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Eric R James

Publications and source records attributed to Eric R James.

5 recordsLinked to original sources
PubMed2005

Glucocorticoid-induced changes in the global gene expression of lens epithelial cells.

AIMS: Clinically, steroid use is accompanied by a risk for posterior subcapsular cataracts (PSCs). PSC possibly involves perturbation of lens epithelial cell proliferation and differentiation; however, the underlying mechanism is unknown. In this study, we aimed to characterize changes in gene expression in human lens epithelial cells exposed to glucocorticoid using DNA microarray hybridization. METHODS: Human lens epithelial cells (HLE B-3) were treated with dexamethasone (1 microM) for 24 or 48 hours or with vehicle (0.01% dimethylsulfoxide) and the derived cRNA was hybridized to U133A microarrays. Data were processed using the Affymetrix program Micro Array Suite, and ontological analyses were performed using the software dChip, filtering to exclude transcripts up- or down-regulated by less than 2-fold. RESULTS: At 24 hours, 57 transcripts were upregulated relative to controls by greater than 2- fold and 50 were downregulated by greater than 2-fold. At 48 hours, 92 transcripts were upregulated and 42 were downregulated. Twenty-two upregulated and 2 downregulated transcripts were shared between the 24-hour and 48-hour data sets. The predominant ontological groupings were: signal transduction, transcription factor activity, cytoskeleton/ECM/adhesion, transport, and cell cycle/development. Alternate ontological interpretations are possible. CONCLUSIONS: The data indicate steroid treatment of lens epithelial cells is associated with significant changes in gene expression in several functional categories and these include transcripts related to cell proliferation.

Cell Line↗
PubMed2004

Parasite cryopreservation by vitrification.

Parasitic protozoa and helminths and parasitic/vector insects each have distinct requirements for cryopreservation. Most parasitic protozoa respond to cryopreservation stresses similarly to other single cell suspensions, but few species are currently routinely cryopreserved by protocols specifically designed for vitrification. With slow equilibrium cooling, some protozoa osmotically dehydrated by solutes concentrated in the residual unfrozen fraction will survive by vitrifying. Several species of helminths, together with insect embryos cannot be cryopreserved by slow cooling protocols and have an absolute requirement for vitrification. Studies incorporating slow cooling and stepped cooling of both protozoa and helminths, particularly the intraerythrocytic stages of malaria and the schistosomula larvae of Schistosoma mansoni have aided in the design of vitrification protocols for parasites. For helminths, the most widely used cryopreservation protocol, originally successful for cryopreserving S. mansoni schistosomula, consists of the addition of ethanediol in two steps, followed by rapid cooling (approximately 5100 degrees C min(-1)) to -196 degrees C. This technique exploits the temperature-dependent differential in permeability of the cryoprotectant additive (CPA) to first permeate into the organism at 37 degrees C followed by a dehydration-mediated internal CPA increase in concentration resulting from incubation in a second higher CPA concentration at 0 degree C. Samples are rapidly warmed/diluted (approximately 14,000 degrees C min(-1)) to recover the organisms from liquid nitrogen storage. Variations on this technique have also been successful in cryopreserving the larvae and adult worms of filariae, muscle stage larvae of Trichinella spp., the infective stages of gastro-intestinal nematode parasites and insect embryos. Other protocols where the dehydration step precedes CPA addition have been used to cryopreserve entomogenous nematode larvae by vitrification. Techniques that utilize high concentrations of CPA cocktails and slower cooling, developed for the vitrification of mammalian embryos, have been applied to the cryopreservation of parasitic protozoa, but with limited success to date. Where cryopreservation by classical slow cooling methods is possible, vitrification has enhanced the levels of survival obtained. And vitrification has enabled the successful cryopreservation of those parasitic species not able to be cryopreserved by traditional methods. Since a limited number of parasitic organisms has been cryopreserved using vitrification protocols, there is considerable scope for further improvement in the cryopreservation techniques used for many parasitic species.

Animals↗
PubMed2003

Presence of a transcriptionally active glucocorticoid receptor alpha in lens epithelial cells.

PURPOSE: The purpose of this study was to determine whether lens epithelial cells (LECs) contain a glucocorticoid receptor (GR) that is transcriptionally active and that is able to induce production of known glucocorticoid-inducible proteins. METHODS: Protein and mRNA were obtained from human, rabbit, and bovine lens epithelia and from cultured human lens epithelial cells (B3, hLECs) and rabbit lens epithelial cells (N/N1003A, rLECs). Paraffin-embedded sections were prepared from human lenses for immunohistochemical localization of GR. RT-PCR was performed to amplify portions of GR, and the products were sequenced. Protein samples were analyzed by Western blot. hLECs and rLECs were transfected with pTAT3-luc and assayed for luciferase activity after treatment with dexamethasone (Dex) and/or RU486. Dex-treated LECs were also analyzed by quantitative real-time PCR and by Western blot for expression of specific mRNA and proteins. RESULTS: By PCR and sequencing, products consistent with GR sequences were obtained from human, rabbit, and bovine lenses and from hLECs and rLECs. The complete GRalpha sequence was obtained from rLECs and was found to be 89% identical with human GR. A 1757-bp 3' fragment of bovine GRalpha cDNA was also amplified from bovine lens. By Western blot, bands of approximately 94 kDa, the expected size of GR, were identified from human, rabbit, and bovine lens samples and from hLECs and rLECs, using anti-GR antibodies. Anti-GR antisera localized GR to both the cytosol of anterior and bow region LECs and to the nuclei of epithelial and early-differentiating lens fiber cells. Luciferase expression was induced in pTAT3-luc-transfected rLECs and hLECs by Dex treatment and this expression was partially (rLECs) or completely (hLECs) blocked by pretreatment with RU486. mRNA levels for type-1 glucocorticoid-induced target genes and also mRNA and protein levels for type-2 genes were upregulated after Dex exposure. CONCLUSIONS: The data confirm the existence of GR in hLECs, indicate that GR is present in rLECs, and resolve the controversy over the presence of GR in bovine lens. The GRalpha in hLECs and rLECs was shown to be transcriptionally active and the expression levels in hLECs of mRNAs and proteins known to be regulated by glucocorticoids were modified in these cells by glucocorticoid treatment.

Amino Acid Sequence↗
PubMed2002

Towards a recombinant antigen vaccine against Onchocerca volvulus.

Various approaches to identify potential vaccine candidates against onchocerciasis resulted in the cloning of recombinant proteins, which confer protection in vaccinated mice. The development of an effective vaccine against onchocerciasis has been the focus of a research program supported by the Edna McConnell Clark Foundation from 1985 to 1999. The approaches used to clone potential protective antigens and the successful vaccination of animals with some of the antigens are summarized here.

Animals↗