RESUMO
Liposomes containing a synthetic recombinant protein were phagocytosed by macrophages, and the internalized protein was recycled to the cell surfaces where it was detected by enzyme-linked immunosorbent assay. The transit time of the liposome-encapsulated protein from initial phagocytosis of liposomes to appearance of protein on the surfaces of macrophages was determined by pulse-chase experiments. The macrophages were pulsed with liposomes containing protein and chased with empty liposomes, and vice versa. The amount and rate of protein antigen expression at the cell surfaces depended on the quantity of encapsulated protein ingested by the macrophages. Although liposomes were rapidly taken up by macrophages, the liposome-encapsulated protein was antigenically expressed for a prolonged period (at least 24 h) on the cell surface. Liposomes were visualized inside vacuoles in the macrophages by immunogold electron microscopy. The liposomes accumulated along the peripheries of the vacuoles and many of them apparently remained intact for a long time (greater than 6 h). However, nonliposomal free protein was also detected in the cytoplasm surrounding these vacuoles, and it was concluded that the free protein in the cytoplasm was probably en route to the macrophage surface. Exposure of the cells to ammonium chloride did not inhibit the appearance of liposomal antigenic epitopes on the cell surface, and this suggests that expression of the liposomal antigenic epitopes at the surface was not a pH-sensitive phenomenon. There was no significant effect of a liposomal adjuvant, lipid A, on the rate or extent of surface expression of the liposomal protein.
Assuntos
Antígenos de Protozoários/metabolismo , Lipossomos/imunologia , Macrófagos/metabolismo , Fagocitose , Plasmodium falciparum/imunologia , Proteínas de Protozoários , Sequência de Aminoácidos , Cloreto de Amônio/farmacologia , Animais , Antígenos Virais/metabolismo , Células da Medula Óssea , Ensaio de Imunoadsorção Enzimática , Cinética , Lipídeo A/farmacologia , Lipossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C3H , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Phosphatidylserine decarboxylase activity has been characterized in membrane preparations from Clostridium butyricum ATCC 19398. A particulate fraction was shown to catalyze the formation of phosphatidylethanolamine and plasmenylethanolamine when vesicles containing phosphatidylserine and plasmenylserine were used as substrate. No plasmenylethanolamine was formed when phosphatidylserine alone was used as substrate. The activity with phosphatidylserine was activated by divalent cations and was optimal under anaerobic conditions. Ionic detergents inhibited phosphatidylethanolamine formation strongly and nonionic detergents inhibited partially. In the presence of Triton X-100, phosphate from [32P]phosphatidylserine appeared in three unidentified lipid products, in addition to phosphatidylethanolamine. The formation of these products was time- and Triton X-100 concentration-dependent. Hydroxylamine inhibited phosphatidylserine decarboxylase, but did not prevent the reactions stimulated by Triton X-100.
Assuntos
Carboxiliases/metabolismo , Clostridium/enzimologia , Carboxiliases/antagonistas & inibidores , Cromatografia em Camada Fina , Clostridium/crescimento & desenvolvimento , Descarboxilação , Detergentes/farmacologia , Fosfatidilserinas/metabolismo , Plasmalogênios/metabolismo , Protoplastos/enzimologiaRESUMO
The plasmalogen content of phospholipids isolated from Megasphaera elsdenii ATCC 17752 decreased markedly in cultures passed serially at intervals of 3 to 6 weeks. From the wild-type ratio of vinyl ether to lipid phosphorus of 0.8, clones were isolated with ratios less than 0.05. Clonal analysis, as well as the reproducibility of the phenomenon and the long time course, suggest that the loss of plasmalogens is an adaptive process. Although small variations in cell morphology and ratios of end products of fermentation were detected, plasmalogen-rich and -deficient cells were virtually indistinguishable with respect to growth rates, range of fermentable carbohydrates, activities of selected enzymes, and electrophoretic patterns in both membrane and soluble proteins. Large decreases in saturated fatty acid production accompanied the decline of plasmalogens.
Assuntos
Fosfolipídeos/metabolismo , Plasmalogênios/análise , Veillonellaceae/metabolismo , Ciclopropanos/metabolismo , Compostos de Vinila/metabolismoRESUMO
Nocardia polychromogenes (saprophytic) and Nocardia asteroides (pathogenic) showed characteristic patterns in changes of cellular lipids during growth. Total lipids and total phospholipids decreased with the age of the culture in the saprophytic strain, whereas in the pathogenic strain total lipids increased throughout the culture period and the total phospholipids decreased in the late stationary phase. The decrease in total phospholipids in saprophytic strain was reflected in the individual phosphatides. In the pathogenic strain, the phosphatidylinositomannoside content doubled in early stationary phase. Differences were observed in fatty acid composition of phosphatides at various stages of growth, but the ratio of saturated to unsaturated fatty acids remained unaltered.
Assuntos
Nocardia asteroides/crescimento & desenvolvimento , Nocardia/crescimento & desenvolvimento , Fosfolipídeos/metabolismo , Nocardia/metabolismo , Nocardia asteroides/metabolismo , Especificidade da EspécieRESUMO
Ca2+-ATPase activity has been characterized in Streptomyces griseus. The enzyme has a pH optimum of 8.5 at 37 degrees C. Its Ca2+ requirement can be substituted by Cd2+, Zn2+ and Mn2+. Mg2+ inhibits the enzyme non-competitively.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Streptomyces griseus/enzimologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Magnésio/farmacologia , Metais/farmacologiaRESUMO
The total lipids increased with decrease in total phospholipids in Mycobacterium tuberculosis H37Rv grown at 30 degrees as compared to 37 degrees. Oleic and elaidic acid supplemented to the growth medium resulted in a higher total lipid and phospholipid content, but the content was lower in cells grown in the presence of palmitoleic acid. Cellular content of mannophosphoinositides decreased, whereas that of phosphatidylethanolamine and cardiolipin increased at the lower temperature. A decrease in mannophosphoinositides and an increase in cardiolipin were noted with all fatty acid supplements; however, phosphatidylethanolamine behaved differently. The unsaturated to saturated fatty acids ratio increased at low temperature with oleic and elaidic acid supplements, but it decreased with palmitoleic acid. Cells from media supplemented with palmitoleic acid were found to be most virulent to guinea-pigs. Possible correlation of altered lipid composition and virulence of M. tuberculosis H37Rv has been discussed.
Assuntos
Lipídeos/análise , Mycobacterium tuberculosis/patogenicidade , Animais , Meios de Cultura , Ácidos Graxos/análise , Ácidos Graxos/farmacologia , Ácidos Graxos Insaturados/análise , Cobaias , Mycobacterium tuberculosis/análise , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fosfolipídeos/análise , TemperaturaRESUMO
Liposomes containing lipid A induced potent humoral immune responses in mice against an encapsulated malaria antigen (R32NS1) containing NANP epitopes. The immune response was not enhanced by lipid A alone or by empty liposomes containing lipid A. Experiments to investigate the adjuvant mechanisms of liposomes and lipid A revealed that liposome-encapsulated R32NS1 was actively presented by bone marrow-derived macrophages to NANP-specific cloned T cells. The degree of presentation was related to the amount of liposomal antigen added per macrophage in the culture medium. At high cell densities, poor presentation occurred when liposomes lacked lipid A but excellent presentation occurred when the liposomes contained lipid A. Liposomes containing lipid A and encapsulated antigen also activated gamma interferon-treated macrophages to produce nitric oxide. Macrophage activation and antigen presentation occurred with liposomes that could not be detected by the Limulus amebocyte lysis assay. Intraperitoneal injection of liposomal lipid A caused a marked increase in the recruitment of immature (peroxidase-positive) macrophages to the peritoneum. On the basis of these experiments, we propose that the mechanism of the adjuvant action of liposomal lipid A is partly due to increased antigen presentation by macrophages and partly due to recruitment of an increased number of macrophages serving as antigen-presenting cells.