Roger L Miesfeld

PMID: 1330656;Abstract:

We have transfected rat ventral prostate (RVP) epithelial cells with a plasmid containing the SV40 large T-antigen in an attempt to establish a panel of cell lines that will be useful in molecular genetic studies of prostate cell function. Since the distribution of cell types in the RVP is dramatically affected by androgen withdrawal and replacement, cells isolated from normal, castrated, or castrated rats that were given daily injections of testosterone were used in these experiments. Cell lines were established in media that were supplemented or depleted of androgens to accommodate the possible requirements of different prostate cell types. Numerous cell lines were isolated which retain characteristics of RVP epithelial cells and five of these cell lines were studied in detail. All five cell lines express the SV40 large T-antigen, supporting the role of this viral protein in immortalization. The RVP cell lines were shown to contain high levels of functional glucocorticoid receptors, but very low levels of androgen binding activity even though androgen receptor RNA could be detected. It was determined that the decreased androgen receptor activity in the RVP cells was apparently due to low receptor expression based on the results of transient transfection assays using androgen receptor cDNA, Taken together, the biochemical, cytological, and morphological characterizations of the RVP cell lines suggest that they may all have been derived from basal prostate epithelial cells despite the initial differences in androgen status of the animal and the level of androgens in the culture media. © 1992 Academic Press, Inc.

PMID: 8022822;PMCID: PMC44244;Abstract:

BCL-2 is a 26-kDa integral membrane protein that represses apoptosis by an unknown mechanism. Recent findings indicate that Ca²⁺ release from the endoplasmic reticulum (ER) mediates apoptosis in mouse lymphoma cells. In view of growing evidence that BCL-2 localizes to the ER, as well as mitochondria and the perinuclear membrane, we investigated the possibility that BCL-2 represses apoptosis by regulating Ca²⁺ fluxes through the ER membrane. A cDNA encoding BCL-2 was introduced into WEHI7.2 cells and two subclones, W.Hb12 and W.Hb13, which express high and low levels of BCL-2 mRNA and protein, respectively, were isolated. WEHI7.2 cells underwent apoptosis in response to treatment with the glucocorticoid hormone dexamethasone, whereas W.Hb12 and W.Hb13 cells were protected from apoptosis, revealing a direct relationship between the level of BCL-2 expression and the degree of protection. Significantly, BCL-2 also blocked induction of apoptosis by thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca²⁺ pump. TG completely inhibited ER Ca²⁺ pumping in both WEHI7.2 and W.Hb12 cells, but the release of Ca²⁺ into the cytosol after inhibition of ER Ca²⁺ pumping was significantly less in W.Hb12 cells than in WEHI7.2 cells, indicating that BCL-2 reduces Ca²⁺ efflux through the ER membrane. By reducing ER Ca²⁺ efflux, BCL-2 interfered with a signal for "capacitative" entry of extracellular Ca²⁺, preventing a sustained increase of cytosolic Ca²⁺ in TG-treated cells. These findings suggest that BCL-2 either directly or indirectly regulates the flux of Ca²⁺ across the ER membrane, thereby abrogating Ca²⁺ signaling of apoptosis.

PMID: 22818079;PMCID: PMC3670763;Abstract:

A collection of Golgicide A (GCA) analogs has been synthesized and evaluated in larval and adult mosquito assays. Commercially available GCA is a mixture of four compounds. One enantiomer (GCA-2) of the major diastereomer in this mixture was shown to be responsible for the unique activity of GCA. Structure-activity studies (SAR) of the GCA architecture suggested that the pyridine ring was most easily manipulated without loss or gain in new activity. Eighteen GCA analogs were synthesized of which five displayed distinct behavior between larval and adult mosquitos, resulting in complete mortality of both Aedes aegypti and Anopheles stephensi larvae. Two analogs from the collection were shown to be distinct from the rest in displaying high selectivity and efficiency in killing An. stephensi larvae. © 2012 Elsevier B.V. All rights reserved.

Transcriptional control of steroid-regulated gene networks by nuclear receptor proteins results in the coordinate expression of a limited number of target genes. Although much is known about the structure and function of steroid receptors, relatively few cell-specific steroid-regulated genes have been isolated and characterized. In this paper we describe results using mRNA differential display reverse transcriptase PCR (DDPCR) to identify and isolate short cDNA sequence tags from thymocyte and prostate cells under various hormone conditions. Using this technique we have isolated several differentially expressed sequence tags (DESTs) from the mouse thymocyte cell line WEHI 7.2. Two of these DESTs, GIG10 and GIG18, are rapidly induced by dexamethasone within 2 h of treatment. GIG10 is a novel sequence and GIG18 is the mouse homologue of a human expressed sequence tag isolated from activated B lymphocytes. We also used DDPCR to isolate DESTs from androgen-modulated rat ventral prostate tissue, one of which we characterized and found to correspond to the 3' end of prostatic spermine binding protein mRNA, a known androgen-regulated gene. Modifications of the original DDPCR protocol, which we found can potentially decrease the frequency of isolating false-positive DESTs, are described and the merits of DDPCR, relative to other differential cDNA cloning strategies, are discussed.

PMID: 20100490;PMCID: PMC2878907;Abstract:

Aedes aegypti utilizes blood for energy production, egg maturation and replenishment of maternal reserves. The principle midgut enzymes responsible for bloodmeal digestion are endoproteolytic serine-type proteases within the S1.A subfamily. While there are hundreds of serine protease-like genes in the A. aegypti genome, only five are known to be expressed in the midgut. We describe the cloning, sequencing and expression profiling of seven additional serine proteases and provide a genomic and phylogenetic assessment of these findings. Of the seven genes, four are constitutively expressed and three are transcriptionally induced upon blood feeding. The amount of transcriptional induction is strongly correlated among these genes. Alignments reveal that, in general, the conserved catalytic triad, active site and accessory catalytic residues are maintained in these genes and phylogenetic analysis shows that these genes fall within three distinct clades; trypsins, chymotrypsins and serine collagenases. Interestingly, a previously described trypsin consistently arose with other serine collagenases in phylogenetic analyses. These results suggest that multiple gene duplications have arisen within the S1.A subfamily of midgut serine proteases and/or that A. aegypti has evolved an array of proteases with a broad range of substrate specificities for rapid, efficient digestion of bloodmeals. © 2010 Elsevier Ltd.