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Analysis of gene regulation in rabbit corneal epithelial cells induced by ultraviolet radiation.

Stevens JJ, Rogers C, Howard CB, Moore C, Chan LM - Int J Environ Res Public Health (2005)

Bottom Line: Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer.Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified.The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Research Laboratory, NIH-Center for Environmental Health, College of Science, Engineering and Technology, Jackson State University, 1400 J R Lynch Street, Jackson, Mississippi 39217, USA. jacqueline.j.stevens@jsums.edu

ABSTRACT
Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s) affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR). The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A+ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.

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Related in: MedlinePlus

Morphology of Corneal Epithelial Cells by Phase Contrast Microscopy. Morphologic changes in corneal epithelial cells exposed to UVB by phase contrast microscopy (40X magnification).A. Untreated confluent corneal epithelial cells (no exposure to UVB); a few cells were sensitive to apoptotic death.B. Morphologic changes in corneal epithelial cells exposed to UVB (0.6J/cm2) for 20 minutes; four apoptotic cells were visualized.C. Morphologic changes in corneal epithelial cells exposed to UVB (0.6J/cm2) for 45 minutes; twenty-six apoptotic cells were visualized.
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f1-ijerph-02-00051: Morphology of Corneal Epithelial Cells by Phase Contrast Microscopy. Morphologic changes in corneal epithelial cells exposed to UVB by phase contrast microscopy (40X magnification).A. Untreated confluent corneal epithelial cells (no exposure to UVB); a few cells were sensitive to apoptotic death.B. Morphologic changes in corneal epithelial cells exposed to UVB (0.6J/cm2) for 20 minutes; four apoptotic cells were visualized.C. Morphologic changes in corneal epithelial cells exposed to UVB (0.6J/cm2) for 45 minutes; twenty-six apoptotic cells were visualized.

Mentions: By phase contrast microscopy, the morphology of the cornea epithelial cells appeared changed after UVB exposure. These cells were visualized at a 40X magnification. The untreated corneal epithelial cells (Figure 1A) were healthy and attached with a cobberstone appearance. These cells have been growing for 3–4 days and a few cells have shown signs of apoptosis. After exposing the cells to UVB for 20 minutes (Figure 1B), the cells became separated and elongated due to a loss of membrane integrity. This event may be a result of primary necrosis or secondary apoptosis [28]. Four cells exhibited apoptosis (Figure 1B). Corneal epithelial cells were exposed for 45 minutes (Figure 1C), which resulted in more separation with some rounding of the cells. Some cells were detached and formed dark bodies within the cells. This may be due to necrosis and other forms of DNA damage such as single strand breaks. Twenty-five cells exhibited apoptosis (Figure 1C).


Analysis of gene regulation in rabbit corneal epithelial cells induced by ultraviolet radiation.

Stevens JJ, Rogers C, Howard CB, Moore C, Chan LM - Int J Environ Res Public Health (2005)

Morphology of Corneal Epithelial Cells by Phase Contrast Microscopy. Morphologic changes in corneal epithelial cells exposed to UVB by phase contrast microscopy (40X magnification).A. Untreated confluent corneal epithelial cells (no exposure to UVB); a few cells were sensitive to apoptotic death.B. Morphologic changes in corneal epithelial cells exposed to UVB (0.6J/cm2) for 20 minutes; four apoptotic cells were visualized.C. Morphologic changes in corneal epithelial cells exposed to UVB (0.6J/cm2) for 45 minutes; twenty-six apoptotic cells were visualized.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814696&req=5

f1-ijerph-02-00051: Morphology of Corneal Epithelial Cells by Phase Contrast Microscopy. Morphologic changes in corneal epithelial cells exposed to UVB by phase contrast microscopy (40X magnification).A. Untreated confluent corneal epithelial cells (no exposure to UVB); a few cells were sensitive to apoptotic death.B. Morphologic changes in corneal epithelial cells exposed to UVB (0.6J/cm2) for 20 minutes; four apoptotic cells were visualized.C. Morphologic changes in corneal epithelial cells exposed to UVB (0.6J/cm2) for 45 minutes; twenty-six apoptotic cells were visualized.
Mentions: By phase contrast microscopy, the morphology of the cornea epithelial cells appeared changed after UVB exposure. These cells were visualized at a 40X magnification. The untreated corneal epithelial cells (Figure 1A) were healthy and attached with a cobberstone appearance. These cells have been growing for 3–4 days and a few cells have shown signs of apoptosis. After exposing the cells to UVB for 20 minutes (Figure 1B), the cells became separated and elongated due to a loss of membrane integrity. This event may be a result of primary necrosis or secondary apoptosis [28]. Four cells exhibited apoptosis (Figure 1B). Corneal epithelial cells were exposed for 45 minutes (Figure 1C), which resulted in more separation with some rounding of the cells. Some cells were detached and formed dark bodies within the cells. This may be due to necrosis and other forms of DNA damage such as single strand breaks. Twenty-five cells exhibited apoptosis (Figure 1C).

Bottom Line: Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer.Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified.The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Research Laboratory, NIH-Center for Environmental Health, College of Science, Engineering and Technology, Jackson State University, 1400 J R Lynch Street, Jackson, Mississippi 39217, USA. jacqueline.j.stevens@jsums.edu

ABSTRACT
Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s) affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR). The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A+ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.

Show MeSH
Related in: MedlinePlus