Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran

Leishmania major is the causative agent and Phlebotomus papatasi is the main vector of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran and elsewhere. Nested PCR protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene) in female P. papatasi. In the current investigation, L. major was found in Natanz, Isfahan province in centre of Iran, in a focus of rural ZCL. Ten (1.8%) out of 549 female P. papatasi was found to be infected with L. major based on the PCR detection and sequencing of parasite ITS-rDNA.Nine (1.8%) out of 498 female P. papatasi infected with L. major came from animal shelters, inside houses and yards. And one (1.9%) out of 51 female P. papatasi infected with L. major came from gerbil borrows. Infection rates were higher for females containing red blood meals, large eggs (semi-mature and mature) than for those without either blood meals or eggs. From the 10 infections detected three different haplotypes of L. major were identified. Two haplotypes were found to be novel. The other haplotypes of L. major was found to be identical to that of isolates of L. major from Iran and in elsewhere using GenBank data. Comparisons of infection rates between habitats will be inaccurate when the proportions of blood-fed and gravid flies differ among sandfly samples.     

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. I. Specificity and binding studies

A stable mouse hybridoma cell line has been developed that produces monoclonal antibody to human plasma apolipoprotein B. This antibody was proven to be specific for apolipoprotein B immunoblotting and an enzyme immunoassay using apolipoprotein B and other apolipoproteins. The antibody bound with comparable affinities to soluble apolipoprotein B, chylomicrons, very-low-density (VLDL) and low-density lipoproteins (LDL). Coupled to agarose, this antibody allowed complete removal of apolipoprotein B-containing lipoproteins from normolipidemic, hypertriglyceridemic and hypercholesterolemic plasma. Desialyzation and deglycosylation had no effect on its binding to LDL. The described antibody had no effect on the receptor-mediated binding of radiolabeled LDL to the human hepatoma cells (HepG2) in culture. Analysis of 25 different samples of human plasma indicated identical expression of the corresponding epitope in these individuals. The described monoclonal antibody, most likely, binds to a rather stable domain of apolipoprotein B that is not altered by the interaction with lipids or polymorphism of the apolipoprotein B. We propose that this antibody be called 'Pan B' antibody.     

Methylamine-treated low density lipoproteins elicit different responses in HepG2 cells and macrophages

Recent results from this laboratory have demonstrated the existence of labile thiolester bonds in apolipoprotein B (ApoB). Thiolester bonds can be cleaved with nucleophiles such as methylamine, resulting in conformational change. The purpose of this study was to explore whether the cellular interactions would be altered after methylamine treatment of low density lipoproteins (LDL). Human hepatoma cells, HepG2, and human monocyte derived macrophages were used for these studies. Fresh LDL were incubated with methylamine under mild alkaline conditions under N₂ and with preservatives for 24 h. The methylamine-treated LDL showed particle size and net charge identical to fresh native LDL. In addition, no oxidative modification of LDL occurred under the experimental conditions. The methylamine-treated LDL were indistinguishable from native LDL in HepG2 cells as judged by binding, degradation, cholesterol accumulation andde novo sterol synthesis. However, methylamine-treated LDL caused an increased accumulation of cholesteryl esters in macrophages which was comparable to the accumulation caused by acetylated LDL. Dual color digital imaging fluorescence microscopy revealed no competition between acetylated and methylamine-treated LDL, suggesting that the excessive uptake of methylamine-treated LDL was not mediated by the scavenger receptor. The increased accumulation of cholesteryl ester in macrophages also did not appear to stem from the classical LDL receptor. These results suggest that a new receptor binding domain is exposed due to the conformational change upon treatment of LDL with methylamine. (Mol Cell Biochem124: 67–79, 1993)Abbreviations LDL low density lipoproteins (d 1.032–1.043 g/ml for this study) - ApoB apolipoprotein B - MA methylamine - TBAR thiobarbituric acid reactive - HepG2 human hepatoma cell line - HMG-CoA reductase, -hydroxy--methylglutaryl CoA reductase - DIFM digital imaging fluorescence microscopy - FITC fluorescence isothiocyanate - ²M ²M-macroglobulin - BSA bovine serum albumin - PBS phosphate buffered saline - ACA -amino caproic acid - SDS-PAGE polyacrylamide gel electrophoresis containing SDS - TCA trichloroacetic acid - LRP lipoprotein receptor-related protein     

Apolipoprotein B-100 is the major form of this apolipoprotein secreted by human intestinal Caco-2 cells

Although the discovery of stop codon has explained the mechanism for the formation of the intestinal marker, apolipoprotein B-48, the dispute regarding the presence of apolipoprotein B-100 in the intestine is still unsettled. To further investigate the characteristics of intestinal apolipoprotein B, the newly developed human colonic adenocarcinoma Caco-2 cells which express functional properties of the differentiated enterocytes, were used. SDS-polyacrylamide gel electrophoresis analyses of the intact culture medium or its lipoproteins of d less than 1.23 g/ml showed the presence of only a single protein band of apolipoprotein B-100 with no detectable apolipoprotein B-48. After immunoblotting with oligoclonal antibodies to the amino-terminal peptide of apolipoprotein B, a trace amount of apolipoprotein B-48 was observed in the isolated lipoproteins, but not in the intact culture medium. These results suggest that apolipoprotein B-100 is the major form of apolipoprotein B secreted by human intestinal cells.     

Structure of apolipoprotein B-100 in low density lipoproteins

There is general consensus that amphipathic alpha-helices and beta sheets represent the major lipid-associating motifs of apolipoprotein (apo)B-100. In this review, we examine the existing experimental and computational evidence for the pentapartite domain structure of apoB. In the pentapartite nomenclature presented in this review (NH(2)-betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3)-COOH), the original alpha(1) globular domain (Segrest, J. P. et al. 1994. Arterioscler. Thromb. 14: 1674;-1685) is expanded to include residues 1;-1,000 and renamed the betaalpha(1) domain. This change reflects the likelihood that the betaalpha(1) domain, like lamprey lipovitellin, is a globular composite of alpha-helical and beta-sheet secondary structures that participates in lipid accumulation in the co-translationally assembled prenascent triglyceride-rich lipoprotein particles. Evidence is presented that the hydrophobic faces of the amphipathic beta sheets of the beta(1) and beta(2) domains of apoB-100 are in direct contact with the neutral lipid core of apoB-containing lipoproteins and play a role in core lipid organization. Evidence is also presented that these beta sheets largely determine LDL particle diameter. Analysis of published data shows that with a reduction in particle size, there is an increase in the number of amphipathic helices of the alpha(2) and alpha(3) domains associated with the surface lipids of the LDL particle; these increases modulate the surface pressure decreases caused by a reduction in radius of curvature. The properties of the LDL receptor-binding region within the overall domain structure of apoB-100 are also discussed. Finally, recent three-dimensional models of LDL obtained by cryoelectron microscopy and X-ray crystallography are discussed. These models show three common features: a semidiscoidal shape, a surface knob with the dimensions of the betaC globular domain of lipovitellin, and planar multilayers in the lipid core that are approximately 35 A apart; the multilayers are thought to represent cholesteryl ester in the smectic phase. These models present a conundrum: are LDL particles circulating at 37 degrees C spheroidal in shape, as generally assumed, or are they semidiscoidal in shape, as suggested by the models? The limited evidence available supports a spheroidal shape.     

Inducing broadcast coral spawning ex situ: Closed system mesocosm design and husbandry protocol

For many corals, the timing of broadcast spawning correlates strongly with a number of environmental signals (seasonal temperature, lunar, and diel cycles). Robust experimental studies examining the role of these putative cues in triggering spawning have been lacking until recently because it has not been possible to predictably induce spawning in fully closed artificial mesocosms. Here, we present a closed system mesocosm aquarium design that utilizes microprocessor technology to accurately replicate environmental conditions, including photoperiod, seasonal insolation, lunar cycles, and seasonal temperature from Singapore and the Great Barrier Reef (GBR), Australia. Coupled with appropriate coral husbandry, these mesocosms were successful in inducing, for the first time, broadcast coral spawning in a fully closed artificial ex situ environment. Four Acropora species (A. hyacinthus, A. tenuis, A. millepora, and A. microclados) from two geographical locations, kept for over 1 year, completed full gametogenic cycles ex situ. The percentage of colonies developing oocytes varied from ~29% for A. hyacinthus to 100% for A. millepora and A. microclados. Within the Singapore mesocosm, A. hyacinthus exhibited the closest synchronization to wild spawning, with all four gravid colonies releasing gametes in the same lunar month as wild predicted dates. Spawning within the GBR mesocosm commenced at the predicted wild spawn date but extended over a period of 3 months. Gamete release in relation to the time postsunset for A. hyacinthus, A. millepora, and A. tenuis was consistent with time windows previously described in the wild. Spawn date in relation to full moon, however, was delayed in all species, possibly as a result of external light pollution. The system described here could broaden the number of institutions on a global scale, that can access material for broadcast coral spawning research, providing opportunities for institutions distant from coral reefs to produce large numbers of coral larvae and juveniles for research purposes and reef restoration efforts.     

Production of apolipoproteins E and A-I by rat hepatocytes in primary culture

Hepatocytes were isolated from normal adult rat livers and cultured in a modified HI-WO/BA medium. A nearly confluent monolayer was established at the plating concentration employed. The hepatocytes synthesized ansd secreted albumin at rates similar to those observed in vivo. The cells secreted triacylglycerol in the absence of fatty acid substrate. Under these conditions the most abundant triacylglycerol molecular species contained 53 carbons. Incubation with oleic acid markedly increased triacylglycerol secretion predominantly in the form containing a total carbon number of 57. Approx. 80% of the secreted cholesterol was in the free form and this was unaffected by oleic acid. Employing monospecific antibodies constant rates of synthesis and secretion of apolipoproteins E and A-I were demonstrated by quantitative electroimmunoassay of the cell culture media. The rates of albumin, apolipoprotein E, and apolipoprotein A-I production were 1480, 170 and 60 microgram/h per g cell protein, respectively.     

Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.