Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism最新文献

The transport of l-carnitine by lactating rat mammary tissue has been examined. l-Carnitine uptake by rat mammary tissue explants isolated from lactating rats, 3–4 days post partum, was via both Na⁺-dependent and Na⁺-independent pathways. The Na⁺-dependent pathway, the predominant route for l-carnitine uptake, was a saturable process: the K_m and V_max were, respectively, 132 μM and 201 pmol/2 h/mg of intracellular water. The Na⁺-independent pathway, which was non-saturable, had a coefficient of 0.26 μl/mg of intracellular water/2 h. The Na⁺-dependent component of l-carnitine uptake by mammary tissue explants was cis-inhibited by d-carnitine and acetyl-l-carnitine, but not by choline or taurine. In contrast, the Na⁺-independent component of l-carnitine uptake was not affected by any of these compounds. The uptake of l-carnitine by mammary tissue isolated from lactating rats, 10–12 days post partum, was qualitatively similar to that by mammary tissue taken from rats during the early stage of lactation. However, l-carnitine uptake was quantitatively lower: this was attributable to a reduction in the Na⁺-dependent component of l-carnitine uptake. l-Carnitine efflux from rat mammary tissue taken from animals 3–4 days post partum, consisted of at least two components; a fast extracellular component and a slow membrane-limited component. Reversing the trans-membrane Na⁺-gradient did not stimulate l-carnitine efflux suggesting that the Na⁺-dependent l-carnitine carrier operates with asymmetrical kinetics. A hyposmotic shock, hence cell-swelling, increased l-carnitine efflux from mammary tissue explants.

Apolipophorin III (apoLp-III) from the locust Locusta migratoria is an exchangeable apolipoprotein that reversibly binds to lipoproteins. During lipid binding the protein has been proposed to undergo a major conformational change. To study the mechanism of lipid binding we have cloned and expressed recombinant protein in bacteria, permitting stable isotope enrichment for heteronuclear NMR spectroscopy and site-directed mutagenesis. The cDNA coding for apoLp-III was subcloned into the pET expression vector and transformed into Escherichia coli cells. Induction of expression resulted in the specific appearance of apoLp-III in the cell culture medium, indicating it escaped the bacteria without lysis. The protein was purified from the cell-free supernatant by reversed-phase HPLC, characterized and compared to the natural protein isolated from locust hemolymph. SDS-PAGE revealed the recombinant protein has a molecular mass of approximately 17 kDa, similar to that of deglycosylated natural apoLp-III. Monoclonal antibodies were used to detect recombinant apoLp-III in the cells as well as in cell-free medium of induced bacterial cultures. Amino acid sequencing and analysis confirmed the identity of the recombinant protein as L. migratoria apoLp-III. Circular dichroism spectroscopy of recombinant and natural apoLp-III showed similar spectra, both displaying high contents of α-helical secondary structure. Denaturation studies of lipid-free apoLp-III with guanidine hydrochloride showed that both proteins have similar denaturation midpoints and ΔG values indicating similar protein stability. The natural and recombinant protein were functional in lipoprotein binding assays. Using recombinant protein, uniformly and specifically labeled with ¹⁵N-amino acids, two dimensional ¹H-¹⁵N heteronuclear single quantum correlation spectra were obtained. The spectra revealed excellent chemical shift dispersion in both the ¹H and ¹⁵N dimensions with a well defined resonance pattern. Studies with ¹⁵N-leucine specifically labeled apoLp-III in the presence and absence of the micelle forming lipid, dodecylphosphocholine, provided evidence for a significant conformational change upon lipid association.

Exposure of rat C6 glioma cells to the β-adrenergic receptor agonist isoproterenol potentiates basal and metabotropic glutamate receptor-stimulated phospholipase C activity in rat C6 glioma cells. After treatment of cells for 24 h with 10 μM isoproterenol, metabotropic glutamate receptors and phospholipase C activity were determined in C6 plasma membranes. Isoproterenol treatment caused an increase of 67% in the total number of binding sites (B_max=12.1±1.8 pmol/mg protein versus B_max=20.27±0.88 pmol/mg protein) with K_d values of the same order (K_d=1250±101 nM versus K_d=1401±211 nM), using l-[³H]glutamate as radioligand. On the other hand, basal, guanylyl imidodiphosphate (Gpp[NH]p)- and trans-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD)-stimulated phospholipase C activities were also significantly increased in membranes from isoproterenol-treated cells compared to control cells, by 337%, 33% and 40% respectively. Moreover, a significant increase of 94% in the steady-state level of phospholipase C β₁ in membranes from isoproterenol-treated cells compared to control was also detected by immunoblot. These results show that metabotropic glutamate receptors and its effector system, phospholipase C, are affected by isoproterenol treatment, showing the existence of cross-talk between these signal transduction pathways.

African swine fever virus (ASFV) enters cells by receptor mediated endocytosis and requires a fusion event between the viral envelope and the limiting membrane of the endosome at low pH. In order to investigate the role of cholesterol in the early stages of ASFV infection, we have studied the effect of the removal of cell and viral membrane cholesterol by cholesterol oxidase treatment of cells and virions, as well as the effect of some inhibitors of cholesterol synthesis on the infectious pathway. In addition, we have investigated viral infection in cholesterol-depleted Vero cells. Both cholesterol-depleted and cholesterol oxidase-treated Vero cells were unaltered in their ability to bind or internalize the virus, but were blocked in ASFV fusion and subsequent virus replication. Our results indicate that ASFV infection is affected by cholesterol in the target membrane.

The squalene-hopene cyclase (SHC) is the only enzyme involved in the biosynthesis of hopanoid lipids that has been characterized on the genetic level. To investigate if additional genes involved in hopanoid biosynthesis are clustered with the shc gene, we cloned and analyzed the nucleotide sequences located immediately upstream of the shc genes from Zymomonas mobilis and Bradyrhizobium japonicum. In Z. mobilis, five open reading frames (ORFs, designated as hpnA–E) were detected in a close arrangement with the shc gene. In B. japonicum, three similarly arranged ORFs (corresponding to hpnC–E from Z. mobilis) were found. The deduced amino acid sequences of hpnC–E showed significant similarity (58–62%) in both bacteria. Similarities to enzymes of other terpenoid biosynthesis pathways (carotenoid and steroid biosynthesis) suggest that these ORFs encode proteins involved in the biosynthesis of hopanoids and their intermediates. Expression of hpnC to hpnE from Z. mobilis as well as expression of hpnC from B. japonicum in Escherichia coli led to the formation of the hopanoid precursor squalene. This indicates that hpnC encodes a squalene synthase. The two additional ORFs (hpnA and hpnB) in Z. mobilis showed similarities to enzymes involved in the transfer and modification of sugars, indicating that they may code for enzymes involved in the biosynthesis of the complex, sugar-containing side chains of hopanoids.

The sphingomyelin pathway, activated by stimuli, such as inflammatory cytokines, results in the formation of ceramide, a second messenger molecule. The purpose of the present study was to examine the mechanism by which macrophage-type nitric oxide synthase (NOS II) is induced by stimulation of the sphingomyelin pathway. When RAW264.7 cells were incubated with sphingomyelinase (SMase), nitrite production, NOS II activity, and NOS II mRNA were increased in a dose-dependent manner. Sphingosine, dihydrosphingosine, N-acetylsphingosine (C2-ceramide), and N-acylsphingosine (natural ceramide) had no effect on nitrite production, suggesting that signal molecules other than these were concomitantly produced by SMase treatment and required for NOS II induction. We then investigated the possible involvement of intracellular reactive oxygen species (ROS) in gene induction. SMase treatment increased the level of intracellular ROS, as assessed by flow cytometric analysis using a ROS-sensitive dye, dichlorofluorescin diacetate. Antioxidants, such as N-acetyl-l-cysteine and α-tocopherol, inhibited gene induction as well as nitrite production by SMase. These results suggest that activation of the sphingomyelin pathway induces gene expression and that the elevated ROS were somehow involved in this process.

Although there is only one documented function carried out by the GM2 activator protein in the lysosome, new information suggests that other less obvious roles may also be played by this protein in vivo. This information includes data demonstrating that the GM2 activator is a secretory, as well as a lysosomal protein, and that cells possess a carbohydrate-independent mechanism to re-capture the activator, with or without bound lipid, from the extracellular fluid. Additionally the GM2 activator has been shown to bind, solubilize and transport a broad spectrum of lipid molecules, such as glycolipids, gangliosides and at least one phosphoacylglycerol, between liposomes. At pH 7 the GM2 activator’s rate of lipid transport is reduced by only 50% from its maximum rate which is achieved at approx. pH 5, suggesting that the GM2 activator may serve as a general intra- and/or inter-cellular lipid transport protein in vivo. Since the late 1970s the lysosomal form of the GM2 activator has been known to act as a substrate-specific co-factor for the hydrolysis of GM2 ganglioside by β-hexosaminidase A. Gangliosides are a class of negatively charged glycolipids particularly abundant in neuronal cells which have been linked to numerous in vivo functions, such as memory formation and signal transduction events. Deficiency of the GM2 activator protein results in the storage of GM2 ganglioside and severe neurological disease, the AB-variant form of GM2 gangliosidosis, usually culminating in death before the age of 4 years. The exact mode-of-action of the GM2 activator in its role as a co-factor, and its specificity for various glycolipids are currently matters of debate in the literature.

The polar lipids from the hydrocarbon using and biosurfactant-producing bacterium Alcanivorax borkumensis were isolated and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The biosurfactant produced by this species is an anionic glucose lipid with a tetrameric oxyacyl side chain. The glycolipids extracted from the cell wall consist of this biosurfactant N-terminally esterified with glycine. Ten different derivatives of this lipid type were identified and their structures elucidated by MSMS. They vary by the chain length of one or two of the four β-hydroxy fatty acids (C₆, C₈ and C₁₀) and by the location of these different fatty acids within the molecule. All compounds are reported here for the first time. In addition to these glycolipids, three different phosphatidylglycerols were identified. While these lipids were found in all strains of A. borkumensis, the relative abundances of the different lipids vary between the strains.

The inflammatory compounds β-glucan, a particulate agonist, and tannin, a soluble agonist, are present in cotton dust and, when inhaled, cause massive arachidonic acid release from alveolar macrophages. Earlier work had shown that these agonists exhibit different effects on arachidonate liberation and release, and that only tannin inhibits the uptake and incorporation of exogenous arachidonic acid, suggesting inhibition of reacylation. Here we have used the time-dependent incorporation of ¹⁸O from H₂¹⁸O-containing media into glycerophospholipid acyl groups as an indicator of acyl turnover in resting and agonist-treated rabbit alveolar macrophages. Highest turnover rates were seen in phosphatidylinositol (∼30% per hour) and in choline phospholipids (10–20% per hour). Both β-glucan and tannin stimulated acyl turnover, especially arachidonic acid turnover, in these and other lipid classes by a factor of 2 or more. We conclude that neither agonist promotes arachidonic acid accumulation in and release from alveolar macrophages by inhibiting reacylation.