Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 pM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 pM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 mu M, respectively. The critical micellar concentration (CMC) of ODPC was 200 mu M. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (Delta H) variation of 7.3 kcal mol(-1). The presence of 25 mu M ODPC decreased T(c) and Delta H to 393 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 mu M destabilized the liposomes (36.3 degrees C. 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition. (C) 2010 Elsevier B.V. All rights reserved.

The neural histogenetic origin of the oral granular cell tumor: An immunohistochemical evidence

Aims: Granular cell tumor (GCT) is a rare neoplasm that can appear in any site of the body, but most are located intraorally. Its histogenetic origin remains unclear. This report analyzes the immunoprofile of 15 cases of granular cell tumors, occurring in 13 women and 2 men and the lesions were located on the tongue or upper lip. Patient age ranged from 7 to 52. Methods: The patients demographic data and the cytological and architectural features of the lesions were analyzed in oral GCTs (n = 15). The lesions were also submitted to a panel of immunohistochemical stains with antibodies against S-100, p75, NSE, CD-68, Ki-67, Synaptofisin, HHF-35, SMA, EMA, Chromogranin, Progesterone, Androgen and Estrogen. Results: Among the fifteen cases analyzed, the most common location was the tongue (84.6%). Histologically, the tumors exhibited cellular proliferation composed mainly by polygonal cells presenting an abundant granular eosinophilic cytoplasm. The nuclei were central, and the cell membranes were moderately clear. No mitotic figures were observed. The immunohistochemical analysis showed positivity in all cases for S-100, p75, NSE and CD-68, and no immunoreactivity for Ki-67, Synaptofisin, HHF-35, SMA, EMA, Chromogranin, Progesterone, Androgen and Estrogen. Conclusion: The immunoprofile of granular cell tumors showed nerve sheath differentiation - lending support to their neural origin - and helping to establish a differential diagnosis between this lesion and other oral granular cell tumors, whether benign or malignant.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Probing the interactions of NK cell receptors with ligand expressed in trans and cis.

Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.

Localization in sucrose gradients of the pyrophosphate-dependent proton transport of maize root membranes.

A maize (Zea mays L. cv LG 11) root homogenate was prepared and centrifuged to sediment the mitochondria. The pellet (6 KP) and the supernatant (6 KS) were collected and fractionated on linear sucrose density gradients. Marker enzymes were used to study the distribution of the different cell membranes in the gradients. The distribution of the ATP- and pyrophosphate-dependent proton pumping activities was similar after 3 hours of centrifugation of the 6 KS or the 6 KP fraction. The pumps were clearly separated from the mitochondrial marker cytochrome c oxidase and the plasmalemma marker UDP-glucose-sterolglucosyl-transferase. The pyrophosphate-dependent proton pump might be associated with the tonoplast, as the ATP-dependent pump, despite the lack of a specific marker for this membrane. However, under all the conditions tested, the two pumps overlapped the Golgi markers latent UDPase and glucan synthase I and the ER marker NADH-cytochrome c reductase. It is therefore not possible to exclude the presence of proton pumping activities on the Golgi or the ER of maize root cells. The two pumps (but especially the pyrophosphate-dependent one) were more active (or more abundant) in the tip than in the basal part of maize roots, indicating that these activities might be important in growth processes.

New cationic nanovesicular systems containing lysine-based surfactants for topical administration: Toxicity assessment using representative skin cell lines

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Cationic nanovesicles have attracted considerable interest as effective carriers to improve the delivery of biologically active molecules into and through the skin. In this study, lipid-based nanovesicles containing three different cationic lysine-based surfactants were designed for topical administration. We used representative skin cell lines and in vitro assays to assess whether the cationic compounds modulate the toxic responses of these nanocarriers. The nanovesicles were characterized in both water and cell culture medium. In general, significant agglomeration occurred after 24 h incubation under cell culture conditions. We found different cytotoxic responses among the formulations, which depended on the surfactant,cell line (3T3, HaCaT, and THP-1) and endpoint assayed (MTT, NRU, and LDH). Moreover, no potential phototoxicity was detected in fibroblast or keratinocyte cells, whereas only a slight inflammatory response was induced, as detected by IL-1a and IL-8 production in HaCaT and THP-1 cell lines, respectively. A key finding of our research was that the cationic charge position and the alkyl chain length of the surfactants determine the nanovesicles resulting toxicity. The charge on the a-amino group of lysine increased the depletion of cell metabolic activity, as determined by the MTT assay, while a higher hydrophobicity tends to enhance the toxic responses of the nanovesicles. The insights provided here using different cell lines and assays offer a comprehensive toxicological evaluation of this group of new nanomaterials.

Electrogenic proton/L-glutamate symport in isolated asparagus sprengeri mesophyll cell

Medium' alkaliniiation occurred -lipon the addition of L-Glu to mechanically isolated Asparagus sprenger-i mesophyll cells suspended in 1 mM CaS04. Alkalinization resulted from the coupled entry of H+ and L-Glu anion into the cells. This H+ IL-Glu symport did not stimulate K+ efflux. K+ efflux has been observed during H~ lamino acid symport in other systems. The stimulation of K+ efflux by proton coupled symport is regarded as an indicator of a plasma membrane depolarizing electrogenic symport process. H+ IL-Glu symport in Asparagus sprengerimesophyl1 cells was investigated to determine whether or not the process was electrogenic. The rate of uptake of 0.25 11M 3H-MTPP+ ( Methyltriphenylphosphonium, methyl-3H ) is a probe for monitoring changes in the membrane potential. 3HMTPP+ uptake was reduced by K+ or CCCP, agents known to depolarize the membrane potential. Uptake of 3H-MTPP+ was also inhibited by L-Glu but not by D-Glu. Conversely, 10 mM external MTPP+ inhibited the uptake of 14C-U-LGlu. Simultaneous measurements of the rates of 14C-U-L-Glu uptake and L-Glu dependent H+ influx showed that the molar stoichiometry of H+ IL-Glu symport was 2 to 1. K+ or Na+ stimulated H+ efflux was completely inhibited by DCCD, DES, oligomycin and antimycin reagents which inhibit ATP driven H+ efflux. The H+ efflux \Vas also stimulate.d by the weak acids, butyric acid and acetic acid, which are known fo-aCidify the cytoplasm. This weak acid stimulated H+ efflux was also completely inhibited by oligomycin. It was calculated that net L-Glu dependent H+ influx increased by 100% in the presence of oligomycin and that despite net medium alkalinization H+ IL-Glu symport stimulates ATP dependent H+ efflux. 11 The data presented in this study indicate that H+ IL-Glu symport is electrogenic. The data also show that ATP dependent Ht efflux rather than K+ efflux is the- process compensating for thi~ electrogenic H+ IL-Glu symport.

Isolation and partial characterization of host cell wall surface glycoproteins: their involvement in agglutination, attachment and appressorium formation by piptocephalis virginiana

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, to the mycoparasite, Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 a.u. mg- t ) as compared with the nonhost extract (21 a.li. mg- t ). SDS-polyacrylamide gel electrophoresis of the cell wall proteins revealed four protein bands, a, b, c, and d (Mr 117, 100, 85 and 64 kd, respectively) at the host surface, but not at the nonhost surface, except for the faint band c. Deletion of proteins b or c from the host cell wall protein extract significantly reduced its agglutinating activity. Proteins band c, obtained as purified preparations by a series of procedures, were shown to be two glycoproteins. Carbohydrate analysis by gas chromatography demonstrated that glucose and Nacetylglucosamine were the major carbohydrate components of the glycoproteins. It was further shown that the agglutinating activity of the pure preparation containing both band c was 500-850 times that of the single glycoproteins, suggesting the involvement of both glycoproteins in agglutination. The results suggest that the glycoproteins band c are the two subunits of agglutinin present at the host cell surface. The two glycoproteins band c purified from the host cell wall protein extract were further examined after various treatments for their possible role in agglutination, attachment and appressorium formation by the mycoparasite. Results obtained by agglutination and attachment tests showed: (1) the two glycoprotein-s are not only an agglutinin responsible for the mycoparasite spore agglutination, but may also serve as a receptor for the specific recognition, attachment and appressorium formation by the mycoparasite; (2) treatment of the rnycoparasite spores with various sugars revealed that arabinose, glucose and N-acetylglucosamine inhibited the agglutination and attachment activity of the glycoproteins, however, the relative percentage of appressorium formation was not affected by the above sugars; (3) the two glycoproteins are relatively stable with respect to their agglutinin and receptor functions. The present results suggest that the agglutination and attachment may be mediated directly by certain sugars present at the host and mycoparasite cell surfaces while the appressorlum formation may be the response of complementary combinations of both sugar and protein, the two parts of the glycoproteins at the interacting surfaces of two fungi.

Probing cell surfaces of host and nonhost species of a mycoparasite, using FITC-labeled lectins

Cell surfaces of susceptible host species (Mortierella pusllla and Cboanepilora cucurbitarum ), resistant host (Pilascolomyces articulosus ), nonhost (Mortierella candelabrum ) and the mycoparasite (Piptocepilalis virginiana) were examined for sugar distribution patterns using light and fluorescent microscopy techniques. The susceptible host, resistant host and the mycoparasite species exhibited a similar sugar distribution profile; they all showed N-acetyl glucosamine and D-glucose on their cell wall surfaces. The nonhost cell wall surface showed a positive binding reaction to FITClectins specific for N-acetyl glucosamine and also for OI.-fucose, N-acetyl galactosamine and galactose. Treatment of these fungi with mild concentrations of proteinases (both commercial as well as the mycoparasiteproteinase) resulted in the revelation of additional sugars on the fungal cell walls. The susceptible host treated with proteinase expressed higher levels of N-acetyl glucosamine and D-glucose. The susceptible host also showed the presence of OI.-fucose, N-acetyl galactosamine and galactose. The proteinasetreated susceptible host cell walls also showed an increase in the levels of attachment with the mycoparasite. Treatment of the resistant host with proteinases revealed OI.-fucose in addition to N-acetyl glucosamine and D-glucose. Treatment of the nonhost cell wall with proteinase resulted in the exposure of low levels of D-glucose, in addition to sugars found on the untreated nonhost cell wall surface. The mycoparasite treated with proteinase revealed OI.-fucose, N-acetyl galactosamine and galactose on its cell surface in addition to the sugars N-acetyl glucosamine and D-glucose. Protoplasts were isolated from hosts and nonhost fungi and their surfaces were examined for sugar distribution patterns. The susceptible host and nonhost protoplast membranes showed all the sugars (N-acetyl glucosamine, D-glucose, (It.-fucose, N-acetyl galactosamine and galactose) tested for. The resistant host protoplast membrane however, had only N-acetyl glucosamine and D-glucose exposed. This sugar distribution profile resembles that exhibited by the untreated resistant host cell wall, as well as that shown by the untreated mycoparasite cell surface. Only susceptible host protoplasts were successful in attaching to the mycoparasite surface. Resistant host protoplasts did not show any interaction with the i mycoparasite cell surface. Both susceptible as well as resistant host protoplasts were incapable of attaching to agarose beads surface-coated with specific carbohydrates. The mycoparasite however, did attach to agarose beads surface-coated with either N-acetyl glucosamine, D-glucose/Dmannose or o:,- methyl-D-mannose. The relevance of the cell wall and the protoplast membrane in the light of the present results, in reacting appropriately to bring about either a susceptible, a resistant or a nonhost response has been discussed.

Elucidation of a mechanism of cell lysis by chorhexidine : a biophysical approach

Chlorhexidine is an effective antiseptic used widely in disinfecting products (hand soap), oral products (mouthwash), and is known to have potential applications in the textile industry. Chlorhexidine has been studied extensively through a biological and biochemical lens, showing evidence that it attacks the semipermeable membrane in bacterial cells. Although extremely lethal to bacterial cells, the present understanding of the exact mode of action of chlorhexidine is incomplete. A biophysical approach has been taken to investigate the potential location of chlorhexidine in the lipid bilayer. Deuterium nuclear magnetic resonance was used to characterize the molecular arrangement of mixed phospholipid/drug formulations. Powder spectra were analyzed using the de-Pake-ing technique, a method capable of extracting both the orientation distribution and the anisotropy distribution functions simultaneously. The results from samples of protonated phospholipids mixed with deuterium-labelled chlorhexidine are compared to those from samples of deuterated phospholipids and protonated chlorhexidine to determine its location in the lipid bilayer. A series of neutron scattering experiments were also conducted to study the biophysical interaction of chlorhexidine with a model phospholipid membrane of DMPC, a common saturated lipid found in bacterial cell membranes. The results found the hexamethylene linker to be located at the depth of the glycerol/phosphate region of the lipid bilayer. As drug concentration was increased in samples, a dramatic decrease in bilayer thickness was observed. Differential scanning calorimetry experiments have revealed a depression of the DMPC bilayer gel-to-lamellar phase transition temperature with an increasing drug concentration. The enthalpy of the transition remained the same for all drug concentrations, indicating a strictly drug/headgroup interaction, thus supporting the proposed location of chlorhexidine. In combination, these results lead to the hypothesis that the drug is folded approximately in half on its hexamethylene linker, with the hydrophobic linker at the depth of the glycerol/phosphate region of the lipid bilayer and the hydrophilic chlorophenyl groups located at the lipid headgroup. This arrangement seems to suggest that the drug molecule acts as a wedge to disrupt the bilayer. In vivo, this should make the cell membrane leaky, which is in agreement with a wide range of bacteriological observations.

Effects of Delta9-tetrahydrocannabivarin on [35S]GTPgammaS binding in mouse brain cerebellum and piriform cortex membranes

BACKGROUND AND PURPOSE: We have recently shown that the phytocannabinoid Delta9-tetrahydrocannabivarin (Delta9-THCV) and the CB1 receptor antagonist AM251 increase inhibitory neurotransmission in mouse cerebellum and also exhibit anticonvulsant activity in a rat piriform cortical (PC) model of epilepsy. Possible mechanisms underlying cannabinoid actions in the CNS include CB1 receptor antagonism (by displacing endocannabinergic tone) or inverse agonism at constitutively active CB1 receptors. Here, we investigate the mode of cannabinoid action in [35S]GTPgammaS binding assays. EXPERIMENTAL APPROACH: Effects of Delta9-THCV and AM251 were tested either alone or against WIN55,212-2-induced increases in [35S]GTPgammaS binding in mouse cerebellar and PC membranes. Effects on non-CB receptor expressing CHO-D2 cell membranes were also investigated. KEY RESULTS :Delta9-THCV and AM251 both acted as potent antagonists of WIN55,212-2-induced increases in [35S]GTPgammaS binding in cerebellar and PC membranes (Delta9-THCV: pA2=7.62 and 7.44 respectively; AM251: pA2=9.93 and 9.88 respectively). At micromolar concentrations, Delta9-THCV or AM251 alone caused significant decreases in [35S]GTPgammaS binding; Delta9-THCV caused larger decreases than AM251. When applied alone in CHO-D2 membranes, Delta9-THCV and AM251 also caused concentration-related decreases in G protein activity. CONCLUSIONS AND IMPLICATIONS: Delta9-THCV and AM251 act as CB1 receptors antagonists in the cerebellum and PC, with AM251 being more potent than Delta9-THCV in both brain regions. Individually, Delta9-THCV or AM251 exhibited similar potency at CB1 receptors in the cerebellum and the PC. At micromolar concentrations, Delta9-THCV and AM251 caused a non-CB receptor-mediated depression of basal [35S]GTPgammaS binding.

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.

Fatty acids as gatekeepers of immune cell regulation

Fatty acids have diverse roles in all cells. They are important as a source of energy, as structural components of cell membranes, as signalling molecules and as precursors for the synthesis of eicosanoids. Recent research has suggested that the organization of fatty acids into distinct cellular pools has a particularly important role in cells of the immune system and that forms of lipid trafficking exist, which are as yet poorly understood. This Review examines the nature and regulation of cellular lipid pools in the immune system, their delivery of fatty acids or fatty acid derivatives to specific locations and their potential role in health and disease.

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)