Intracellular dissemination of peroxidative stress. Internalization, transport, and lethal targeting of a cholesterol hydroperoxide species by sterol carrier protein-2-overexpressing hepatoma cells

Sterol carrier protein-2 (SCP-2) plays a crucial role in the trafficking and metabolism of cholesterol and other lipids in mammalian cells. Lipid hydroperoxides generated under oxidative stress conditions are relatively long-lived intermediates that damage cell membranes and play an important role in redox signaling. We hypothesized that SCP-2-facilitated translocation of lipid hydroperoxides in oxidatively stressed cells might enhance cytolethality if highly sensitive sites are targeted and detoxification capacity is insufficient. We tested this using a clone (SC2A) of rat hepatoma cells that overexpress mature immunodetectable SCP-2. When challenged with liposomal cholesterol-7alpha-hydroperoxide (7alpha-OOH), SC2A cells were found to be much more sensitive to viability loss than vector control (VC) counterparts. Correspondingly, SC2A cells imported [14C]7alpha-OOH more rapidly. The clones were equally sensitive to tert-butyl hydroperoxide, suggesting that the 7alpha-OOH effect was SCP-2-specific. Fluorescence intensity of the probes 2',7'-dichlorofluorescein and C11-BODIPY increased more rapidly in SC2A than VC cells after 7alpha-OOH exposure, consistent with more rapid internalization and oxidative turnover in the former. [14C]7alpha-OOH radioactivity accumulated much faster in SC2A mitochondria than in VC, whereas other subcellular fractions showed little rate difference. In keeping with this, 7alpha-OOH-stressed SC2A cells exhibited a faster loss of mitochondrial membrane potential and development of apoptosis. This is the first reported evidence that peroxidative stress damage can be selectively targeted and exacerbated by an intracellular lipid transfer protein.     

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3α-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7α-hydroxylase, sterol 27α-hydroxylase, Na⁺/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.     

Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 A resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [(3)H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.     

Role of an Ancestral D-Bifunctional Protein Containing Two Sterol-Carrier Protein-2 Domains in Lipid Uptake and Trafficking in Toxoplasma

The inability to synthesize cholesterol is universal among protozoa. The intracellular pathogen Toxoplasma depends on host lipoprotein-derived cholesterol to replicate in mammalian cells. Mechanisms of cholesterol trafficking in this parasite must be important for delivery to proper organelles. We characterized a unique d-bifunctional protein variant expressed by Toxoplasma consisting of one N-terminal d-3-hydroxyacyl-CoA dehydrogenase domain fused to two tandem sterol carrier protein-2 (SCP-2) domains. This multidomain protein undergoes multiple cleavage steps to release free SCP-2. The most C-terminal SCP-2 carries a PTS1 that directs the protein to vesicles before processing. Abrogation of this signal results in SCP-2 accumulation in the cytoplasm. Cholesterol specifically binds to parasite SCP-2 but with 10-fold lower affinity than phosphatidylcholine. In mammalian cells and Toxoplasma, the two parasite SCP-2 domains promote the circulation of various lipids between organelles and to the surface. Compared with wild-type parasites, TgHAD-2SCP-2–transfected parasites replicate faster and show enhanced uptake of cholesterol and oleate, which are incorporated into neutral lipids that accumulate at the basal end of Toxoplasma. This work provides the first evidence that the lipid transfer capability of an ancestral eukaryotic SCP-2 domain can influence the lipid metabolism of an intracellular pathogen to promote its multiplication in mammalian cells.     

Macrophage mitochondrial damage from StAR transport of 7-hydroperoxycholesterol: Implications for oxidative stress-impaired reverse cholesterol transport

StAR family proteins in vascular macrophages participate in reverse cholesterol transport (RCT). We hypothesize that under pathophysiological oxidative stress, StARs will transport not only cholesterol to macrophage mitochondria, but also pro-oxidant cholesterol hydroperoxides (7-OOHs), thereby impairing early-stage RCT. Upon stimulation with dibutyryl-cAMP, RAW264.7 macrophages exhibited a strong time-dependent induction of mitochondrial StarD1 and plasma membrane ABCA1, which exports cholesterol. 7α-OOH uptake by stimulated RAW cell mitochondria (like cholesterol uptake) was strongly reduced by StarD1 knockdown, consistent with StarD1 involvement. Upon uptake by mitochondria, 7α-OOH (but not redox-inactive 7α-OH) triggered lipid peroxidation and membrane depolarization while reducing ABCA1 upregulation. These findings provide strong initial support for our hypothesis.     

Lethal damage to murine L1210 cells by exogenous lipid hydroperoxides: protective role of glutathione-dependent selenoperoxidases

The effect of selenium deprivation on the viability of murine L1210 cells exposed to various exogenous lipid hydroperoxides has been investigated. Selenoperoxidase activities of cells grown for longer than 1 week in 1% serum with no added selenium [Se(-) cells] were less than 10% of the activities of selenium-satisfied controls [Se(+) cells] or selenium-repleted counterparts [Se(-/+) cells]. The enzymes measured were classical glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). Se(-) cells exhibited a compensatory increase in catalase activity. Dye exclusion and clonal survival assays indicated that Se(-) and Se(+) cells were relatively insensitive to photochemically generated phospholipid hydroperoxides in liposomal form. However, both cell types were sensitive to liposomal cholesterol hydroperoxides, e.g., 7-hydroperoxycholesterol (7-OOH), Se(-) being much more so (LD50 approximately 10 microM) than Se(+) (LD50 approximately 75 microM). By contrast, 7-hydroxycholesterol over a comparable concentration range was minimally toxic to Se(-) and Se(+) cells. Cell killing by 7-OOH was inhibited by desferrioxamine and by butylated hydroxytoluene, suggesting that iron-mediated free radical reactions are involved. The involvement of glutathione in cytoprotection was confirmed by showing that Se(+) cells were more sensitive to 7-OOH after treating with buthionine sulfoximine, an inhibitor of GSH synthesis. Cellular detoxification of 7-OOH is provisionally attributed to PHGPX rather than GPX, since 7-OOH and other cholesterol hydroperoxides were found to be good substrates for PHGPX in a cell free system, but were unreactive with GPX.     

An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome

The level of 7-dehydrocholesterol (7-DHC) is elevated in tissues and fluids of Smith-Lemli-Opitz syndrome (SLOS) patients due to defective 7-DHC reductase. Although over a dozen oxysterols have been identified from 7-DHC free radical oxidation in solution, oxysterol profiles in SLOS cells and tissues have never been studied. We report here the identification and complete characterization of a novel oxysterol, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), as a biomarker for 7-DHC oxidation in fibroblasts from SLOS patients and brain tissue from a SLOS mouse model. Deuterated (d₇)-standards of 7-DHC and DHCEO were synthesized from d₇-cholesterol. The presence of DHCEO in SLOS samples was supported by chemical derivatization in the presence of d₇-DHCEO standard followed by HPLC-MS or GC-MS analysis. Quantification of cholesterol, 7-DHC, and DHCEO was carried out by isotope dilution MS with the d₇-standards. The level of DHCEO was high and correlated well with the level of 7-DHC in all samples examined (R = 0.9851). Based on our in vitro studies in two different cell lines, the mechanism of formation of DHCEO that involves 5α,6α-epoxycholest-7-en-3β-ol, a primary free radical oxidation product of 7-DHC, and 7-cholesten-3β,5α,6β-triol is proposed. In a preliminary test, a pyrimidinol antioxidant was found to effectively suppress the formation of DHCEO in SLOS fibroblasts.     

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium⁺ uptake into KG-l cells suspended in sucrose medium (EC₅₀ of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium⁺ uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC₅₀ of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC₅₀ of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium⁺ uptake into KG-1 cells. ATP-induced ethidium⁺ uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium⁺ uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1²⁺ and propidium²⁺ uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells.     

A new N-terminal recognition domain in caveolin-1 interacts with sterol carrier protein-2 (SCP-2)

Although plasma membrane domains, such as caveolae, provide an organizing principle for signaling pathways and cholesterol homeostasis in the cell, relatively little is known regarding specific mechanisms, whereby intracellular lipid-binding proteins are targeted to caveolae. Therefore, the interaction between caveolin-1 and sterol carrier protein-2 (SCP-2), a protein that binds and transfers both cholesterol and signaling lipids (e.g., phosphatidylinositides and sphingolipids), was examined by yeast two-hybrid, in vitro binding and fluorescence resonance energy transfer (FRET) analyses. Results of the in vivo and in vitro assays identified for the first time the N-terminal amino acids (aa) 1-32 amphipathic alpha helix of SCP-2 functionally interacted with caveolin-1. This interaction was independent of the classic caveolin-1 scaffolding domain, in which many signaling proteins interact. Instead, SCP-2 bound caveolin-1 through a new domain identified in the N-terminal domain of caveolin-1 between aa 34-40. Modeling studies suggested that electrostatic interactions between the SCP-2 N-terminal aa 1-32 amphipathic alpha-helical domain (cationic, positively charged face) and the caveolin-1 N-terminal aa 33-59 alpha helix (anionic, negatively charged face) may significantly contribute to this interaction. These findings provide new insights on how SCP-2 enhances cholesterol retention within the cell as well as regulates the distribution of signaling lipids, such as phosphoinositides and sphingolipids, at plasma membrane caveolae.     

Sterol carrier protein-2 alters high density lipoprotein-mediated cholesterol efflux

Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on "reverse cholesterol transport" has not been explored. As shown herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of [(3)H]cholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (t(b)(12) near 1 min) and vesicular (t(d)(12) = 10-20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t(b)(12) decreased 36%, while the slower t(d)(12) increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and t(d)(12) were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.     

An Integrin α4β7?IgG Heterodimeric Chimera Binds to MAdCAM-1 on High Endothelial Venules in Gut-Associated Lymphoid Tissue

Lymphocyte homing is regulated by a multistep process mediated by sequential adhesive interactions between circulating lymphocytes and high endothelial venules (HEVs). In gut-associated lymphoid tissue (GALT), the initial interactive step, “tethering and rolling,” is partly mediated by integrin α4β7 expressed on GALT-homing lymphocytes and its ligand MAdCAM-1, which is exclusively expressed on HEVs in GALT. To probe functional MAdCAM-1 in tissue sections, we developed a soluble integrin α4β7 heterodimeric IgG chimera by joining the extracellular region of mouse integrin α4 and β7 subunits to a human IgG Fc domain. Western blot analysis revealed that co-transfection of HEK 293T cells with expression vectors encoding integrin α4•IgG and β7•IgG results in the formation of α4β7•IgG heterodimeric chimeras. This complex preferentially binds to CHO cells expressing MAdCAM-1 and, to a lesser extent, to cells expressing VCAM-1, but not to cells expressing ICAM-1. Moreover, α4β7•IgG specifically binds to HEVs in GALT in situ in a divalent cation–dependent fashion and inhibits lymphocyte binding to HEVs in GALT. These findings indicate that α4β7•IgG can be used as a probe for functional MAdCAM-1 expressed on HEVs in GALT and could potentially serve as an anti-inflammatory drug inhibiting GALT-specific lymphocyte migration.     

7-Hydroperoxycholesterol as a marker of oxidative stress in rat kidney induced by paraquat

The in vivo paraquat-induced oxidative stress in rat tissue was studied by analyzing cholesterol-derived hydroperoxide as an index of lipid peroxidation. Paraquat (10 mg/kg) was administered i.p. to rats. Rats were sacrificed and lung, liver, and kidney were collected 2, 24 h, and 5 d after paraquat injection. Lipids were extracted and analyzed by HPLC with post-column chemiluminescence. We found that two cholesterol-derived hydroperoxides, 7α-hydroperoxycholest-5-en-3β-ol (7α-OOH) and 7β-hydroperoxycholest-5-en-3β-ol (7β-OOH) were present in lungs of control animals (0.06 and 0.06 nmol/g, respectively), in livers (6.5 and 15.8 nmol/g, respectively) and in kidneys (3.7 and 8.9 nmol/g, respectively). In liver paraquat increased lipid peroxidation approximately by 60% over the levels of control animals only at 2 h after paraquat treatment. In kidney, augmented lipid peroxidation, 7α-OOH and 7β-OOH (by 70% and 147%, respectively) above levels was found at 2 h after paraquat treatment. Interestingly, these increase remained in kidney of rats 5 d after a single dose of paraquat. In contrast, cholesterol-derived hydroperoxides were not affected in lung of paraquat dosed rats. This is the first report on 7α-OOH and 7β-OOH accumulations in rat liver and kidney, and it seems to reflect greater oxidative stress in the pathology of kidney of rats treated with acute paraquat at low dose.     

Colocalization of prostaglandin F2�� receptor FP and prostaglandin F synthase-I in the spinal cord

Prostaglandin F_2α is synthesized by prostaglandin F synthase, which exists in two types, prostaglandin F synthase I (PGFS I) and prostaglandin F synthase II (PGFS II). Prostaglandin F_2α binds to its specific receptor, FP. Our previous immunohistochemical study showed the distinct localization of prostaglandin F synthases in rat spinal cord. PGFS I exists in neuronal somata and dendrites in the gray substance, and PGFS II exists in ependymal cells and tanycytes surrounding the central canal. Both enzymes are also present in endothelial cells of blood vessels in the white and gray substances of the spinal cord. In this study, we found that FP localizes in neuronal somata and dendrites but not in ependymal cells, tanycytes, or endothelial cells. Immunohistochemical analysis of serial sections showed the colocalization of FP and PGFS I. FP immunoreactivity was intense in spinal laminae I and II of the dorsal horn, a connection site of pain transmission, and was similar to that of PGFS I in neuronal elements. These findings suggest that prostaglandin F_2α synthesized in the neuronal somata and dendrites exert an autocrine action there.—Suzuki-Yamamoto, T., K. Toida, Y. Sugimoto, and K. Ishimura. Colocalization of prostaglandin F_2α receptor FP and prostaglandin F synthase-I in the spinal cord.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

HSV-2 infection of dendritic cells amplifies a highly susceptible HIV-1 cell target

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α₄β₇ on lymphocytes. α₄β₇ can be engaged by HIV-1 on the cell-surface and CD4⁺ T cells expressing high levels of this integrin (α₄β₇ ^high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α₄β₇ ^high CD4⁺ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c⁺ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α₄β₇ expression on CD4⁺ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α₄β₇ ^highCD4⁺ T cells. These factors may play a role in increasing the susceptibility to HIV-1.     

Lipid binding to sterol carrier protein-2 is inhibited by ethanol

Sterol carrier protein-2 (SCP-2) is an intracellular lipid carrier protein that binds cholesterol, phospholipids, fatty acids and other ligands. It has been reported that expression of SCP-2 was increased in brain nerve endings or synaptosomes of chronic ethanol-treated mice and it was shown that cholesterol homeostasis was altered in brain membranes of chronic ethanol-treated animals. Ethanol may interfere with the capacity of SCP-2 to bind cholesterol as well as other lipids. This hypothesis was tested using recombinant SCP-2 and fluorescent-labeled cholesterol, phosphatidylcholine (PC), and stearic acid. The association constants (K_a) of the ligand-SCP-2 complex were in the following order: NBD-cholesterol>NBD-PC>NBD-stearic acid. Ethanol, beginning at a concentration of 25 mM, significantly reduced the affinity of NBD-cholesterol and NBD-PC for SCP-2. Effects of ethanol on the K_a of NBD-stearic acid was significant only at the highest concentration that was examined (200 mM). Ethanol significantly increased the B_max of NBD-cholesterol for SCP-2 but did not have a significant effect on the B_max of NBD-PC. Similar results were found for effects of ethanol on the K_as and B_maxs using pyrene-labeled cholesterol and PC. In conclusion, ethanol beginning at a physiological concentration of 25 mM inhibited binding of cholesterol and PC to SCP-2. However, effects of ethanol on lipid binding to SCP-2 were dependent on the type of lipid. Ethanol in vivo may interfere with lipid binding to SCP-2 and disrupt lipid trafficking within cells.