The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines.

Studies show that lipid-free apoA-I stimulates release of cholesterol and phospholipid from fibroblasts and macrophages. ATP-binding cassette 1 (ABC1) is implicated in this release and has been identified as the genetic defect in Tangier disease, evidence that ABC1 is critical to the biogenesis of high density lipoprotein. We quantified levels of ABC1 mRNA, protein, and cholesterol efflux from J774 mouse macrophages +/- exposure to a cAMP analog. Up-regulating ABC1 mRNA correlated to increased cholesterol efflux in a dose- and time-dependent manner. mRNA levels rose after 15 min of exposure while protein levels rose after 1 h, with increased efflux 2-4 h post-treatment. In contrast to cells from wild-type mice, peritoneal macrophages from the Abc1 -/- mouse showed a lower level of basal efflux and no increase with cAMP treatment. The stimulation of efflux exhibits specificity for apoA-I, high density lipoprotein, and other apolipoproteins as cholesterol acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin. We have studied a number of cell types and found that while other cell lines express ABC1 constitutively, only J774 and elicited mouse macrophages show a substantial increase of mRNA and efflux with cAMP treatment. ApoA-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment.

High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1.

Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from Abc1 knockout (-/-) mice revealed an approximately 70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/- and -/- compared with their littermate controls. The inactivation of the Abc1 gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of Abc1-/- mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that Abc1-/- mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.

Binding and functional effects of transcription factors Sp1 and Sp3 on the proximal human lecithin:cholesterol acyltransferase promoter.

Human lecithin:cholesterol acyltransferase (LCAT) circulates in plasma bound to high density lipoproteins (HDL) and modulates the rate by which cholesteryl ester is transported to the liver. So far, little is known about the regulation of the expression of the LCAT gene. In this study we have defined the cis-elements, identified the trans-acting factors and demonstrated their functional effects and significance in determining transcriptional activity of the proximal LCAT promoter. Using deletion mutants having 5' variable ends (from nucleotides -72 to -27), we have identified the presence of two non-consensus GC-rich regions that stimulate transcription in HepG2 and HeLa cells. These regions designated sites A (-29 to -47) and B (-49 to -65) contain the CCTCC core sequence which in electromobility shift analysis is critical for the formation of two DNA-protein complexes designated I and II. Site-directed mutagenesis suggests that both sites are equally important in promoter activity, and that cooperative interactions between both sites are not required for activity. Electromobility shift and supershift experiments using oligonucleotides spanning sites A and B identified Sp1 and Sp3 as the transcription factors interacting at these sites. To determine the significance and functional effects that Sp1 and Sp3 have in regulating LCAT promoter activity, we performed transfection experiments in Drosophila SL-2 cells as they lack endogenous Sp1 and Sp3. Sp1 but not Sp3 activates the human LCAT promoter and when Sp1 is co-transfected along with Sp3, Sp3 functions as a dose-dependent repressor of Sp1-mediated activation. These findings indicate that Sp1 is capable of transactivating a reporter gene linked to the LCAT promoter containing Sp binding sites and suggests that the levels of Sp3 or the nuclear Sp1/Sp3 ratio may play an important role in determining the transcriptional activity of the LCAT promoter in vivo.

Susceptibility of wild mammals to infection with Naegleria fowleri.

Animals of 4 families of small wild mammals were live-trapped and inoculated intranasally with Naegleria fowleri to determine patterns of susceptibility. Of the 7 species of animals examined, only rodents were susceptible to N. fowleri. Susceptible animals were eastern gray squirrel, hispid cotton rat, muskrat, and house mouse. Mammals that were not susceptible at a dose of 10(6) were opossum, raccoon, and eastern cottontail rabbit. Perhaps rodents and humans share a common anatomical or physiological determinant that makes them susceptible to infection with N. fowleri.

Bovine leukosis virus transmission with mouthparts from Tabanus abactor after interrupted feeding.

A successful attempt was made to mechanically transmit bovine leukosis virus (BLV) from a BLV-infected cow with a normal lymphocyte count to sheep by inoculation with horse fly (Tabanus abactor) mouthparts. After interrupted natural feeding, horse flies were anesthetized with CO2. Mouthparts were severed and pooled into a tissue grinder containing medium. Five inocula containing the mouthparts of 10 flies each, and 5 inocula containing the mouthparts of 20 flies each, were prepared and inoculated SC in the right axilla of 10 BLV antibody-negative sheep. Five additional sheep served as controls. Serum samples were collected at 2-week intervals and tested by agar gel immunodiffusion for BLV antibodies. One sheep injected with 20 mouthparts developed antibodies to BLV at 10 weeks after inoculation. Six months after inoculation with fly mouthparts, 1 BLV antibody-negative sheep was randomly selected from each treatment group and injected, in the left axilla, with 3 ml of blood from the donor cow to confirm susceptibility of the sheep. All 3 sheep developed antibodies to BLV within 4 weeks.

Identification of horse flies (Diptera: Tabanidae) by analysis of cuticular hydrocarbons.

Thin-layer chromatography of the cuticular lipids of horse flies from Oklahoma revealed that hydrocarbon was the major lipid class present. The hydrocarbon fraction was composed of n-alkanes and methyl branched alkanes with only a small amount of alkenes present. Gas chromatography of the isolated cuticular hydrocarbons from a single species, Tabanus abactor Philip, showed no major differences in the profiles between individuals or between the sexes. Analysis of extracts of fresh, frozen, and pinned specimens yielded nearly identical hydrocarbon profiles. Profiles of several species were examined and found to be unique for each. Three species with similar morphological characteristics and similar geographical ranges. Tabanus abdominalis F., T. limbatinevris Macquart, and T. sulcifrons Macquart, were differentiated easily by comparison of the hydrocarbon profiles.

Trypanosoma cruzi from wild raccoons in Oklahoma.

Trypomastigotes of Trypanosoma cruzi were detected in the blood of 5 of 8 wild adult raccoons which were live-trapped in Tulsa, Okla. Organisms were isolated in diphasic blood agar medium and maintained in Vero cell cultures. Inoculated mice exhibited transient parasitemias without tissue involvement. Amastigote forms occurred within Vero cells. This is the first report of naturally occurring T cruzi infection of wild mammals in Oklahoma.