Susceptibility of the anaerobic gram-negative non-sporulating rod, Bilophila wadsworthia to beta-lactams, beta-lactamase inhibitors, meropenem, metronidazole, clindamycin and quinolones.

The susceptibility of eighty-seven strain of Bilophila wadsworthia to five beta-lactams, two beta-lactamase inhibitors, meropenem, metronidazole, clindamycin and two quinolones was determined. Tests were performed by the modified reference agar dilution technique using triphenyltetrazolium chloride for endpoint reading. The test strains showed a reduced susceptibility to the beta-lactams, penicillin G (MIC90 4 micrograms/ml), ampicillin (MIC90 32 micrograms/ml), piperacillin (MIC90 64 micrograms/ml), cephalothin (MIC90 2 micrograms/ml and cefotaxim (MIC90 4 micrograms/ml). The activity of ampicillin was increased by addition of the beta-lactamase inhibitor, sulbactam (MIC90 2 micrograms/ml), as was the activity of piperacillin by the addition of tazobactam (MIC90 4 micrograms/ml) 90.8% of the strains were found to produce beta-lactamase by the nitrocefin tube method. All strains were shown to be highly susceptible to meropenem, metronidazole and clindamycin (MICs < or = 1 microgram/ml). Sparfloxacin (MIC90 1 microgram/ml) and ciprofloxacin (MIC90 0.5 microgram/ml) were found to be active against most of the strains tested.

Decreased reverse cholesterol transport from Tangier disease fibroblasts. Acceptor specificity and effect of brefeldin on lipid efflux.

Tangier disease is characterized by HDL hypercatabolism and increased deposition of cholesterol in tissues. Tangier disease skin fibroblasts have decreased apoA-I-mediated cholesterol and phospholipid efflux, which may lead to the excess accumulation of cellular cholesterol. The mechanism of apolipoprotein-mediated cholesterol efflux and the apolipoprotein acceptor specificity for cholesterol efflux from normal and Tangier disease fibroblasts was investigated. Normal cells readily effluxed cholesterol and phospholipid to apoA-I and to all of the other apolipoproteins tested (apoA-II, AIV, C-I, C-II, C-III). In contrast, Tangier cells were almost completely defective in cholesterol efflux to apoA-I and to all of the other apolipoproteins tested. HDL was also less effective, by approximately 50%, in stimulating cholesterol efflux from Tangier cells compared with normal cells. In addition, Tangier cells also showed significantly reduced phospholipid efflux to both apolipoproteins and HDL. A similar rate of cholesterol efflux, however, was observed from normal and Tangier cells when phospholipid vesicles or cyclodextrin were used as acceptors. In contrast to normal cells, only phospholipid vesicles and cyclodextrin and not apoA-I or HDL depleted intracellular cholesteryl esters from Tangier cells. Brefeldin, an inhibitor of intracellular vesicular trafficking, decreased HDL-mediated cholesterol efflux by approximately 40% but almost completely blocked both cholesterol and phospholipid efflux to apoA-I from normal cells. Brefeldin also inhibited cholesteryl ester depletion by apoA-I and HDL from normal cells. Brefeldin, however, had no significant effect on cholesterol efflux from Tangier cells to HDL. In summary, Tangier cells were found to be defective in both cholesterol and phospholipid efflux to HDL and apoA-I. The defect in apolipoprotein-mediated lipid efflux was not specific for apoA-I but also occurred for other apolipoproteins, and brefeldin blocked HDL-mediated lipid efflux from normal but not Tangier disease cells. On the basis of these results, a model is proposed whereby decreased cholesterol efflux by apolipoproteins in Tangier cells is the result of a defect in a brefeldin-sensitive pathway of lipid efflux.

Identification of novel differentially expressed hepatic genes in cholesterol-fed rabbits by a non-targeted gene approach.

Several key genes involved in cholesterol metabolism are known to be directly regulated by cholesterol. The possible indirect effect, however, of increased levels of cellular cholesterol on gene expression and its possible role in cholesterol metabolism and atherosclerosis has not been thoroughly explored. In order to determine the overall effect of cholesterol on gene expression, we isolated differentially expressed genes from a PCR-based subtraction library prepared from the liver of chow-fed and cholesterol-fed rabbits. A total of nine upregulated and four down-regulated cDNA fragments were isolated. As determined by Northern blot analysis, the expression of the isolated cDNAs began to change as early as the first week on the cholesterol-rich diet or as late as 4 weeks, which corresponded with hepatic cholesterol accumulation. Three of the cDNAs were identified by DNA sequence homology, whereas the remaining cDNAs had no significant homology match. CYP1A1, a cytochrome P450 isoenzyme, was found to be down-regulated in hepatocytes by cholesterol feeding. Osteopontin and Mac-2, which are produced by macrophages, were found to be up-regulated in Kupffer cells by cholesterol feeding. Overall these results demonstrate the usefulness of the subtraction library approach for identifying new candidate genes for exploring the pathogenesis of atherosclerosis.

O-linked glycosylation modifies the association of apolipoprotein A-II to high density lipoproteins.

O-linked glycosylation is a common post-translational modification of apolipoproteins, but no structural or functional role for it has been identified. We examined the biosynthesis of apolipoprotein (apo) A-II in Hep G2 cells and in glycosylation-defective Chinese hamster ovary (CHO) cell mutants transfected with apoA-II cDNA. Three monomeric isoforms of apoA-II with an apparent molecular mass of 8.5, 9.8, and 11.4 kDa were synthesized by Hep G2 cells and transfected wild-type CHO cells. The 9.8- and 11.4-kDa isoforms were sialylated but not the 8.5-kDa isoform. Transfected 1dlD cells, which are defective in the biosynthesis of galactose and N-acetylgalactosamine, only produced the 8.5-kDa isoform; however, when grown in media supplemented with these sugars, ldlD cells produced all three isoforms of apoA-II. Pulse-chase analysis of ldlD cells showed that glycosylation was not necessary for secretion of apoA-II. Glycosylation did modify the association of apoA-II with nascent high density lipoprotein (HDL) secreted by Hep G2 cells. The sialylated isoforms were lipid-poor and were present in the lipoprotein-deficient density range, whereas the nonsialylated 8.5-kDa isoform was associated with LpA-I, A-II lipoprotein particles in the HDL density range. ApoA-II from transfected ldlD cells, regardless of glycosylation, were lipid-poor. When preincubated with HDL from serum, however, sialylated apoA-II from both ldlD cells and Hep G2 cells associated with lipoprotein particles within the HDL3 density, whereas nonsialylated apoA-II was found throughout the HDL density range. In summary, O-linked glycosylation is not necessary for the secretion of apoA-II but does modify the association of apoA-II to HDL and may, therefore, play an important role in the metabolism of HDL.

Apolipoprotein B upstream suppressor site: identification of an element which can decrease apolipoprotein B transcription.

Elevated plasma levels of apolipoprotein B (apoB) may predispose to development of premature coronary atherosclerosis. We have identified the first well localized domain of the apoB gene which can effect negative regulation of its transcription. This region binds trans-activating factors present only in apoB producing cell lines. Mutagenesis of this region causes up-regulation of its transcriptional activity. We have termed this element apoB upstream suppressor site (aBUSS) and its trans-activators the apoB repressor proteins (ARP). aBUSS and ARP may play important roles in the transcriptional modulation of apoB.

Homozygous hypobetalipoproteinemia: transcriptional regulation and 5'-flanking sequence analysis in an apolipoprotein B deficiency state.

Apolipoprotein (apo) B is the principal apolipoprotein of chylomicrons, very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Patients with homozygous hypobetalipoproteinemia (HBL), characterized by apoB deficiency, have markedly decreased levels of hepatocyte mRNA as well as intracellular B apolipoprotein, and a virtual absence of plasma apoB. We have cloned, sequenced and analyzed the 5' regulatory region of the human apoB gene from -899 to +121 bp in normal and hypobetalipoproteinemic subjects. TATA and CAAT boxes were located at -30 and -61, respectively, and two GC-like boxes were identified at positions +56 and +108. The analysis of the HBL sequence revealed two substitutions at positions -838 and -517, when compared to the normal sequence. These substitutions were not present in any known apoB regulatory elements. The transcriptional activities of the homozygous hypobetalipoproteinemic and normal regulatory regions were compared by chloramphenicol acetyltransferase (CAT) assays in Hep G2 cells, and were found to be the same. Therefore, we conclude that the 5' regulatory region of the HBL apoB gene in this kindred is normal, and the two base substitutions do not affect promoter activity of the apoB gene. These studies suggest that a coding region abnormality in the apoB gene may lead to HBL.

Tissue-specific expression of apolipoprotein A-I (ApoA-I) is regulated by the 5'-flanking region of the human ApoA-I gene.

We have isolated and characterized a 2.5-kilobase pairs genomic DNA fragment which includes the 5'-flanking region and the first and second exons of the human apolipoprotein (apo) A-I gene. The major transcriptional start site was determined by primer extension analysis and is 235 base pairs (bp) upstream from the AUG translational start codon in liver and 234 bp upstream in the intestine. TATA box-like and CAT box-like sequences and two GC box sequences are present in the intestine 30, 108, 220, and 440 bp upstream, respectively, from the transcriptional start site. Fragments of 570 bp (-487 to +71) and 2.15 kilobase pairs (-2067 to +99) containing the 5'-flanking region of the apoA-I gene were fused upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene. These constructs, designated pA-I(0.6)CAT and pA-I(2.2)CAT, respectively, were introduced into human oral epithelial cells (KB), mouse NIH 3T3 cells, Chinese hamster ovary (CHO) cells, human hepatoma cells (Hep G2), human duodenal epithelial cells (Hutu 80), and human colonic epithelial cells (Caco-2) by calcium phosphate coprecipitation. When compared with control vectors, highly efficient CAT expression of both the pA-I(0.6)CAT and pA-I(2.2)CAT constructs were observed only in cells derived from the liver (Hep G2) and intestine (Caco-2), which is consistent with the tissue specificity of expression of the native gene. Analysis of deletion mutants of the human apoA-I 5'-flanking region revealed that: 1) the region from -250 to -199 bp, from -487 to -413 bp, and -1021 to -691 bp upstream from the transcriptional start site contain sequences required for maximum gene expression; and 2) the regions from -2067 to -1476 bp and -199 to -80 bp contain the sequences required for tissue-specific repression of apoA-I gene expression in non-apoA-I producing cells.

Inhibition of thrombin-induced platelet aggregation by uteroglobin.

Uteroglobin, a steroid-dependent, small molecular weight (15K) protein in the rabbit, inhibited thrombin-induced aggregation of both rabbit and human gel-filtered platelets (GFP). GFP aggregation by arachidonic acid was not affected by uteroglobin. There were no effects of uteroglobin on thrombin-induced clotting of plasma or purified fibrinogen, or inhibition of thrombin by antithrombin III. Additionally, preliminary results suggest that uteroglobin does not interfere with binding of thrombin to platelets. We suggest that inhibition of platelet aggregation by uteroglobin may function in preventing thrombosis and ensuring free flow of blood through the microvasculature of the uterus and the placenta and may induce some of the antimotility effects of progesterone on the uterus.

Uteroglobin inhibits phospholipase A2 activity.

Although progesterone is known to produce quiescence in the mammalian uterus, the mechanism of this effect is not clearly understood. Here, we report that uteroglobin, a progesterone-induced small molecular weight (16K) protein, inhibits phospholipase A2(PLA2) derived from porcine pancreas as well as from the RAW 264.7 macrophage cell line. We speculate that progesterone may exert its antimotility effects on the uterus via uteroglobin which, by inhibiting PLA2, decreases arachidonic acid release and subsequently reduces prostaglandin levels in this organ. This may explain why progesterone is so vital for the maintenance of pregnancy in almost all mammals.

Effect of beta-FNA on opiate delta receptor binding.

beta-FNA, the beta-fumaramate methyl ester of naltrexone, has been shown to antagonize irreversibly the actions of morphine on the guinea pig ileum and mouse vas deferens bioassays but does not affect the actions of delta-receptor ligands on the mouse vas deferens bioassay, suggesting that the compound does not irreversibly bind to the delta receptor. In this paper we examine the effect of beta-FNA on the binding of the prototypic delta agonists, Leu-enkephalin and D-Ala2-D-Leu5-enkephalin, its metabolically stable analogue, and show that treatment of membranes with beta-FNA does lead to alterations in the in vitro properties of delta receptors.

Preparation of rat brain membranes greatly enriched with either type-I-delta or type-II-delta opiate binding sites using site directed alkylating agents: evidence for a two-site allosteric model.

Although it is widely accepted that radiolabeled prototypic delta receptor agonists label two binding sites in vitro, the mechanism by which mu ligands inhibit peptide binding as well as the identity of the binding sites remains unsettled (Rothman and Westfall, Mol. Pharmacol. 21:538-547, 1982 ; Bowen et al., Proc. Natl. Acad. Sci. U.S.A. 78:4818-4822, 1981). Using the site directed, receptor selective alkylating agents, BIT and FIT (Rice et al., Science 220:314-316, 1983), we describe the preparation of membranes devoid of high affinity binding sites and demonstrate that the mu agonist oxymorphone noncompetitively inhibits the binding of [3H]DADL to the residual lower affinity binding sites.

Binding of radiolabeled opiates to slide-mounted sections of molded minced rat brain: a novel method for conducting radioreceptor assays.

To facilitate the quantitative study of the opiate receptor, we have developed a novel variation of the ligand binding technique. The binding of 3H-opiates to rat brain membranes is compared with the binding to slide-mounted sections of molded minced rat brain. The latter method is characterized by a high signal and a superior signal-to-noise ratio over a wide range of ligand concentrations. The results are discussed in reference to other ligand binding methodologies.