Limits...
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.

Show MeSH

Related in: MedlinePlus

Protein expression of 80 kDa dimer and 40 kDa monomer subunits of alcohol dehydrogenase in cultured corneal fibroblasts. C1-C5 are normal corneal fibroblast cell lines, and K1–K3 are keratoconus corneal fibroblast cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2666775&req=5

f2: Protein expression of 80 kDa dimer and 40 kDa monomer subunits of alcohol dehydrogenase in cultured corneal fibroblasts. C1-C5 are normal corneal fibroblast cell lines, and K1–K3 are keratoconus corneal fibroblast cell lines.

Mentions: Western blot analysis of the cell lysates of corneal fibroblasts using ADH1B mouse polyclonal antibody detected 40 kDa monomer subunits and 80 kDa dimer forms of the enzyme (Figure 2). Mean adjusted densitometry of the alcohol dehydrogenase (ADH) bands was 0.14 in the KC group versus 0.44 in the normal fibroblasts (p=0.03; Table 3).


Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts.

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

Protein expression of 80 kDa dimer and 40 kDa monomer subunits of alcohol dehydrogenase in cultured corneal fibroblasts. C1-C5 are normal corneal fibroblast cell lines, and K1–K3 are keratoconus corneal fibroblast cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Protein expression of 80 kDa dimer and 40 kDa monomer subunits of alcohol dehydrogenase in cultured corneal fibroblasts. C1-C5 are normal corneal fibroblast cell lines, and K1–K3 are keratoconus corneal fibroblast cell lines.
Mentions: Western blot analysis of the cell lysates of corneal fibroblasts using ADH1B mouse polyclonal antibody detected 40 kDa monomer subunits and 80 kDa dimer forms of the enzyme (Figure 2). Mean adjusted densitometry of the alcohol dehydrogenase (ADH) bands was 0.14 in the KC group versus 0.44 in the normal fibroblasts (p=0.03; Table 3).

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