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Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts.

Mootha VV, Kanoff JM, Shankardas J, Dimitrijevich S - Mol. Vis. (2009)

Bottom Line: Microarray analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B) in KC fibroblasts (p=0.04).Immunohistochemistry also showed decreased immunostaining against alcohol dehydrogenase in the KC stromal sections compared to those obtained from Fuchs' endothelial corneal dystrophy samples.Decreased alcohol dehydrogenase in KC corneal fibroblasts represents a strong marker and possible mediator of keratoconus.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. vinod.mootha@utsouthwestern.edu

ABSTRACT

Purpose: To identify differentially expressed genes in keratoconus (KC) corneal fibroblasts.

Methods: Stromal keratocytes (having a fibroblast morphology) from KC keratoplasty specimens and eye bank donor corneas were isolated and expanded using a serum containing medium. RNA was isolated from three KC fibroblast cultures and five eye bank donor cornea fibroblast cultures. The targets from the cultured fibroblasts were hybridized to the Affymetrix U133 Plus 2.0 microarrays. Western blot analyses of cell lysates were performed to examine protein levels of interest in the two groups. Protein levels of select differentially expressed genes were further examined by immunohistochemistry. Keratocyte staining of archived KC keratoplasty specimens were graded using a 0 to 3+ scale and compared to five archived whole globes having normal corneas as well as to 10 Fuchs' dystrophy keratoplasty specimens.

Results: Microarray analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B) in KC fibroblasts (p=0.04). Decreased alcohol dehydrogenase in KC fibroblasts was confirmed by western blot analysis of early passage primary keratocyte cell lysates. Immunohistochemistry using a monoclonal mouse immunoglobulin G (IgG) against human liver alcohol dehydrogenase revealed a dramatic difference in protein staining in the keratocytes of the KC group compared to the normal cornea group. Immunohistochemistry also showed decreased immunostaining against alcohol dehydrogenase in the KC stromal sections compared to those obtained from Fuchs' endothelial corneal dystrophy samples.

Conclusions: Decreased alcohol dehydrogenase in KC corneal fibroblasts represents a strong marker and possible mediator of keratoconus.

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Alcohol dehydrogenase is a dimeric zinc metalloenzyme that catalyzes the reversible oxidation of alcohols to aldehydes.
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f4: Alcohol dehydrogenase is a dimeric zinc metalloenzyme that catalyzes the reversible oxidation of alcohols to aldehydes.

Mentions: ADH1B and ADH1C respectively code for the beta and gamma polypeptide of the class I human alcohol dehydrogenase enzyme. ADH5 encodes for the chi polypeptide of the class III human alcohol dehydrogenase. Alcohol dehydrogenase (ADH) is a dimeric zinc metalloenzyme with 40 kDa subunits that catalyzes the reversible oxidation of alcohols to aldehydes (Figure 4) [25]. Enzymes within one class form homodimers and heterodimers with each other [25]. The ADH genes are expressed in a tissue specific pattern in the body and are important in detoxification pathways with the substrate ranging from methanol to long chain alcohols and sterols (retinol) [25].


Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts.

Mootha VV, Kanoff JM, Shankardas J, Dimitrijevich S - Mol. Vis. (2009)

Alcohol dehydrogenase is a dimeric zinc metalloenzyme that catalyzes the reversible oxidation of alcohols to aldehydes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666775&req=5

f4: Alcohol dehydrogenase is a dimeric zinc metalloenzyme that catalyzes the reversible oxidation of alcohols to aldehydes.
Mentions: ADH1B and ADH1C respectively code for the beta and gamma polypeptide of the class I human alcohol dehydrogenase enzyme. ADH5 encodes for the chi polypeptide of the class III human alcohol dehydrogenase. Alcohol dehydrogenase (ADH) is a dimeric zinc metalloenzyme with 40 kDa subunits that catalyzes the reversible oxidation of alcohols to aldehydes (Figure 4) [25]. Enzymes within one class form homodimers and heterodimers with each other [25]. The ADH genes are expressed in a tissue specific pattern in the body and are important in detoxification pathways with the substrate ranging from methanol to long chain alcohols and sterols (retinol) [25].

Bottom Line: Microarray analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B) in KC fibroblasts (p=0.04).Immunohistochemistry also showed decreased immunostaining against alcohol dehydrogenase in the KC stromal sections compared to those obtained from Fuchs' endothelial corneal dystrophy samples.Decreased alcohol dehydrogenase in KC corneal fibroblasts represents a strong marker and possible mediator of keratoconus.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. vinod.mootha@utsouthwestern.edu

ABSTRACT

Purpose: To identify differentially expressed genes in keratoconus (KC) corneal fibroblasts.

Methods: Stromal keratocytes (having a fibroblast morphology) from KC keratoplasty specimens and eye bank donor corneas were isolated and expanded using a serum containing medium. RNA was isolated from three KC fibroblast cultures and five eye bank donor cornea fibroblast cultures. The targets from the cultured fibroblasts were hybridized to the Affymetrix U133 Plus 2.0 microarrays. Western blot analyses of cell lysates were performed to examine protein levels of interest in the two groups. Protein levels of select differentially expressed genes were further examined by immunohistochemistry. Keratocyte staining of archived KC keratoplasty specimens were graded using a 0 to 3+ scale and compared to five archived whole globes having normal corneas as well as to 10 Fuchs' dystrophy keratoplasty specimens.

Results: Microarray analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B) in KC fibroblasts (p=0.04). Decreased alcohol dehydrogenase in KC fibroblasts was confirmed by western blot analysis of early passage primary keratocyte cell lysates. Immunohistochemistry using a monoclonal mouse immunoglobulin G (IgG) against human liver alcohol dehydrogenase revealed a dramatic difference in protein staining in the keratocytes of the KC group compared to the normal cornea group. Immunohistochemistry also showed decreased immunostaining against alcohol dehydrogenase in the KC stromal sections compared to those obtained from Fuchs' endothelial corneal dystrophy samples.

Conclusions: Decreased alcohol dehydrogenase in KC corneal fibroblasts represents a strong marker and possible mediator of keratoconus.

Show MeSH
Related in: MedlinePlus