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Our recent studies have shown that the de novo spingolipids play a role in apoptosis of photosensitized cells. To elucidate the involvement of the de novo sphingolipids in reactive oxygen species (ROS) production and mitochondrial depolarization during apoptosis, the stess inducer photodynamic therapy (PDT) with the photosensitizer Pc 4 was used. In Jurkat cells PDT-triggered ROS production or mitochondrial membrane potential (&#61508 ; ; &#61561 ; ; m) loss was not prevented by the de novo sphingolipid synthesis inhibitor ISP-1. However, PDT-C16-ceramide led to enhanced mitochondrial depolarization and DEVDase activation. The superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) protected Jurkat cells from ROS generation and apoptosis, but not from &#61508 ; ; &#61561 ; ; m reduction. Spinghanine or C16-ceramide counteracted MnTBAP-induced protection from apoptosis in Jurkat, as well as CHO cells. In LY-B cells, CHO-derived mutants deficient in serine palmitoyltransferase (SPT) activity and the de novo sphingolipid synthesis, mitochondrial depolarization, but not ROS generation, was suppressed post-PDT. In LY-B cells transfected with the SPT component LCB1, &#61508 ; ; &#61561 ; ; m collapse post-PDT was restored. The data support that: MnTBAP protects against apoptosis via steps downstream of &#61508 ; ; &#61561 ; ; m loss ; de novo sphingolipids are not required for ROS generation, but can play a role in &#61508 ; ; &#61561 ; ; m dissipation in photosensitized apoptotic cells.

Serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the biosynthesis of sphingolipids, comprises two different subunits, LCB1 and LCB2. LCB1 has a single highly hydrophobic domain near the N terminus. Chinese hamster ovary cell mutant LY-B cells are defective in SPT activity because of the lack of expression of an endogenous LCB1 subunit. Stable expression of LCB1 having an epitope tag at either the N or C terminus restored SPT activity of LY-B cells, suggesting that the epitope tag did not affect the localization or topology of LCB1. Indirect immunostaining showed that the N- and C-terminal epitopes are oriented toward the lumenal and cytosol side, respectively, at the endoplasmic reticulum. Interestingly, there was far less LCB2 in LY-B cells than in wild-type cells, and the amount of LCB2 in LY-B cells was restored to the wild-type level by transfection with LCB1 cDNA. In addition, overproduction of the LCB2 subunit required co-overproduction of the LCB1 subunit. These results indicated that the LCB1 subunit is most likely an integral protein having a single transmembrane domain with a lumenal orientation of its N terminus in the endoplasmic reticulum and that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells.

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is approximately 25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum-infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID(50) values for SM/LCPL-PLC activities and the parasite growth at 3-5 microM and approximately 7 microM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

Membrane microdomains enriched in cholesterol and sphingolipids modulate a number of signal transduction pathways and provide a residence for heterotrimeric G proteins, their receptors, and their effectors. We investigated whether signaling through G(s) was dependent on these membrane domains, characterized by their resistance to detergents, by depleting cells of cholesterol and sphingolipids. For cholesterol depletion, rat salivary epithelial A5 cells were cultured under low-cholesterol conditions, and then treated with the cholesterol chelator methyl-beta-cyclodextrin. For sphingolipid depletion, LY-B cells, a mutant CHO cell line that is unable to synthesize sphingolipids, were incubated under low-sphingolipid conditions. Depletion of cholesterol or sphingolipid led to a loss or decrease, respectively, in the amount of Galpha(s) from the detergent-resistant membranes without any change in the cellular or membrane-bound amounts of Galpha(s). The cAMP accumulation in response to a receptor agonist was intact and the level slightly increased in cells depleted of cholesterol or sphingolipids compared to that in control cells. These results indicate that localization of Galpha(s) to detergent-resistant membranes was not required for G(s) signaling. Analysis of the role of lipid rafts on the kinetics of protein associations in the membrane suggests that compartmentalization in lipid rafts may be more effective in inhibiting protein interactions and, depending on the pathway, ultimately inhibit or promote signaling.

Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM). N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1R,3S)-HPA-12, [corrected] which resembles natural ceramide stereochemically, was found to be the most active, although (1R,3S)-HPA-12 [corrected] did not affect ER-to-Golgi trafficking of protein. Interestingly, (1R,3S)-HPA-12 [corrected] inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1R,3S)-HPA-12 [corrected] inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway.

Malaria is clinically manifested only when the human malaria parasites in the genus Plasmodium enter the obligatory intraerythrocytic life cycle. Elucidation for the roles of the serum, the key nutrient, and its components is then deemed essential for thorough understanding of the proliferation of Plasmodium cells at the erythrocytic stage. Fractionation and analysis of serum and its components was performed by chromatography, solvent extraction, and subsequent reconstitution experiments. Only fractions containing serum albumin (SA) from the serum and purified intact bovine serum albumin (BSA) showed comparable growth promoting activity with human serum (HS). Delipidated BSA can only effect parasite growth after reconstitution with lipids extracted from intact BSA. Fatty acid (FA) species in the neutral lipid fraction from intact BSA proved likewise when reconstituted with delipidated BSA. Furthermore, the involved FA species have to come in a pair of one saturated and one unsaturated, with palmitic and oleic acids as the best combination. The results were further substantiated by morphological analysis as well as biochemical analysis of the DNA synthesis during the intraerythrocytic development. This study can be a basis to explore the molecular mechanism of lipid traffic within the parasitized red blood cell (RBC), which can be an important adjunct to the development of drugs for malaria therapy.

Sphingolipid metabolism and metabolites are important in various cellular events in eukaryotes. However, little is known about their function in plasmodial parasites. Here we demonstrate that neutral sphingomyelinase (SMase) involved in the sphingomyelin (SM) catabolism is retained by the intraerythrocytic parasite Plasmodium falciparum. When assayed in a neutral pH buffer supplemented with Mg2+ and phosphatidylserine, an activity for the release of the phosphocholine group from SM was detected in parasite-infected, but not in uninfected, erythrocyte ghosts. The SMase activity in the parasite-infected erythrocyte ghosts was enhanced markedly by anionic phospholipids including unsaturated but not saturated phosphatidylserine. Mn2+ could not substitute for Mg2+ to activate SMase in parasite-infected erythrocyte ghosts, whereas both Mn2+ and Mg2+ activated mammalian neutral SMase. The specific activity level of SMase was higher in isolated parasites than in infected erythrocyte ghosts; further fractionation of lysates of the isolated parasites showed that the activity was bound largely to the membrane fraction of the parasites. The plasmodial SMase seemed not to hydrolyse phosphatidylcholine or phosphatidylinositol. The plasmodial SMase, but not SM synthase, was sensitive to scyphostatin, an inhibitor of mammalian neutral SMase, indicating that the plasmodial activities for SM hydrolysis and SM synthesis are mediated by different catalysts. Our finding that the malaria parasites possess SMase activity might explain why the parasites seem to have an SM synthase activity but no activity to synthesize ceramide de novo.

Orientia tsutsugamushi is one of the obligate intracellular bacteria, and causes Tsutsugamushi disease (scrub typhus). The whole genome sequence of Boryong strain revealed some characteristics of this bacterium, such as a type IV secretion system, many histidine kinases, SpoT, Tra, and ankyrin repeatand tetratricopeptide repeat (TPR)-containing proteins [1]. Additionally, the structure of the genome of O. tsutsugamushi was completely different from those of other rickettsia that have been previously reported. We performed a combination assay of SDS-PAGE and LC-MSMS (GeLC-MSMS) to identify expressed proteins and study the protein profile of this bacterium. The Kuroki strain of O. tsutsugamsuhi, which is very close to the Boryong strain, was propagated in L929 cells and purified by the Renografin density method. About 50 lg of the purified rickettsia was subjected to SDS-PAGE, and the gel was cut into about 40 pieces. Then, the pieces of gel were subjected to in-gel digestion with trypsin or endoproteinase Lys-C. Trypsin digests proteins at the arginine and lysine sites, whereas Lys-C digests only at the lysine site. Finally, the digested peptides were subjected to LC-MSMS analysis (Magic2002 Michrom Biosource Inc., Auburn, CA, USA and LCQ-DECAXP Thermo Fisher Scientific, San Jose, CA, USA). Two different procedures were used, involving different sizes of column (InertsilODS3; GL Science Inc., Shinjuku, Tokyo, Japan) and acetonitrile concentration gradient times: a 0.1 mm · 5 cm column and a 60-min gradient time; and a 0.1 mm · 15 cm column and a 120-min gradient time. For data analysis, proteins were identified using the TurboSequest algorithm in the Bioworks 3.1 package software (Thermo fisher Scientific) and the O. tsutsugamushi database (NCBI, National Center for Biotechnology Information, Bethesda, MD, USA). The criteria for positive identification of peptides were cross-correlation number (Xcorr) >1.0 for singly charged ions, Xcorr >1.5 for doubly charged ions, and Xcorr >2.0 for triply charged ions. Only the best-matched peptides were considered. The strategy using both trypsin and Lys-C for in-gel digestion and two sets of columns and gradient times for LC-MSMS had the great advantage of identifying proteins, especially proteins of lower molecular mass (

Exposure of Chinese hamster CHO-K1 transfectant cells expressing mouse CD14 (CHO/CD14 cells) to lipopolysaccharide (LPS) induced rapid elevation of the cellular diacylglycerol (DAG) and choline/phosphocholine levels and nuclear translocation of nuclear factor kappaB (NFkappaB). When cells were incubated with short-chain DAG analogues or bacterial phospholipase C, NFkappaB activation occurred even without the LPS stimulus. Treatment of CHO/CD14 cells with tricyclo[5.2.1.0(2.6)]decyl-(9[8])xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C and phospholipase D, almost completely inhibited not only the LPS-dependent production of DAG and choline/phosphocholine but also the LPS-dependent NFkappaB activation. In contrast, treatment of cells with 1-(6-?[3-methoxyoestra-1,3, 5(10)-trien-17beta-yl]-1H-pyrrole-2,5-dione ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122), an inhibitor of phosphatidylinositol-specific phospholipase C in vitro, did not affect the LPS-dependent activation of NFkappaB. Production of DAG and activation of NFkappaB after the LPS stimulus were observed in mouse macrophage-like J774.1 cells, and this response to LPS by J774. 1 cells was also inhibited by D609. These results suggest that the production of DAG from phosphatidylcholine was upstream of NFkappaB activation in response to a CD14-mediated LPS stimulus.

Chinese hamster ovary (CHO) K1 cells, typical nonprofessional phagocytes, exhibited intense phagocytosis of latex beads when incubated under serum-free conditions. Under the serum-free conditions, the recognition mechanism of latex beads by cells was investigated. Exogenous heparin and heparan sulfate but not chondroitin sulfate effectively inhibited the binding of latex beads to cells. The binding of latex beads to cells was also inhibited by treatment of cells with heparitinase more effectively than by treatment of cells with chondroitinase. Furthermore, CHO mutant cells defective in biosyntheses of both heparan sulfate and chondroitin sulfate proteoglycans almost completely lacked binding activity of latex beads. Another mutant, which is deficient in heparan sulfate proteoglycans but rather overproduces chondroitin sulfate proteoglycans, also showed lower binding activity, compared with wild-type cells. Coculture of these proteoglycan-less mutants and the wild-type cells did not restore the binding activity of the mutant cells, suggesting that membrane-bound rather than secretory proteoglycans were responsible for the binding of latex beads. These results indicated that heparan sulfate proteoglycans at the cell surface were involved in the binding step for phagocytosis of latex beads by CHO cells.

The orientation of the carboxyl terminal region of the Escherichia coli proline carrier in the cytoplasmic membrane was studied. The β-galactosidase moiety of the PutP-LacZ fusion protein [(1987) J. Biol. Chem. 262, 14100-14104] was exposed outside the inside-out vesicles and inside the right-side-out vesicles. A site-directed antibody raised against a synthetic peptide corresponding to the putative carboxyl terminal region of the carrier reacted preferentially with the inside-out vesicles prepared from a wild-type proline carrier overproducing strain and less with the right-side-out vesicles. These results indicate that the carboxyl terminus of the proline carrier is exposed to the cytoplasmic side of the membrane.Proline carrier, orientation; Carboxyl terminus; Fusion protein, PutP-LacZ; Antibody, site-directed; ELISA, competitive

Proline carrier, a product of the put P gene of Escherichia coli, was identified as a 35 kDa cytoplasmic membrane protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its identification was based on the following evidence: First, the density of the band corresponding to a 35 kDa protein correlated with the proline-binding activity of cytoplasmic membranes from put P-deficient and put P-amplified strains. Second, by the differential labeling method, the 35 kDa protein was specifically labeled with radioactive N-ethyl-maleimide. The 35 kDa protein was found to aggregate on heat treatment and to show abnormal mobility on SDS-PAGE.

Proline carrier of Escherichia coli was extracted from the carrier-overproducing membranes with dodecylmaltoside in the presence of phospholipid. The solubilized carrier showed the same proline binding activity as that in normal membranes. As judged from determinations of the binding activity in the micellar state as a marker of active carrier and the radioactivity of N-[ethyl-2- 3 H ] ethylmaleimide-labeled carrier as a marker of carrier polypeptide, 80% of the carrier molecules in the membranes were extracted. Optimal conditions for reconstitution of the solubilized carrier were established. By a combination of freeze-thawing, sonication and dilution procedures, 70% of the solubilized carrier molecules were incorporated into proteoliposomes and the restored active transport of proline showed an apparent K t of 1 μM and turnover number of 0.6 s−1. The transport of proline was driven by a membrane potential in a Na+ (or Li+)-dependent manner.

Sphingolipids are ubiquitous constituents of membrane lipids in eukaryotes. Sphingolipid metabolites modulate various cellular events including proliferation, differentiation, and apoptosis. In addition, sphingolipids, along with cholesterol, form detergent-resistant membrane microdomains, so called 'lipid-rafts', which are implicated in signal transduction and membrane trafficking. Sphingolipids are also relevant to infectious diseases. Various types of pathogens exploit the sphingolipids of host cells as membrane receptors. Sphingolipid metabolites regulate pathogen infection and host defense: for instance, a specific glycosphingolipid acts as an endogenous ligand for activation of natural killer T cells. Lipid-rafts of host cells serve as platforms also for infection signaling and entry of intracellular parasites. Moreover, some post-infectious autoimmune diseases result from production of antibodies cross-reacting with mammalian sphingolipids. Differences in the pathways of sphingolipid metabolism between mammals and non-mammals are good clues for rational development of new anti-infectious disease drugs. This review summarizes recent advances in sphingolipid biology related to infectious diseases.

Strain SPB-1 is a temperature-sensitive Chinese hamster ovary cell mutant defective in sphingoid base biosynthesis. To examine the effects of sphingolipid deficiency on a glycosyl phosphatidylinositol-anchored protein, we constructed transfectants expressing mouse CD14 from the SPB-1 and the wild type cells. When the cells were precultured in a sphingolipid-deficient medium at 39 degrees C, CD14 expressed in the SPB-1 cells was hypersensitive to phosphatidylinositol-specific phospholipase C (PI-PLC), compared with that in the wild type cells. Moreover, after exposure of the cells to fumonisin B1, an inhibitor of ceramide synthase, CD14, even in the wild type cells, became hypersensitive to PI-PLC. The PI-PLC hypersensitivity in the SPB-1 cells was almost completely suppressed when the cells were cultured in the presence of exogenous sphingosine or sphingomyelin. On the other hand, the hypersensitivity induced by fumonisin B1 was suppressed by exogenous sphingomyelin but not by sphingosine. The suppression by exogenous glucosylceramide was partial under both conditions. Likewise, CD14 molecules in membranes prepared from the sphingolipid-deficient cells were more sensitive to PI-PLC than those from the control cells. These results indicated that a deficiency in cellular complex sphingolipids caused the PI-PLC hypersensitivity of CD14, suggesting the interaction of CD14 with sphingolipids in membranes.

We devised an in situ assay method for the activity of serine palmitoyltransferase (SPT) that catalyzes the first step in sphingolipid biosynthesis and isolated a temperature-sensitive mutant of Chinese hamster ovary cells with thermolabile SPT. This mutant stopped growing at 40 degrees C after several generations, although the cells grew at 33 and 37 degrees C at rates similar to those of the parent. The SPT activity in cell homogenates of the mutant grown at low temperatures was 4-8% of that in the parent homogenates. When the cells were cultured for several generations at 40 degrees C, the activity in the mutant homogenate became negligible. When cell homogenates were incubated at 45 degrees C before enzyme assay, mutant SPT was more markedly inactivated than parental SPT, indicating that mutant SPT had become thermolabile. The rates of de novo synthesis of sphingolipids in the mutant were much slower at 40 degrees C than at lower temperatures, in contrast to those in the parent. The sphingomyelin content in the mutant cultivated at 40 degrees C for several generations was also less than that at low temperatures. These results indicate that SPT functions in the main pathway for sphingolipid biosynthesis. The temperature-sensitive growth of the mutant defective in sphingolipid synthesis suggests that sphingolipid(s) plays an essential role in cell growth.

Studies on human cell hybrids of a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that the tumorigenicity of these cells is under the control of a putative tumor suppressor on chromosome 11, although the nature of this suppressor remains unknown. We examined the expression of caveolin-1, a protein component of caveolae of the plasma membrane in these cell hybrids. The non-tumorigenic cell hybrid, CGL1, and normal fibroblast WI38 cells expressed 21-24 kDa caveolin-1, whereas in tumorigenic hybrid CGL4 as well as in the parental HeLa cells, the level of caveolin-1 was markedly reduced. Caveolin-1 expression was also reduced in gamma-ray-induced tumorigenic clones (GIMs) isolated from CGL1 cells, whereas non-tumorigenic irradiated cells expressed the same level of caveolin-1 as CGL1 cells. In accordance with these changes, the cellular level of caveolin-1 mRNA was reduced in the tumorigenic CGL4 cells and GIMs without any detectable changes in the caveolin-1 gene. However, the in vivo tumor growth of CGL4 cells was not altered when caveolin-1 was stably overexpressed through the transfection of a human caveolin-1 cDNA. These results suggest that reduction of caveolin-1 expression is necessary but not sufficient for emergence of the tumorigenic phenotypes of HeLa cell hybrids. Possible roles of the putative tumor suppressor in the control of gene expression are also discussed.

Background Progressive familial intrahepatic cholestasis type 1 (PFIC1), an inherited liver disease caused by mutations in ATP8B1, progresses to severe cholestasis with a sustained intractable itch. Currently, no effective therapy has been established for PFIC1. Decreased function of the bile salt export pump (BSEP) in hepatocytes is suggested to be responsible for the severe cholestasis observed in PFIC1. We found a previously unidentified pharmacological effect of 4-phenylbutyrate (4PB) that increases the expression and function of BSEP. Here, we tested 4PB therapy in three patients with PFIC1. Methods The therapeutic potency of 4PB in these patients was tested by oral administration of this drug with gradually increasing dosage (200, 350, and 500 mg/kg/day) for 6 months. Biochemical, histological, and clinical data were collected. Results 4PB therapy had no beneficial effect on the patients’ liver functions, as assessed by biochemical and histological analyses, despite an increase in hepatic BSEP expression. However, therapy with 4PB at a dosage of 350 or 500 mg/kg/day significantly relieved the intractable itch. Serum levels of potential pruritogens in cholestasis were much higher than the reference ranges during the 4PB therapy. Conclusions 4PB therapy may be a new medication for patients with intractable cholestatic pruritus and may improve quality of life for patients and their families.