Biomedical subjects

Jody A Rada

Publications and source records attributed to Jody A Rada.

4 recordsLinked to original sources

PubMed2004

Microarray analysis of gene expression in human donor sclera.

PURPOSE: To develop gene expression profiles of human sclera to allow for the identification of novel, uncharacterized genes in this tissue-type, and to identify candidate genes for scleral disorders. METHODS: Total RNA was isolated from 6 donor sources of human sclerae, and reverse transcribed into cDNA using a T7-(dT) 24 primer. The resulting cDNA was in vitro transcribed to produce biotin-labeled cRNA, fragmented, and mixed with hybridization controls before a 16 h hybridization step with oligonucleotide probes on 6 Affymetrix U95A chips. The chips were scanned twice at 570 nM and the data collected using GeneChip software. Array analyses were carried out with Microarray Suite, version 5.0 (Affymetrix), using the expression analysis algorithm to run an absolute analysis after cell intensities were computed. All arrays were scaled to the same target intensity using all probe sets. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to validate the microarray results. RESULTS: There were 3,751 genes with "present" calls assigned independently to all six human scleral samples. These genes could be clustered into 4 major categories; transcription (10%), metabolism (8.8%), cell growth and proliferation (5.4%), and extracellular matrix (2%). Many extracellular matrix proteins, such as collagens 6A3 and 10A1, thrombospondins 2 and 4, and dystroglycan have not previously been shown to be expressed in sclera. RT-PCR results confirmed scleral expression in 7 extracellular matrix genes examined. CONCLUSIONS: This study demonstrated the utility of gene microarray technology in identifying global patterns of scleral gene expression, and provides an extended list of genes expressed in human sclera. Identification of genes expressed in sclera contributes to our understanding of scleral biology, and potentially provides positional candidate genes for scleral disorders such as high myopia.

Adult↗

PubMed2003

Identification of genes expressed in a human scleral cDNA library.

PURPOSE: Clones established from a human scleral cDNA library were systematically sequenced. Public database sequence comparisons were performed to generate a profile of genes expressed in the human sclera and identify candidate genes for inherited diseases with scleral involvement. METHODS: A directionally cloned pCMV-PCR cDNA library was constructed from RNA isolated from scleras of human donor eyes with known plano refractive history. Plasmid DNA was extracted from randomly selected cDNA clones, and the insert sequences were determined by 5' end single-pass sequencing. Expressed sequence tags (ESTs) were generated and analyzed with the GenBank BLASTN program to identify sequence homologies to known genes. RESULTS: A total of 609 ESTs underwent BLAST analysis. Of these, 341 (56%) matched 228 known human genes and 4 non-human genes, 252 matched uncharacterized ESTs, and 16 showed no significant homology to human or non-human known sequences. The most redundant connective tissue-related genes were alphaA-crystalline, Xalpha-1 collagen, and beta-5 integrin. Other extracellular matrix gene matches were biglycan, syndecan, decorin, fibromodulin, proline arginine-rich end leucine-rich repeat protein, transgelin, TIMP-1, and fibulin 1. Human scleral expression of all but decorin and biglycan has not previously been reported. CONCLUSIONS: This effort provides the first partial list of genes expressed in human sclera. Identification of genes expressed in the sclera contributes to our understanding of scleral biology, and potentially provides positional candidate genes for scleral disorders such as high myopia.

Adult↗

PubMed2003

Melatonin receptor expression in the cornea and sclera.

The scornea and sclera have been shown to exhibit circadian rhythms in cellular proliferation, wound healing and extracellular matrix synthesis. The distribution of melatonin Mel1a and Mel1c receptors was examined in the cornea and sclera of the Xenopus laevis eye in order to determine whether melatonin may potentially influence the growth and/or development of these ocular tissues. Sections of adult X. laevis eyes were analyzed by immunocytochemistry and confocal microscopy, using antibodies prepared against specific peptide sequences of the Xenopus Mel1a and Mel1c receptor proteins. Antibodies were pre- incubated with their appropriate antigenic peptides to control for non-specific labelling. Analysis of the distribution of Mel1a and Mel1c receptor immunoreactivity in the Xenopus eye revealed that both the Mel1a and Mel1c receptors were located in the outer fibrous layer (OFL) of the sclera, with Mel1c labelling being the most prominent. Similarly, Mel1a and Mel1c (Mel1c mostly) were also located in cells of the inner fibrous layer (IFL) with Mel1c being most abundant. The chondrocytes of the cartilaginous layer also appeared to express Mel1a, Mel1c, or both receptors. Both Mel1a and Mel1c receptor immunoreactivity were observed in the corneal epithelium and endothelium. Whereas the Mel1a antibody labelled the entire corneal epithelial layer, the Mel1c antibody labelled only the most superficial layer of epithelial cells. Cell processes of fibroblasts of the corneal stroma were immunoreactive for either Mel1a or Mel1c receptors. The identification of Mel1a and Mel1c receptors in restricted distributions in the cornea and sclera suggests that melatonin may play a role in the cellular physiology of these ocular tissues.

Animals↗

PubMed2002

Altered collagen fibril formation in the sclera of lumican-deficient mice.

PURPOSE: To better understand the role of lumican (corneal keratan sulfate proteoglycan) in the scleral extracellular matrix, collagen fibril size, shape, and organization were evaluated in the sclera of wild-type mice and in mice homozygous or heterozygous for a null mutation in the lumican gene. METHODS. Anterior and posterior sclera from 6-month-old wild-type (lum+/lum+) and lumican-deficient mice (lum+/lum- and lum-/lum-) were analyzed by transmission electron microscopy. In addition, lumican was characterized in the sclera of wild-type and lumican-deficient mice by Western blot analyses. RESULTS: Lumican was present in the mouse sclera as an approximately 48-kDa core protein containing short glycosaminoglycan side chains consisting of moderate- to low-sulfated keratan sulfate. The wild-type mouse sclera consisted of irregularly arranged lamellae of collagen fibrils with an average diameter of 47.37 +/- 0.648 nm in the anterior sclera and 54.68 +/- 0.342 nm the posterior sclera. Collagen fibrils in the sclera of lumican mutant mice (lum+/lum- and lum-/lum-) were significantly larger in diameter in anterior (72.61 +/- 0.445 and 84.47 +/- 0.394 nm, respectively) and posterior (75.92 +/- 0.361 and 80.90 +/- 0.490 nm, respectively) scleral regions compared with wild-type mice (P < 0.001). CONCLUSIONS: The results of the present study indicate that null mutations in one or both alleles of the lumican gene result in significant defects in scleral collagen fibril formation that could lead to alterations in ocular shape and size and severely affect vision.

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