Jeffrey J. Seilhamer

The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway

The ABC1 transporter was identified as the defect in Tangier disease by a combined strategy of gene expression microarray analysis, genetic mapping, and biochemical studies. Patients with Tangier disease have a defect in cellular cholesterol removal, which results in near zero plasma levels of HDL and in massive tissue deposition of cholesteryl esters. Blocking the expression or activity of ABC1 reduces apolipoprotein-mediated lipid efflux from cultured cells, and increasing expression of ABC1 enhances it. ABC1 expression is induced by cholesterol loading and cAMP treatment and is reduced upon subsequent cholesterol removal by apolipoproteins. The protein is incorporated into the plasma membrane in proportion to its level of expression. Different mutations were detected in the ABC1 gene of 3 unrelated patients. Thus, ABC1 has the properties of a key protein in the cellular lipid removal pathway, as emphasized by the consequences of its defect in patients with Tangier disease.

A gene expression profile of Alzheimer's disease

Postmortem analysis of brains of patients with Alzheimers disease (AD) has led to diverse theories about the causes of the pathology, suggesting that this complex disease involves multiple physiological changes. In an effort to better understand the variety and integration of these changes, we generated a gene expression profile for AD brain. Comparing affected and unaffected brain regions in nine controls and six AD cases, we showed that 118 of the 7050 sequences on a broadly representative cDNA microarray were differentially expressed in the amygdala and cingulate cortex, two regions affected early in the disease. The identity of these genes suggests the most prominent upregulated physiological correlates of pathology involve chronic inflammation, cell adhesion, cell proliferation, and protein synthesis (31 upregulated genes). Conversely, downregulated correlates of pathology involve signal transduction, energy metabolism, stress response, synaptic vesicle synthesis and function, calcium binding, and cytoskeleton (87 downregulated genes). The results support several separate theories of the causes of AD pathology, as well as add to the list of genes associated with AD. In addition, approximately 10 genes of unknown function were found to correlate with the pathology.

Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation

The pattern of expression of an endogenous lectin, galectin-1, was examined in human lymphoid tissue. Galectin-1 was detected in the endothelial cells lining specialized vessels, termed high endothelial venules, in activated lymphoid tissue, but not in a resting lymph node. Cultured endothelial cells (human aortic and umbilical vein endothelial cells (HAECs and HUVECs)) expressed galectin-1. Activation of the cultured endothelial cells increased the level of galectin-1 expression, as determined by ELISA, Northern blot analysis and high throughput cDNA sequencing. These results suggest that galectin-1 expressed by endothelial cells may bind to and affect the trafficking of cells emigrating from blood into tissues.

Human and canine gene homologs of porcine brain natriuretic peptide

A cDNA clone encoding porcine brain natriuretic peptide (BNP) precursor was used to probe a canine genomic DNA library for homologous sequences. A unique clone was obtained (D1) which encoded a peptide homologous to porcine BNP. A 4.0 kB Hind III fragment of D1 containing the entire BNP precursor coding region was hybridized against blots of EcoRI digested human genomic DNA. A 7.0 kB hybridizing band was observed, which was subsequently subcloned and sequenced. This human clone, H1, also contained a sequence homologous to porcine BNP precursor. Transcription of the human BNP gene was confirmed by detection of BNP-specific sequences amplified from human atrial RNA by polymerase chain reaction. These results suggest that both BNP and ANP are present and function in a wide variety of mammalian species.

The application of Shannon entropy in the identification of putative drug targets

A major challenge in the field of functional genomics is the development of computational techniques for organizing and interpreting large amounts of gene expression data. These methods will be critical for the discovery of new therapeutic drug targets. Here, we present a simple method for determining the most likely drug target candidates from temporal gene expression patterns assayed with reverse-transcription polymerase chain reaction (RT-PCR) and DNA microarrays.

Gene Expression Microarray Data Analysis for Toxicology Profiling

Abstract: When dealing with thousands of genes, all potentially interesting, it is desirable to rank the genes according to their degree of participation in a physiological process. Therefore, genes with the highest Shannon entropy and ERL can be selected as the best toxicity target candidates, permitting preclinical scientists to focus their research and resources on those genes.

Complexity of Inflammatory Responses in Endothelial Cells and Vascular Smooth Muscle Cells Determined by Microarray Analysis

Abstract: To better understand the molecular basis of vascular cell system behavior in inflammation, we used gene expression microarrays to analyze the expression of 7,075 genes and their response to IL‐1β and TNFα in cultures of coronary artery endothelium and smooth muscle derived from a single coronary artery. The most noticeable difference between the cell types was the considerably greater magnitude and complexity of the transcriptional response in the endothelial cells. Two hundred and nine genes were regulated in the endothelium and only 39 in vascular smooth muscle. Among the 209 regulated genes in the endothelium, 99 have not been previously associated with endothelial cell activation and many implicate the endothelium in unconventional roles. For example, the induced genes include several that have only been associated with leukocyte function (e.g., IL‐7 receptor, EBI‐3 receptor) and others related to antiviral and antibacterial defense (e.g., oligoadenylate synthetase, LMP7, toll‐like receptor 4, complement component 3). In addition, 43 genes likely to participate in signal transduction (eg. IL‐18 receptor, STK2 kinase, STAF50, ANP receptor, VIP receptor, RAC3, IFP35) were regulated providing evidence that a major effect of TNFα and IL‐1β is to alter the potential of the endothelial cell to respond to various other external stimuli.

Cloning, expression and partial characterization of a novel rat phospholipase A2

We report the cloning of a novel rat cDNA encoding a Ca(2+)-dependent, low molecular weight phospholipase A2 (PLA2). A rat RNA blot hybridized with the cDNA exhibited a putative 2.4 kb transcript in heart. When the cDNA was expressed in human 293s cells, PLA2 activity accumulated in the culture medium. This conditioned medium optimally hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli at neutral to alkaline pH with 10 mM or greater Ca2+. When single substrates were tested, L-alpha-palmitoyl-2-oleoyl phosphatidylcholine was more efficiently hydrolyzed than L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine or L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol.

GALECTIN-1 IS EXPRESSED BY THYMIC EPITHELIAL CELLS IN MYASTHENIA GRAVIS

Galectin-1, a member of a family of carbohydrate binding proteins, is synthesized by thymic epithelial cells in normal juvenile thymus, and mediates adhesion of immature T cells to thymic epithelium. Because cell adhesion molecules are proposed to play a role in the thymic hyperplasia and neoplasia seen in the autoimmune disease myasthenia gravis, we examined the expression of galectin-1 in myasthenic thymi. We detected abundant galectin-1 expression in thymic epithelial cells in 27 hyperplastic and neoplastic thymi from patients with myasthenia gravis. Primary cultures of neoplastic epithelial cells from a thymoma continued to express galectin-1, and bound immature T cells; T cell binding was inhibited by the addition of the β-galactosides lactose and thiodigalactoside, suggesting that galectin-1 on the thymoma cells and a saccharide ligand on the T cells participated in cell-cell adhesion. Expression of galectin-1 by thymic epithelial cells may play a role in the thymic pathology seen in myasthenia gravis.

A novel LBP-BPI fusion protein with in vivo efficacy and longer half life

Bactericidal/permeability-increasing protein (BPI) is a neutrophil azurophilic granule protein which binds to and neutralizes the biological activity of Gram-negative bacterial endotoxin both in vitro and in vivo. The therapeutic utility of BPI is hampered by its remarkably short circulating half life. BPI shares 44% sequence identity with lipopolysaccharide binding protein (LBP), an acute phase protein which facilitates endotoxin-mediated cellular responses and has a long circulating half-life. BPI and LBP are members of a family of proteins which regulate LPS activity in vivo. Genetically engineered variants of BPI and LBP were designed to maintain the endotoxin neutralizing properties of BPI but to have an extended circulating half life. One of these, LBP-BPI, is comprised of the N-terminal half of LBP, linked to the C-terminal half of BPI. Like BPI, LBP-BPI had endotoxin neutralizing activity in vitro and in vivo and gave a significant protective effect against lethal endotoxin challenge in mice (90% survival in treated group vs 10% survival in untreated controls P < 0.001). Neither LBP nor the 25 kDa N-terminal fragments of BPI and LBP were protective in this model of lethal endotoxin challenge. Pharmacokinetic studies in CD-1 mice showed that the LBP-BPI variant is cleared 74 times more slowly than BPI. Longer half-life versions of BPI have a broader window of protection and require lower overall doses to maintain therapeutically effective circulating concentrations. Molecules such as the LBP-BPI variant will have greater therapeutic utility than the native BPI molecule by virtue of these properties.