Lipidomic strategies to study structural and functional defects of ABC-transporters in cellular lipid trafficking

The majority of the human ATP-binding cassette (ABC)-transporters function in cellular lipid trafficking and in the regulation of membrane lipid composition associating their dysfunction with human disease phenotypes related to sterol, phospholipid and fatty acid homeostasis. Based on findings from monogenetic disorders, animal models, and in vitro systems, major clues on the expression, function and cellular localization of human ABC-transporters have been gained. Here we review novel lipidomic technologies including quantitative mRNA expression monitoring by realtime RT-PCR and DNA-microarrays, lipid mass spectrometry, cellular fluorescence imaging and flow cytometry as promising tools to further define regulatory networks, lipid species patterns and subcellular domains important for ABC-transporter-mediated lipid trafficking.     

Studies of Polymorphic DNA Fingerprinting and Lipid Pattern of Mycobacterium tuberculosis Patient Isolates in Japan

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two-dimensional thin-layer chromatography (2-D TLC) with silica gel, since M. tuberculosis has a lipid-rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2-O-methyl-L -rhamnose and L-rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome-lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.     

A statistical method for identifying differential gene-gene co-expression patterns

MOTIVATION: To understand cancer etiology, it is important to explore molecular changes in cellular processes from normal state to cancerous state. Because genes interact with each other during cellular processes, carcinogenesis related genes may form differential co-expression patterns with other genes in different cell states. In this study, we develop a statistical method for identifying differential gene-gene co-expression patterns in different cell states. RESULTS: For efficient pattern recognition, we extend the traditional F-statistic and obtain an Expected Conditional F-statistic (ECF-statistic), which incorporates statistical information of location and correlation. We also propose a statistical method for data transformation. Our approach is applied to a microarray gene expression dataset for prostate cancer study. For a gene of interest, our method can select other genes that have differential gene-gene co-expression patterns with this gene in different cell states. The 10 most frequently selected genes, include hepsin, GSTP1 and AMACR, which have recently been proposed to be associated with prostate carcinogenesis. However, genes GSTP1 and AMACR cannot be identified by studying differential gene expression alone. By using tumor suppressor genes TP53, PTEN and RB1, we identify seven genes that also include hepsin, GSTP1 and AMACR. We show that genes associated with cancer may have differential gene-gene expression patterns with many other genes in different cell states. By discovering such patterns, we may be able to identify carcinogenesis related genes.     

New Insights into the Stratum Corneum Lipid Organization by X-Ray Diffraction Analysis

The characteristic 13-nm lamellar phase that is formed by lipids in the outermost layer of the skin, the stratum corneum (SC), is very important for the barrier function of the skin. To gain more insight into the molecular organization of this lamellar phase, we performed small-angle x-ray diffraction (SAXD) using various lipid mixtures mimicking the lipid composition in SC. In the SAXD pattern of each mixture, at least seven diffraction orders were observed, attributed to the lamellar phase with a repeat distance ranging from 12.1 to 13.8 nm. Using the sampling method based on the variation in repeat distance, we selected phase angles for the first six diffraction orders. Using these phase angles for the lamellar phase, a high-resolution electron density distribution could be calculated. Subsequently, from SAXD patterns of isolated SC, the electron density distribution of the lamellar phase was also calculated and appeared to be very similar to that in the lipid mixtures. This demonstrates that the lipid mixtures serve as an excellent model for the lipid organization in SC, not only with respect to the repeat distance, but also in terms of the electron density distribution within the unit cell.     

Fixation methods for the study of lipid droplets by immunofluorescence microscopy.

The study of proteins associated with lipid droplets in adipocytes and many other cells is a rapidly developing area of inquiry. Although lipid droplets are easily visible by light microscopy, few standardized microscopy methods have been developed. Several methods of chemical fixation have recently been used to preserve cell structure before visualization of lipid droplets by light microscopy. We tested the most commonly used methods to compare the effects of the fixatives on cellular lipid content and lipid droplet structure. Cold methanol fixation has traditionally been used before visualization of cytoskeletal elements. We found this method unacceptable for study of lipid droplets because it extracted the majority of cellular phospholipids and promoted fusion of lipid droplets. Cold acetone fixation is similarly unacceptable because the total cellular lipids are extracted, causing collapse of the shell of lipid droplet-associated proteins. Fixation of cells with paraformaldehyde is the method of choice, because the cells retain their lipid content and lipid droplet structure is unaffected. As more lipid droplet-associated proteins are discovered and studied, it is critical to use appropriate methods to avoid studying artifacts.     

The cell biology of hepatitis C virus (HCV) lipid addiction: Molecular mechanisms and its potential importance in the clinic

Hepatitis C virus (HCV) is a major cause of chronic hepatitis associated with liver steatosis, commonly evolving to cirrhosis or hepatocellular carcinoma. The World Health Organisation (WHO) estimates that there are around 170 million chronic HCV carriers worldwide. The virus has a highly variable sequence, allowing definition of seven genotypes with different geographical distributions. Both clinical outcome and response to antiviral therapy are strongly influenced by HCV genotype. Importantly, several recent papers have suggested that the lipid profile of infected patients is strongly indicative of the various clinical outcomes of HCV infection. Furthermore, viral molecular and cellular studies have shown a tight link between cellular lipid metabolism and almost every step of the HCV infectious cycle. In the present review we summarise the current knowledge establishing the interplay between the molecular features of HCV replication, the cellular lipid biology and the lipid profiles observed in the serum of infected patients.     

Biochemical and chemotaxonomic characterization of Pseudomonas stutzeri genomovars

A total of 48 strains representing the seven Pseudomonas stutzeri genomovars (DNA/DNA homology groups) were studied for cellular fatty acid composition, physiological characteristics and protein profiles. All strains were found to be homogeneous with respect to their fatty acid patterns. Numerical analysis of physiological properties demonstrated a considerable phenotypic heterogeneity within the genomovars. Characterization of the individual genomic groups on the basis of biochemical tests was not possible. In a numerical study of cellular protein patterns, two main clusters were obtained, one representing genomovars 1, 6 and 7 characterized by a high G + C content (mean value > 64 mol%), the other representing genomovars 2, 3, 4 and 5 with a low G + G content (< 64 mol%). The standardized cellular protein patterns have potential for differentiation of the genomic groupings within the species Ps. stutzeri as currently circumscribed.     

Rapid analysis of cellular lipids without extraction

A method is described where cell suspension obtained by scraping of monolayers was directly applied on silica gel plates. Extraction and separation of different lipid classes were simultaneously obtained during chromatography. In the range of validity of the method (no more than 80 micrograms of cellular protein tested for neutral lipids and 30 micrograms for phospholipids), this technique allows rapid lipid analysis of small samples of cultured cells, bypassing all the time- and solvent-consuming extraction and evaporation steps. The method appears to be also suitable for measurement of enzymes of lipid metabolism such as acyl coenzyme A-cholesterol-acyltransferase.     

Enhanced cellular uptake and metabolic stability of lipo-oligoarginine peptides

Developing efficient cellular delivery vectors is crucial for designing novel therapeutic agents to enhance their plasma membrane permeability and metabolic stability in cells. Previously, we engineered cell penetrating peptide vectors named as "lipo-oligoarginine peptides" (LOAPs) by conjugating a proper combination of fatty acid and oligoarginine that translocated into cell easily without adverse effect on cell viability. In the present study, we report a systemic evaluation of cellular uptake and metabolic stability of LOAPs in Jurkat cells by introducing different combination of D-Arg residues in the peptide backbone. The cellular uptake and intracellular fate, cell viability, and metabolic stability and proteolytic degradation patterns of various LOAPs consisted of different combination of L- and D-Arg sequences were confirmed by flow cytometry, cytotoxicity assay, and analytical RP-HPLC with MALDI-TOF mass. All investigated LOAPs penetrated the cell efficiently with low cellular toxicity. The LOAPs having D-Arg residues at their N-termini seemed to have better metabolic stability than the LOAPs having C-terminal D-Arg residues. The metabolic degradation patterns were similar among all investigated LOAPs. The major hydrolytic site was between lauroyl group and β-Ala residue. Without the lipid chain, the oligoarginine peptide was pumped out ofcells easily. The results presented in this study suggest that structurally modified LOAPs could be used as a novel CPP design toward improved therapeutic application.     

Lipid profiling of Synechococcus sp. PCC7002 and two related strains by HPLC coupled to ESI-(ion trap)-MS/MS

The lipid profiles of Synechococcus sp. PCC7002 and two related 16S rDNA (99% identity) strains were established by a new method of high-performance liquid chromatography coupled to electrospray-mass spectrometry (HPLC-MS). Lipids were analysed in the positive and negative ionization mode, and fragmentation patterns are reported. No differences in the lipid profile between the three strains could be observed, but the relative content of some species differed. Major lipid species were found to be 1-octadecatrienoyl-2-hexadecanoyl-3-(6'-sulfo-alpha-D-quinovosyl)-sn-glycerol [SQDG (18:3/16:0)] and 1-octadecatrienoyl-2-hexadecenoyl-3-beta-D-monogalactosyl-sn-glycerol [MGDG (18:3/16:1)]. Ten species of SQDG, six species of PG (phosphatidyl-glycerol), seven species of MGDG, and two species of DGDG (digalactosyl-diacyl-glycerol) were detected. A PG species (m/z 761) containing hydroxylinolenic acid or oxophytodienoic acid acyl ester (C18H32O3), and SQDG species containing C17:1 and C17:3 fatty acyl esters are reported for the first time in cyanobacteria. The method also allowed the separation of two pairs of closely related isobaric MGDG species (m/z 770 and m/z 772 in positive ionization).     

Differential gene expression in primary and recurrent carotid stenosis

Apoptosis of the cellular components of complex atherosclerotic plaque may lead to plaque instability and rupture. In this study, five primary plaques and one recurrent fibrointimal lesion obtained from patients undergoing carotid endarterectomy for symptomatic carotid stenosis > or = 70% were analyzed by immunohistochemistry and cDNA microarray to identify gene expression patterns that may determine plaque susceptibility or resistance to apoptosis. Immunohistochemistry showed expression of active caspase 3, an effector of apoptosis, in macrophages and lymphocytes surrounding the lipid core, in smooth muscle cells in the fibrous cap, and media of primary plaques as well as in occasional smooth muscle cells in the recurrent lesion. Among the genes demonstrating increased expression in primary plaques were IGFR2, DR4, DAPK1, Bak, and ERK 1 and 2 and those showing decreased expression included the TNF receptors 1 and 2, akt1, and IGFBP3. When comparing the recurrent lesion to the normal tissue, the expression of 13 genes was decreased by 3-fold, including IGFBP2 and IGFBP3, and none were increased by more than 1.5-fold. The analysis of gene expression patterns in primary and recurrent stenotic lesions provides a powerful approach to identify the signaling pathways that alter cellular apoptotic patterns in such lesions.     

Immunocytochemistry of the Microtubules of fat-laden cells. Brown fat cells and adrenocortical cells in primary monolayer culture

Cytoplasmic microtubules of fat-laden cells containing fine lipid droplets, such as brown fat cells and adrenocortical cells, were studied in relation to the metabolism of intracellular lipid. In these cells, the amount and distribution of lipid droplets reflect the state of inherent cellular function. Materials used were primary monolayer culture of fetal rat brown fat cells and that of bovine adrenocortical cells. The method was the immunocytochemistry with anti-tubulin antibody. When brown fat cells were being lipolyzed or the steroidogenesis of adrenocortical cells were being stimulated, the cytoplasmic microtubules in the cells were organized in a radial pattern in response to the behavior of the lipid droplets. It is assumed that the microtubules were in the regulation of cellular function in terms of the metabolism of lipid droplets in these cells. We have devised, in the course of the current study, a double fluorescence technique as an observational method whereby microtubules were observed immunocytochemically and lipid droplets by a secondary fluorescence with phosphine E staining.     

Analysis of phospholipids and ornithine-containing lipids from Mesorhizobium spp

Polar lipid compositions of seven strains belonging to the four species of the Mesorhizobium genus were described. The lipid patterns of Mesorhizobium strains were very similar. Only quantitative differences were observed. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were found to be the major phospholipids of the analysed bacteria. In addition, two methylated derivatives of PE were observed: phosphatidyl-N,N-dimethylethanolamine (DMPE) and phosphatidyl-N-monomethylethanolamine (MMPE). Polar head groups of those phospholipids were predominately acylated with lactobacillic (19:0 cyclopropane) acid. Ornithine-containing lipid (OL) was also identified. 3-hydroxy fatty acids found in the lipid preparations were derived exclusively from the ornithine lipid. 3-hydroxylactobacillic was the main acyl residue amide linked to the ornithine.     

Effect of cerulenin on cellular autolytic activity and lipid metabolism during inhibition of protein synthesis in Streptococcus faecalis.

Cellular autolytic activity as well as lipid and lipoteichoic acid metabolism have been studied in cultures of Streptococcus faecalis receiving various combinations of the following treatments: chloramphenicol addition, starvation for an essential amino acid (valine), and cerulenin treatment. Lipoteichoic acid initially accumulated in chloramphenicol-treated and amino acid-starved cells and decreased relative to the cellular mass in cerulenin-treated cells. The relative phosphatidylglycerol content of amino acid-starved cultures or of cultures treated with either antibiotic rapidly decreased upon initiation of each treatment. In all cases, cerulenin initially stimulated diphosphatidylglycerol synthesis. Pretreatment of cultures with cerulenin prevented the inhibition of cellular synthesis autolysis normally observed during chloramphenicol treatment, but did not affect amino acid starvation-induced inhibition of autolytic activity. Variations in the levels of the nonionic lipid fraction, predominantly diglycerides, correlated best with the patterns of autolytic activity observed during chloramphenicol treatment, whereas variations in the levels of diphosphatidylglycerol and lipoteichoic acid correlated best with the patterns of autolytic activity observed during amino acid starvation. Components of the nonionic lipid fraction were demonstrated to inhibit autolytic activity 50% in whole cell and in cell wall assays at 60 and 120 nmol/mg (dry weight), respectively.     

Lipid peroxidation in isolated hepatocytes.

Intracellular lipid peroxidation was initiated by the addition of ADP-complexed ferric iron to isolated rat hepatocytes and the reaction monitored by the thiobarbituric acid method or by measurement of the formation of conjugated dienes. Both the production of malondialdehyde (thiobarbituric-acid-reacting substances) and of conjugated dienes was dependent, on the ADP-Fe-3+ concentration in a dose-related fashion. Malondialdehyde formation stopped spontaneously within 20 min after the initiation of the reaction and the plateau reached was also related to the ADP-Fe-3+ concentration. Control experiments revealed that more than 90% of the malondialdehyde accumulating during the incubation period could be ascribed to intracellular production. The cellular NADPH/NADP+ ratio was always high and only slightly decreased upon ADP-Fe-3+-induced lipid peroxidation which, however, was associated with a marked decrease in the cellular glutathione concentration. The rate of accumulation of malondialdehyde as well as the final level reached during ADP-Fe-3+-initiated lipid peroxidation was increased by the addition of chloral hydrate. This apparent stimulatory effect could, however, be ascribed to the inhibition of the mitochondrial oxidation of the malondialdehyde formed during cellular lipid peroxidation, thus allowing more malondialdehyde to accumulate during the process. ADP-Fe-3+-induced cellular lipid peroxidation was associated with a decrease in the concentration of glutathione. Also, lowering of the intracellular glutathione level by the addition of diethyl maleate or by simply preincubating the hepatocytes (up to 50 min) promoted the ADP-Fe-3+ malondialdehyde production and formation of conjugated dienes. Furthermore, when cellular glutathione concentration had been lowered by preincubation of the hepatocytes, significant malondialdehyde production could be observed even at ADP-Fe-3+ concentrations which were too low to induce measurable lipid peroxidation in fresh hepatocytes. It is thus concluded that glutathione has an important role in the cell defence against lipid peroxidation and suggested that the isolated hepatocytes provide a suitable experimental model system for the characterization of this and other possible cellular defence mechanisms and how they are affected by the nutritional status of the donor animal.     

Prediction of lipid accumulation-degradation in oleaginous micro-organisms growing on vegetable oils

Oleaginous micro-organisms (yeasts, moulds), in culture media having the carbon source as limited factor, degrade reserve lipids and produce new biomass, after the onset of carbon exhaustion from the medium. In this paper the process of lipid accumulation-degradation in oleaginous micro-organisms, growing on a vegatable oil was simulated. The model was integrated with 4 different methods and the parameters were optimised with the least squares method. It was found that the degradation of endocellular carbon pool is a very slow process characterised, however, by a good yield in fat-free biomass. Low values of the specific growth rates of the fat-free microbial mass, both from consumption of extra cellular and endocellular carbon pools, favourite the production of microbial lipid. The maximum of the specific rate of lipid accumulation is positively affected by the low values of the specific growth rate of the fat-free microbial mass from consumption of extra cellular carbon pool, but remained unaffected by the specific growth rate of the fat-free microbial mass from consumption of endocellular carbon pool. On the other hand, lipid production and specific rate of lipid accumulation are positively influenced by the high values of the specific rate of storage lipid formation. In conclusion, this numerical model can be used in the laboratory as pilot for planing further experimental work.     

Ultrastructure of lipid bodies in Tilletia caries teliospores.

The ultrastructure of lipid bodies within developing, dormant, and germinating Tilletia caries (DC). Tul. (race T-16) teliospores was studied by freeze-etching and thin-sectioning techniques. When teliospores were prefixed in sodium cacodylate-buffered glutaraldehyde-acrolein for 24 h before further processing, most of the lipid bodies appeared to have a uniformly osmiophilic matrix. Some of these lipid bodies were surrounded by thin electron-dense lines that appeared to be half-unit membranes. Occasionally this membrane seemed to be absent, allowing for a direct interface between lipid and cytosol. Irregular electron-dense patterns were occasionally observed in lipid bodies of developing, dormant, and germinating teliospores. A lamellar substructure with 6- to 10-nm center-to-center spacing was visible in the electron-dense patterns at high magnifications. Irregular fracture patterns were visible in freeze-etch replicas.