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Biomedical subjects

E R Smith

Publications and source records attributed to E R Smith.

At least 19 recordsLinked to original sources
PubMed2001

Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells.

F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Linking global histone acetylation to the transcription enhancement of X-chromosomal genes in Drosophila males.

It has become well established for several genes that targeting of histone acetylation to promoters is required for the activation of transcription. In contrast, global patterns of acetylation have not been ascribed to any particular regulatory function. In Drosophila, a specific modification of H4, acetylation at lysine 16, is enriched at hundreds of sites on the male X chromosome due to the activity of the male-specific lethal (MSL) dosage compensation complex. Utilizing chromatin immunoprecipitation, we have determined that H4Ac16 is present along the entire length of X-linked genes targeted by the MSL complex with relatively modest levels of acetylation at the promoter regions and high levels in the middle and/or 3' end of the transcription units. We propose that global acetylation by the MSL complex increases the expression of X-linked genes by facilitating transcription elongation rather than by enhancing promoter accessibility. We have also determined that H4Ac16 is absent from a region of the X chromosome that includes a gene known to be dosage-compensated by a MSL-independent mechanism. This study represents the first biochemical interpretation of the very large body of cytological observations on the chromosomal distribution of the MSL complex.

Acetylation↗
PubMed2001

Disassociation of MAPK activation and c-Fos expression in F9 embryonic carcinoma cells following retinoic acid-induced endoderm differentiation.

Retinoic acid induces cell differentiation and suppresses cell growth in a wide spectrum of cell lines, and down-regulation of activator protein-1 activity by retinoic acid contributes to these effects. In embryonic stem cell-like F9 teratocarcinoma cells, which are widely used to study retinoic acid actions on gene regulation and early embryonic differentiation, retinoic acid treatment for 4 days resulted in suppression of cell growth and differentiation into primitive and then visceral endoderm-like cells, accompanied by a suppression of serum-induced c-Fos expression. The MAPK (ERK) pathway was involved in mitogenic signaling in F9 cells stimulated with serum. Surprisingly, although c-Fos expression was reduced, the MAPK activity was not decreased by retinoic acid treatment. We found that retinoic acid treatment inhibited the phosphorylation of Elk-1, a target of activated MAPK required for c-Fos transcription. In F9 cells, the MAPK/MEK inhibitor PD98059 suppressed Elk-1 phosphorylation and c-Fos expression, indicating that MAPK activity is required for Elk-1 phosphorylation/activation. Phosphoprotein phosphatase 2B (calcineurin), the major phosphatase for activated Elk-1, is not the target in the disassociation of MAPK activation and c-Fos expression since its inhibition by cyclosporin A or activation by ionomycin had no significant effects on serum-stimulated c-Fos expression and Elk-1 phosphorylation. Thus, we conclude that retinoic acid treatment to induce F9 cell differentiation uncouples Ras/MAPK activation from c-Fos expression by reduction of Elk-1 phosphorylation through a mechanism not involving the activation of phosphoprotein phosphatase 2B.

Animals↗
PubMed2001

Disabled-2 exerts its tumor suppressor activity by uncoupling c-Fos expression and MAP kinase activation.

Disabled-2 (Dab2) is a putative tumor suppressor in breast and ovarian cancers. Its expression is lost in a majority of tumors, and homozygous deletions have been identified in a small percentage of tumors. Dab2 expression is absent or very low in the majority of breast and ovarian cancer cell lines, including MCF-7 and SK-Br-3 breast cancer cells. Transfection and expression of Dab2 in MCF-7 and SK-Br-3 cells suppress tumorigenicity. The cells reach a much lower saturation density and have reduced ability to form colonies on agar plates. In examining the signal transduction pathway of Dab2-transfected cells, we found that serum-stimulated c-Fos expression was greatly suppressed; however, the effects of Dab2 on MAPK family kinases were not as consistent. In MCF-7 and SK-Br-3 cells, although c-Fos expression was suppressed, the Erk1/2, JNK, and p38(MAPK) activities were unchanged or even increased. Serum-stimulated c-Fos expression is dependent on MAPK/Erk activity because the MEK inhibitor PD98059 suppresses Erk activity and c-Fos expression. Therefore, Dab2 appears to uncouple MAPK activation and c-fos transcription. Thus, we conclude that Dab2 re-expression suppresses tumorigenicity by reducing c-Fos expression at a site downstream of the activation of MAPK family kinases. Because Dab2 is frequently lost in cancer, the uncoupling of MAPK activation and c-Fos expression may be a favored target for inactivation in tumorigenicity.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Chromosomal location of murine disabled-2 gene and structural comparison with its human ortholog.

Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The three spliced forms, p96, p93, and p67 of murine Dab2 cDNAs were first isolated as phosphoproteins functioning in the macrophage CSF-1 signal transduction pathway. Subsequently, the involvement of Dab2 in ovarian cancer development has been investigated: Dab2 expression is lost or greatly diminished in breast and ovarian cancers, and gene deletions have been found. Regulation of Disabled-2 expression is also found to be important in development and physiological functions. Structural information of the murine Dab2 gene is essential for studies of transcription regulation and gene function in mouse models. In this study, the mouse Dab2 gene coding sequence was identified and sequenced from three lambda phage clones containing the gene. Two BAC clones of mouse genomic DNA were also used to identify the sequences of the non-coding first exon and promoter. The first exon is separated from the second exon by a large (15 kb) intron. The mouse gene is about 40 kb in size and consists of 15 exons, producing a 3.6 kb message. The translation initiation site resides in the middle of the second exon. The mouse Dab2 gene structure is very similar to that of its human ortholog in exon/intron sizes and promoter sequences. The chromosomal localization of mouse Dab2 was mapped by FISH to chromosome 15A2, a site of syntax with the human 5p12 where human Dab2 gene resides. The information on the mouse Dab2 gene structure and promoter will be invaluable in studies of the involvement of Dab2 gene in cancer, expression, physiological function, and development in mouse models.

Animals↗
PubMed2001

GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endosomal compartment.

Glycosylphosphatidylinositol (GPI) anchoring is important for the function of several proteins in the context of their membrane trafficking pathways. We have shown previously that endocytosed GPI-anchored proteins (GPI-APs) are recycled to the plasma membrane three times more slowly than other membrane components. Recently, we found that GPI-APs are delivered to endocytic organelles, devoid of markers of the clathrin-mediated pathway, prior to their delivery to a common recycling endosomal compartment (REC). Here we show that the rate-limiting step in the recycling of GPI-APs is their slow exit from the REC; replacement of the GPI anchor with a transmembrane protein sequence abolishes retention in this compartment. Depletion of endogenous sphingolipid levels using sphingolipid synthesis inhibitors or in a sphingolipid-synthesis mutant cell line specifically enhances the rate of endocytic recycling of GPI-APs to that of other membrane components. We have shown previously that endocytic retention of GPI-APs is also relieved by cholesterol depletion. These findings strongly suggest that functional retention of GPI-APs in the REC occurs via their association with sphingolipid and cholesterol-enriched sorting platforms or 'rafts'.

Animals↗
PubMed2001

Influence of insulin and testosterone on adrenocortical steroidogenesis in vitro: preliminary studies.

OBJECTIVE: The mechanisms underlying the adrenal androgen (AA) excess of polycystic ovary syndrome (PCOS) remain unclear, although it is possible that the adrenocortical dysfunction may be a response to other, extraadrenal factors. Consistent with the pathophysiology of PCOS and with in vivo data in normal and PCOS women, we have hypothesized that insulin inhibits and that T stimulates AA secretion in vitro. DESIGN: In vitro experimental study. SETTING: University medical center. PATIENT(S): Normal human adrenals (n = 4 women, ages 25-57 years) were obtained with consent at the time of organ donation. INTERVENTION(S): Fresh adrenal tissue minces were incubated in serum-free medium with 10-microM pregnenolone substrate and 1-microM ACTH-(1-24). Challenge doses of 0.2, 1, 5, 20, and 100 nM of insulin and 1, 10, 100, 1,000, and 10,000 nM of T were added, and the media were sampled after 8 hours of incubation at 37 degrees C, 4% CO2. Dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS), and cortisol (F) were measured by radioimmunoassay (significant effects compared with the case of zero-dose control). MAIN OUTCOME MEASURE(S): The production of DHEA, DHEAS, and F in the media of the adrenal minces was compared between different subjects and at different concentrations of T and insulin. RESULT(S): Analysis of the combined data from all donors indicated that insulin stimulated DHEAS and suppressed DHEA production but had no consistent effect on F. Similar analyses of the combined data indicated that T had no significant predictable effect on the production of DHEAS, DHEA, or F. When examining donor data individually, insulin and T did elicit significant increases and/or decreases in steroid production within subjects, although no consistent trends were observed. CONCLUSION(S): On the basis of these data, it is clear that extra-adrenal factors such as insulin and T have some adrenal regulatory capacity. In general, insulin stimulated DHEAS and decreased DHEA production, suggesting that it increases adrenocortical sulfotransferase activity. However, although in the individual subjects studied, both insulin and T frequently altered the production of DHEAS, DHEA or F, these effects did not appear to be uniform or consistent from subject to subject. Expanded studies are required to confirm these results.

Adrenal Cortex↗
PubMed2001

An infectious transfer and expression system for genomic DNA loci in human and mouse cells.

The recent completion of the human genome sequence allows genomics research to focus on understanding gene complexity, expression, and regulation. However, the routine-use genomic DNA expression systems required to investigate these phenomena are not well developed. Bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) have proved excellent tools for the human genome sequencing projects. We describe a system to rapidly and efficiently deliver and express BAC and PAC library clones in human and mouse cells by converting them into infectious amplicon vectors. We show packaging and intact delivery of genomic inserts of >100 kilobases with efficiencies of up to 100%. To demonstrate that genomic loci transferred in this way are functional, the complete human hypoxanthine phosphoribosyltransferase (HPRT) locus contained within a 115-kilobase BAC insert was shown to be expressed when delivered by infection into both a human HPRT-deficient fibroblast cell line and a mouse primary hepatocyte culture derived from Hprt-/- mice. Efficient gene delivery to primary cells is especially important, as these cells cannot be expanded using antibiotic selection. This work is the first demonstration of infectious delivery and expression of genomic DNA sequences of >100 kilobases, a technique that may prove useful for analyzing gene expression from the human genome.

Animals↗
PubMed2001

Ventricular interaction and external constraint account for decreased stroke work during volume loading in CHF.

The slope of the stroke work (SW)-pulmonary capillary wedge pressure (PCWP) relation may be negative in congestive heart failure (CHF), implying decreased contractility based on the premise that PCWP is simply related to left ventricular (LV) end-diastolic volume. We hypothesized that the negative slope is explained by decreased transmural LV end-diastolic pressure (LVEDP), despite the increased LVEDP, and that contractility remains unchanged. Rapid pacing produced CHF in six dogs. Hemodynamic and dimension changes were then measured under anesthesia during volume manipulation. Volume loading increased pericardial pressure and LVEDP but decreased transmural LVEDP and SW. Right ventricular diameter increased and septum-to-LV free wall diameter decreased. Although the slopes of the SW-LVEDP relations were negative, the SW-transmural LVEDP relations remained positive, indicating unchanged contractility. Similarly, the SW-segment length relations suggested unchanged contractility. Pressure surrounding the LV must be subtracted from LVEDP to calculate transmural LVEDP accurately. When this was done in this model, the apparent decrease in contractility was no longer evident. Despite the increased LVEDP during volume loading, transmural LVEDP and therefore SW decreased and contractility remained unchanged.

Animals↗
PubMed2001

A diagnostic dilemma of fever and back pain postpartum.

A 26-year-old woman presented with fever, chills, and back pain 6 weeks postpartum. An infused CT scan of the abdomen and pelvis with IV contrast confirmed septic pelvic vein thrombophlebitis as the diagnosis. To the best of our knowledge, this is the first case report describing such a massive thrombophlebitis extending from the superior vena cava to the femoral vein inferiorly responsive to conventional anticoagulation therapy. This exceptional case reminds us to entertain septic pelvic thrombophlebitis in the differential of any patient who presents with fever and back pain of unknown etiology.

Adult↗
PubMed2001

Ventricular interaction: from bench to bedside.

Decreased right ventricle (RV) output results in decreased left ventricle end-diastolic volume (LVEDV) and output by series interaction. Direct ventricular interaction may also have a major effect on LV function. Thus, decreased LVEDV caused by reduced RV output may be further reduced by a leftward septal shift and pericardial constraint. This has been shown to be true in acute and chronic pulmonary hypertension and is now also apparent in severe congestive heart failure. The use of intracavitary LV end-diastolic pressure (LVEDP) to assess LVEDV is inappropriate if pressure surrounding the LV is increased: the surrounding pressure should be subtracted from LVEDP to calculate the effective distending (transmural) pressure which governs preload. If the surrounding pressure increases more than LVEDP, transmural LVEDP and LVEDV will decrease despite the increased LVEDP. Thus, the use of filling pressure to reflect changes in LVEDV has led to erroneous conclusions regarding changes in myocardial compliance and contractility. It is now clear that volume loading may reduce LVEDV and stroke work in pulmonary embolism, chronic lung disease and severe congestive heart failure despite increased LVEDP. The decreased stroke work is a result of reduced LV preload, not decreased contractility as would be suggested if filling pressure is used to reflect preload.

Blood Pressure↗