PubMed Health⌕ Search

Biomedical subjects

A R Shepard

Publications and source records attributed to A R Shepard.

At least 19 recordsLinked to original sources
PubMed2001

Optimal procedure for extracting RNA from human ocular tissues and expression profiling of the congenital glaucoma gene FOXC1 using quantitative RT-PCR.

PURPOSE: To develop methods for obtaining high quality RNA from human donor eyes and to determine the expression profile of the congenital glaucoma gene FOXC1 in human ocular tissues. METHODS: To obtain high quality RNA from donor eyes, several different preservation methods were tested including storing eyes on ice, freezing eyes, and placing eyes in the commercial fixative RNAlaterTM prior to dissection and RNA extraction. Nine different ocular tissues from human donors were dissected and examined. Pigment-free total RNA was isolated and used for quantitative real-time RT-PCR using FOXC1 and GAPDH (internal standard) primers to assess the quality and expression of FOXC1. RESULTS: An expression profile of FOXC1 in human ocular tissues was determined using quantitative PCR of RNA isolated using a simple and effective procedure for ocular tissue preservation and pigment-free RNA isolation. Higher quality RNA was obtained from human donor eyes preserved in RNAlaterTM compared to RNA extracted from eyes stored on ice or frozen at -80 degrees C. RNA extraction techniques that removed interfering pigment from ocular tissues produced RNA that could be easily amplified by PCR. In the adult human eye, expression of FOXC1 was greatest in the trabecular meshwork (TM) followed by the optic nerve head, choroid/RPE, ciliary body, cornea, and iris. FOXC1 expression levels were much lower in other non-ocular human tissues, such as liver, muscle, lung, heart, and kidney. CONCLUSIONS: Using an optimized donor eye preservation method and tissue RNA isolation procedure, we show that the FOXC1 transcription factor gene, which is known to be associated with developmental glaucoma, also may have an important role in the adult eye.

Aged↗
PubMed2001

Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor.

Until recently, very little was known about the molecular mechanisms responsible for the development of glaucoma, a leading cause of blindness worldwide. Mutations in the glaucoma gene myocilin (MYOC, GLC1A) are associated with elevated intraocular pressure and the development of autosomal dominant juvenile glaucoma and a subset of adult-onset glaucoma. MYOC is expressed in the trabecular meshwork (TM), a tissue responsible for drainage of aqueous humor from the eye, and the tissue involved in elevated intraocular pressure associated with glaucoma. To better understand the role of MYOC in glaucoma pathogenesis, we examined the expression of normal and mutant myocilin in cultured ocular (TM) and non-ocular cells as well as in the aqueous humor of patients with and without MYOC glaucoma. Normal myocilin was secreted from cultured cells, but very little to no myocilin was secreted from cells expressing five different mutant forms of MYOC. In addition, no mutant myocilin was detected in the aqueous humor of patients harboring a nonsense MYOC mutation (Q368X). Co-transfection of cultured cells with normal and mutant myocilin led to suppression of normal myocilin secretion. These studies suggest that MYOC glaucoma is due either to insufficient levels of secreted myocilin or to compromised TM cell function caused by congestion of the TM secretory pathway.

Animals↗
PubMed2001

Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues.

PURPOSE: To examine the intracellular and extracellular expression of myocilin in the human and primate trabecular meshwork (TM) in the presence and absence of glucocorticoids. METHODS: Myocilin expression was examined in cultured human TM cells by Northern blot analysis and myocilin antibody-mediated immunoprecipitation. Myocilin expression was quantified using high-resolution two-dimensional polyacrylamide gel electrophoresis of radiolabeled proteins from human TM cells, TM tissue explants, and perfused human anterior segments cultured with and without dexamethasone (DEX) for 14 to 21 days, as well as TM tissue from pigtailed monkeys treated orally for 1 year with cortisone acetate. Immunofluorescence with anti-myocilin antibodies was used to localize cellular and extracellular expression of myocilin in cultured human TM cells. RESULTS: Glucocorticoid treatment caused a significant induction of myocilin mRNA, a tetrad of cell-associated proteins, and 8 to 20 secreted proteins (molecular mass [M(r)] 56 and 59 kDa and isoelectric point [pI] 5.2 and 5.3) in some, but not all the cultured human TM cells and explanted tissues. Western immunoblot analysis using anti-myocilin peptide antibodies identified these proteins as encoded by the MYOC gene. There was significant induction of the myocilin proteins in three perfusion-cultured human eyes, in which DEX-induced elevated intraocular pressure developed. Monkeys treated 1 year with cortisol acetate showed steroid glaucoma-like morphologic changes in the TM that correlated with the induction of myocilin in the TM. Immunofluorescence analysis of cultured TM cells localized myocilin intracellularly in discrete perinuclear and cytoplasmic vesicular deposits as well as extracellularly on the cell surface associated with the extracellular matrix. In several DEX-treated TM cell lines, there were significant levels of myocilin secreted into the media. Enzymatic deglycosylation of proteins in the TM media converted the higher molecular weight isoforms of myocilin (approximately 57 kDa) to the lower molecular weight isoforms ( approximately 55 kDa). CONCLUSIONS: Although the function of myocilin is unknown, induction of these TM proteins was found in eyes in which glucocorticoid-induced ocular hypertension developed. Therefore, myocilin may play an important pathogenic role in ocular hypertension in addition to its role in certain forms of POAG.

Aged↗
PubMed2001

Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells.

PURPOSE: To characterize the glucocorticoid responsiveness of the glaucoma gene MYOC (myocilin/TIGR) in cultured human trabecular meshwork (TM) cells. METHODS: MYOC expression in two independently derived human TM cell lines was quantified by Western immunoblot analysis of protein levels and quantitative PCR analysis of mRNA levels. Promoter activity was measured indirectly with the luciferase reporter gene in a dual luciferase reporter assay. RESULTS: Application of the synthetic glucocorticoid dexamethasone (Dex) to cultured TM cells at 100 nM resulted in a delayed (8-16 hours) induction of myocilin. The concentration dependence (median effective concentration [EC(50)], approximately 10 nM) and reversal by the glucocorticoid antagonist, RU486, implicates the glucocorticoid receptor (GR). In an interesting observation, RU486 alone acted as a partial agonist to MYOC expression. Treatment of TM cells with the protein synthesis inhibitor cycloheximide abolished the Dex induction, suggesting an indirect effect of the GR on MYOC expression. In addition, the RNA synthesis inhibitor actinomycin D also blocked Dex induction, indicating that the Dex effect was due to increased MYOC transcription. Analysis of up to 2700 nucleotides (nt) of the MYOC gene 5'-flanking region in luciferase reporter constructs showed no Dex induction, despite the presence of multiple putative glucocorticoid response element (GRE)-like half-sites in the MYOC promoter and the presence of an intact cellular GR-mediated signaling system. CONCLUSIONS: MYOC is a delayed secondary glucocorticoid-responsive gene. Characterization of the transcription factors that mediate the secondary response will shed new light on the pathophysiology of steroid-induced ocular hypertension and glaucoma.

Cells, Cultured↗
PubMed2000

Kv3.3 potassium channels in lens epithelium and corneal endothelium.

Human Kv3.3/KCNC3 is a Shaw-type potassium channel that has been mapped to chromosome 19q13.3-13.4. Complete mouse and rat Kv3.3 cDNA coding sequences have been published, yet the human Kv3.3 cDNA has remained incomplete for years. We report here for the first time the amino acid sequence for hKv3.3 and the electrophysiological behavior of the encoded channel in transiently transfected mammalian cells. In addition, we report the occurrence of Kv3.3 message in rabbit corneal endothelial cells and the properties of the currents when the corneal channel is expressed. The hKv3.3 gene is highly GC-rich (69%) and contains numerous GC runs which made DNA sequencing and PCR amplification especially problematic. The full-length sequence contains two possible start codons. The encoded 757 amino acid hKv3. 3 protein is about 93% identical to mouse and rat Kv3.3 in the first 659 amino acids before the C-terminal domains diverge greatly as a result of alternative splicing. The rabbit cornea Kv3.3 is a close sequence match for hKv3.3 even in the C-terminal domain. However, we have not yet found a cornea sequence which contains the first potential start codon from hKv3.3. Electrophysiologically, the hKv3. 3 channel produces an A-current although expression of constructs which lack the 5' region of the first start codon inactivate much more slowly than full-length constructs. This short hKv3.3 construct also shows changes in activation.

Amino Acid Sequence↗
PubMed2000

Kir2.1 Potassium channels and corneal epithelia.

PURPOSE: To determine the existence of inward rectifier (Kir2.1) sequences in mRNA from corneal epithelium and endothelium. METHODS: cDNA library construction, cloning, PCR, patch clamp. RESULTS. Both the corneal epithelium and endothelium contain mRNA for Kir2.1 inwardly rectifying potassium channels. When the cDNA is transfected into Chinese hamster ovary cells, the channel currents match those expected from Kir2.1 inward rectifiers. CONCLUSIONS: The mRNA for Kir2.1 potassium channels exists in corneal epithelium and endothelium. Therefore, Kir2.1 inwardly rectifying potassium channels probably make some contributions to the resting voltages of cornea epithelium and endothelium. Previous data, however, suggest that they are probably not the dominant contributors in these preparations.

Amino Acid Sequence↗
PubMed1999

Electrically silent potassium channel subunits from human lens epithelium.

We describe the cloning and characterization of the first human members, hKv9.1 and hKv9.3, of the electrically silent delayed-rectifying-like K+ channel subfamily. Their modulatory effects on the electrically active subfamily member hKv2.1 are also quantified. The hKv9 K+ channels were isolated from a human lens epithelium cDNA library, but both hKv9.1 mRNA and hKv9.3 mRNA were found to coexist with the mRNA for hKv2.1 in a large number of human tissues. The hKv9.1 gene is composed of a minimum of five exons, with at least two alternatively spliced exons in the 5'-untranslated region (UTR). In contrast, the hKv9.3 gene is intronless across the coding region, 3'-UTR, and all of the analyzed 5'-UTR. Radiation hybrid mapping localized the hKv9.1 gene to 20q12 and the hKv9.3 gene to 2p24. Each electrically silent subunit, when coexpressed with hKv2.l, slows deactivation and inactivation compared with hKv2. 1 expressed alone. In addition, each results in an increment in the single channel conductance.

Amino Acid Sequence↗
PubMed1998

Inwardly rectifying potassium channels in lens epithelium are from the IRK1 (Kir 2.1) family.

Lens epithelial cells from many species contain inwardly rectifying K+ channels. The channels are highly selective for K+ over Na+. They have a conductance of 27-30 pS in symmetrical 150 mM K+ solutions. The conductance to inwardly flowing current depends on the external [K+], being 1/2 maximal at about 50 mM and maximal by 110-150 mM. The amino acid sequences from lens epithelium (eight different species) show at least 98% sequence homology to each other and to the potassium channel known as IRK1 (Kir 2.1). Cloned channels from chick, rabbit, and human lens epithelium all make functional channels when their cDNA is transfected into HEK-293 or tsA-201 cells. Human lens inward rectifiers when engineered as fusion proteins with green fluorescent protein (GFP) also make functional channels. In addition, their localization in the membrane and in intracellular organelles can be demonstrated by fluorescence microscopy.

Amino Acid Sequence↗
PubMed1998

Molecular biology and electrophysiology of calcium-activated potassium channels from lens epithelium.

PURPOSE: We structurally and functionally characterized the alpha and beta subunits of the human lens epithelium Ca(++)-activated potassium channel (BK). METHODS: The two subunits were sequenced following RT-PCR with multiple primer pairs. The subunits were cloned using a PCR approach and were expressed in tsA-201 cells for patch clamp recording. Green fluorescence protein-channel subunit fusion proteins were characterized by patch clamping and were imaged by fluorescence microscopy. RESULTS: Alpha subunits alone make a large single-channel conductance, potassium-selective channel with modest Ca++ sensitivity. Beta subunits alone make no channel but, when coexpressed with the alpha subunit, make a channel with increased Ca++ sensitivity, although still less than for natural channels of this type. GFP-BK subunit fusion proteins continue to function and result in a fluorescing channel, which can be localized by fluorescence microscopy. The alpha subunit codes for a "minimal" BK channel in that none of its potential alternative splicing sites contains an "extra" exon. CONCLUSIONS: The Ca(++)-activated potassium channel known as BK has the nucleotide sequences of its alpha and beta subunits represented in messenger RNA of cultured human lens epithelium. It is the first identified channel, to date, which imparts internal Ca++ dependence to lens epithelial potassium conductance.

Amino Acid Sequence↗
PubMed1998

Ion transporters and receptors in cDNA libraries from lens and cornea epithelia.

PURPOSE: To construct tissue-specific cDNA libraries containing copies of genes of ion transporting and receptor proteins in lens and cornea epithelia. To screen the libraries for the cDNA of these molecules and to deduce the protein sequence from the cDNA sequence. METHOD: Lens and corneal cDNA libraries are not commercially available and are difficult to prepare from microdissected single animal eyes or cadaver human eyes due to the small amount of tissue. To overcome these problems, we refined an approach for preparing high efficiency, non-PCR amplified plasmid cDNA libraries, where only nanogram quantities of poly (A) RNA are required. Plasmids were electrotransformed into DH10B bacteria and routinely yielded >10(6) independent colonies with typically >90% recombinants. Randomly selected colonies were subjected to colony PCR analysis to determine the libraries average insert size (typically approximately 1-kb). Primers to known genes were used in PCR amplification to check for representation of the genes in the cDNA libraries. RESULTS: Using libraries from rabbit cornea epithelium and endothelium, cultured human lens epithelium, and alphaTN4 cells, we have found the libraries to contain the cDNA for 3 common housekeeping proteins expected to be at high copy numbers in all cells. In addition, we identified 22 rare proteins and for many we determined the complete coding regions. These include K+ channels, Cl- channels, Ca++ channels, Na+ channels, three exchangers, the Na+-HCO3- cotransporter, and three kinds of receptors. CONCLUSION: Cornea and lens libraries have been constructed from single cornea or lens preparations. The libraries often contain the cDNA sequences for ion transporting and receptor proteins which are expected to be relatively rare in these cells. Many of these cDNA's have now been sequenced and the encoded proteins identified.

Animals↗
PubMed1997

Magnetic bead capture of cDNAs from double-stranded plasmid cDNA libraries.

We have developed a cDNA library screening method which allows the simultaneous screening of >10 ( 12 ) double-stranded plasmid cDNA molecules with minimal a priori sequence knowledge. A biotinylated, gene-specific oligonucleotide probe along with abutting 'blocking' oligos is hybridized to the plasmid cDNA library and the target plasmid retrieved with paramagnetic streptavidin beads and transformed into Escherichia coli. Multiple rounds of enrichment with a target plasmid represented at 0.002-0.0001% resulted in over one-third positive clones. Our method will be useful for isolating even the rarest cDNAs starting from ESTs, isolated exons or homologous sequence information.

Bacterial Proteins↗
PubMed1995

An initiator element is required for maximal human chorionic somatomammotropin gene promoter and enhancer function.

Previous studies have indicated that cell-specific expression of the human chorionic somatomammotropin (hCS) gene may be mediated by a placental-specific enhancer (CSEn). In the current studies, we have analyzed the promoter elements that are required for enhancer and promoter function in choriocarcinoma cells (BeWo). Mutation of both hCS GHF1 sites had no effect on promoter or enhancer activity. In contrast, mutation of the Sp1 site diminished basal and CSEn-stimulated transcription by approximately 75% and approximately 56%, respectively, indicating that Sp1 was necessary but not sufficient for maximal basal and enhancer-mediated transcription. Deletion and site-specific mutation of the proximal promoter region indicated that the TATA box and an initiator site (InrE) located between nucleotides -15/+1 of the hCS promoter were required for maximal promoter and enhancer function. Mutations of the InrE were associated with reduced basal and enhancer-stimulated activities and altered transcription initiation sites. A protein of 70-kDa mass, that was preferentially expressed in human choriocarcinoma cells (BeWo and JEG-3), bound specifically to the InrE. The data suggest that an initiator present in high concentrations in placental cells contributes to the control of cell-specific hCS gene expression at the promoter level and is required for maximal enhancer function.

Base Sequence↗
PubMed1994

Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells.

We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.

Animals↗
PubMed1993

Molecular mechanisms of thyroid hormone action.

This review summarizes our current understanding of the mechanisms of thyroid hormone action. Topics included are the structures of thyroid hormone response elements (TREs), thyroid hormone receptor (TR)-TRE interactions, and TR association with other proteins to illustrate their importance in modulating both positive and negative transcriptional activation of specific genes. Certain studies focusing on other members of the steroid/thyroid hormone receptor superfamily are covered to provide a current view of how this class of receptors stabilizes transcription initiation complexes.

Animals↗
PubMed1992

Thyroid hormone responsiveness in human growth hormone-related genes. Possible correlation with receptor-induced DNA conformational changes.

Triiodothyronine (T3) induces the transcription of the human chorionic somatomammotropin (hCS) promoter transfected into rat pituitary (GC) cells, but does not stimulate the homologous human growth hormone (hGH) promoter. As demonstrated by forward and reverse mutagenesis, this differential T3 responsiveness is due to subtle structural differences in a T3 response element located between nucleotides -64 and -44 of the 5'-flanking DNA of the hGH and hCS promoters. Synthetic hCS(-70/-40) DNA binds thyroid hormone receptors with a 4-fold higher affinity than the corresponding hGH T3 response element, indicating that small differences in receptor binding properties are reflected by major differences in T3 responsiveness. Analysis of circular permutation fragments containing the native hGH and hCS or mutated hCS(-70/-40) sequences demonstrates that the thyroid hormone receptor induces DNA bending. The extent of bending shows a possible correlation with the function of these sequences, suggesting that the receptor-induced changes in DNA conformation may be required for thyroid hormone receptor action.

Animals↗