocyte nuclei with no Phalloidin stain overlying Phalloidin stained, undamaged cells. DOI measurements showed that slight irritants damaged 50% of the epithelium, extending at times into the anterior stroma ( 50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant (p<0.007) correlation (r=0.785). These data suggest that fluorescent staining of fixed and sectioned tissue using biomarkers can be used to objectively identify the depth of injury caused by ocular irritants.

Joseph Ling - One of the best experts on this subject based on the ideXlab platform.

  • measuring depth of injury doi in an isolated rabbit eye irritation test ire using biomarkers of cell death and viability
    Toxicology in Vitro, 2010
    Co-Authors: James V Jester, Joseph Ling, J W Harbell
    Abstract:

    Abstract While DOI is a mechanistic correlate to the ocular irritation response, attempts to measure DOI in alternative tests have been limited to qualitative histopathologic assessment by veterinarian pathologists. The purpose of this study was to determine whether DOI could be measured objectively by fluorescent staining for biomarkers of cell death and viability using an ex vivo isolated rabbit eye (IRE) test. A panel of nine materials characterized by in vivo DOI were selected that caused slight (3% acetic acid and 5% SDS), mild (acetone, sodium hypochlorite and 10% acetic acid), moderate (cyclohexanol and parafluoroanaline) and severe (8% sodium hydroxide and 10% benzalkonium chloride) irritation. Materials were then tested using a modified IRE test with 3 h recovery and then processed for cyrosectioning and staining using a TUNEL assay to detect cell death, Phalloidin to detect intracellular f-actin and DAPI staining to detect nuclei. Control eyes treated with water showed intense Phalloidin staining of the corneal epithelium and stromal keratocytes but no TUNEL labeling. In general, eyes treated with mild, moderate and severe irritants showed regions of TUNEL labeled epithelial and keratocyte nuclei with no Phalloidin stain overlying Phalloidin stained, undamaged cells. DOI measurements showed that slight irritants damaged  50% of the epithelium, extending at times into the anterior stroma ( 50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant ( p r  = 0.785). These data suggest that fluorescent staining of fixed and sectioned tissue using biomarkers can be used to objectively identify the depth of injury caused by ocular irritants.

  • measuring depth of injury doi in an isolated rabbit eye irritation test ire using biomarkers of cell death and viability
    Toxicology in Vitro, 2010
    Co-Authors: James V Jester, Joseph Ling, J W Harbell
    Abstract:

    While DOI is a mechanistic correlate to the ocular irritation response, attempts to measure DOI in alternative tests have been limited to qualitative histopathologic assessment by veterinarian pathologists. The purpose of this study was to determine whether DOI could be measured objectively by fluorescent staining for biomarkers of cell death and viability using an ex vivo isolated rabbit eye (IRE) test. A panel of nine materials characterized by in vivo DOI were selected that caused slight (3% acetic acid and 5% SDS), mild (acetone, sodium hypochlorite and 10% acetic acid), moderate (cyclohexanol and parafluoroanaline) and severe (8% sodium hydroxide and 10% benzalkonium chloride) irritation. Materials were then tested using a modified IRE test with 3h recovery and then processed for cyrosectioning and staining using a TUNEL assay to detect cell death, Phalloidin to detect intracellular f-actin and DAPI staining to detect nuclei. Control eyes treated with water showed intense Phalloidin staining of the corneal epithelium and stromal keratocytes but no TUNEL labeling. In general, eyes treated with mild, moderate and severe irritants showed regions of TUNEL labeled epithelial and keratocyte nuclei with no Phalloidin stain overlying Phalloidin stained, undamaged cells. DOI measurements showed that slight irritants damaged 50% of the epithelium, extending at times into the anterior stroma ( 50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant (p<0.007) correlation (r=0.785). These data suggest that fluorescent staining of fixed and sectioned tissue using biomarkers can be used to objectively identify the depth of injury caused by ocular irritants.

Rolf K Reed - One of the best experts on this subject based on the ideXlab platform.

  • effect of the cytoskeletal fixation agent Phalloidin on transcapillary albumin transport and interstitial fluid pressure in anaphylaxis in the wistar rat
    Microcirculation, 2002
    Co-Authors: Aurora Bronstad, Alison Reith, Ansgar Berg, Rolf K Reed
    Abstract:

    Objective:Interstitial fluid pressure (Pif) plays an important role in controlling interstitial fluid volume. In the early phase of rapid edema formation, such as in dextran-induced anaphylaxis in the Wistar rat, Piffalls from −0.5 mm Hg to a value between −5 and −10 mm Hg. It is believed that Pifis controlled by the interaction between connective tissue cells and the extracellular matrix. This hypothesis was tested by studying dextran-induced edema formation and the subsequent changes in Pifin response to a pretreatment with Phalloidin, a reagent that fixes the actin filaments within the cell. Methods:Pifwas measured in anesthetized female Wistar rats by using a micropuncture technique. The rats were pretreated with Phalloidin followed by dextran. Albumin extavasation (Ealb) was measured as the extravascular space of 125I-labeled human serum albumin (HSA) after 25 minutes. Total tissue water (TTW) was calculated as the difference between wet weight and dry weight divided by dry weight. Localization of ph...

Edward D Korn - One of the best experts on this subject based on the ideXlab platform.

  • jasplakinolide a cytotoxic natural product induces actin polymerization and competitively inhibits the binding of Phalloidin to f actin
    Journal of Biological Chemistry, 1994
    Co-Authors: Michael R Bubb, Adrian M Senderowicz, Edward A Sausville, Kimberly L K Duncan, Edward D Korn
    Abstract:

    Jasplakinolide, a naturally occurring cyclic peptide from the marine sponge, Jaspis johnstoni, has both fungicidal and antiproliferative activity. We now report that this peptide is a potent inducer of actin polymerization in vitro. The peptide has a much greater effect on Mg(2+)-actin than on Ca(2+)-actin. Competitive binding studies using rhodamine-Phalloidin suggest that jasplakinolide binds to F-actin competitively with Phalloidin with a dissociation constant of approximately 15 nM. This compares favorably to the previously reported IC50 of 35 nM for the antiproliferative effect of jasplakinolide on PC3 prostate carcinoma cells. The binding curve suggests that nearest neighbor positive cooperativity influences the binding of jasplakinolide (and perhaps also Phalloidin) to F-actin. These results imply that jasplakinolide may exert its cytotoxic effect in vivo by inducing actin polymerization and/or stabilizing pre-existing actin filaments.