RESUMEN
Cells are dynamic systems with complex mechanical properties, regulated by the presence of different species of proteins capable to assemble (and disassemble) into filamentous forms as required by different cells functions. Giant unilamellar vesicles (GUVs) of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) are systems frequently used as a simplified model of cells because they offer the possibility of assaying separately different stimuli, which is no possible in living cells. Here we present a study of the effect of acting protein on mechanical properties of GUVs, when the protein is inside the vesicles in either monomeric G-actin or filamentous F-actin. For this, rabbit skeletal muscle G-actin is introduced inside GUVs by the electroformation method. Protein polymerization inside the GUVs is promoted by adding to the solution MgCl2 and the ion carrier A23187 to allow the transport of Mg+2 ions into the GUVs. To determine how the presence of actin changes the mechanical properties of GUVs, the vesicles are deformed by the application of an AC electric field in both cases with G-actin and with polymerized F-actin. The changes in shape of the vesicles are characterized by optical microscopy and from them the bending stiffness of the membrane are determined. It is found that G-actin has no appreciable effect on the bending stiffness of DMPC GUVs, but the polymerized actin makes the vesicles more rigid and therefore more resistant to deformations. This result is supported by evidence that actin filaments tend to accumulate near the membrane.
Asunto(s)
Actinas/química , Dimiristoilfosfatidilcolina/química , Electricidad , Liposomas Unilamelares/química , Citoesqueleto de Actina/química , Actinas/metabolismo , Animales , Calcimicina/química , Cloruro de Magnesio/química , Cloruro de Magnesio/metabolismo , Microscopía , Músculo Esquelético/metabolismo , Conejos , Tensión Superficial , Liposomas Unilamelares/metabolismo , ViscosidadRESUMEN
Available assays for measuring cellular manganese (Mn) levels require cell lysis, restricting longitudinal experiments and multiplexed outcome measures. Conducting a screen of small molecules known to alter cellular Mn levels, we report here that one of these chemicals induces rapid Mn efflux. We describe this activity and the development and implementation of an assay centered on this small molecule, named manganese-extracting small molecule (MESM). Using inductively-coupled plasma-MS, we validated that this assay, termed here "manganese-extracting small molecule estimation route" (MESMER), can accurately assess Mn in mammalian cells. Furthermore, we found evidence that MESM acts as a Mn-selective ionophore, and we observed that it has increased rates of Mn membrane transport, reduced cytotoxicity, and increased selectivity for Mn over calcium compared with two established Mn ionophores, calcimycin (A23187) and ionomycin. Finally, we applied MESMER to test whether prior Mn exposures subsequently affect cellular Mn levels. We found that cells receiving continuous, elevated extracellular Mn accumulate less Mn than cells receiving equally-elevated Mn for the first time for 24 h, indicating a compensatory cellular homeostatic response. Use of the MESMER assay versus a comparable detergent lysis-based assay, cellular Fura-2 Mn extraction assay, reduced the number of cells and materials required for performing a similar but cell lethality-based experiment to 25% of the normally required sample size. We conclude that MESMER can accurately quantify cellular Mn levels in two independent cells lines through an ionophore-based mechanism, maintaining cell viability and enabling longitudinal assessment within the same cultures.
Asunto(s)
Ionóforos/química , Manganeso/análisis , Animales , Calcimicina/química , Calcimicina/farmacología , Calcio/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fura-2/química , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ionomicina/química , Ionomicina/farmacología , Ionóforos/farmacología , Masculino , Manganeso/química , Manganeso/metabolismo , Manganeso/toxicidad , Espectrometría de Masas/métodos , RatonesRESUMEN
NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca2+ into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.
Asunto(s)
Citocinas/metabolismo , Estrés del Retículo Endoplásmico , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Peptidoglicano/sangre , Calcimicina/química , Calcimicina/farmacología , Calcio/química , Calcio/metabolismo , Quimiocina CXCL1/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Interleucina-8/metabolismo , Proteína Adaptadora de Señalización NOD1/deficiencia , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/deficiencia , Proteína Adaptadora de Señalización NOD2/genética , Transducción de Señal/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A1-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A1 and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Mastocitos/metabolismo , Poliaminas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Animales , Calcimicina/química , Calcio/química , Catepsina D/química , Exocitosis , Histamina/química , Liberación de Histamina , Inmunohistoquímica , Masculino , Mastocitos/citología , Ratones , Microscopía Fluorescente , Proteínas R-SNARE/metabolismo , Ratas , Ratas Wistar , Vesículas Secretoras/metabolismo , Serotonina/química , Espermidina/metabolismo , Espermina/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 3 de Membrana Asociada a Vesículas/metabolismoRESUMEN
A novel acid-promoted rearrangement is disclosed. In the previously unknown transformation, an allyl group migrated to an in situ formed carbocation stabilized by an electron-rich aryl or heteroaryl group, resulting in a stereoselective intramolecular Grob fragmentation. The outcome of the rearrangement observed with an array of substrates can be satisfactorily rationalized using a working hypothesis with the aid of a six-membered transition state similar to those proposed for the anionic oxy-Cope or oxonia-Cope rearrangements, but involving only one instead of two double bonds.
Asunto(s)
Calcimicina/análogos & derivados , Acetatos/química , Calcimicina/síntesis química , Calcimicina/química , Difosfonatos/química , Peróxido de Hidrógeno/química , Policétidos/química , EstereoisomerismoRESUMEN
The synthesis of (-)-demethyl (C-11) cezomycin was achieved through an efficient route that features the use of a Kulinkovich reaction to couple two multifunctionality-containing fragments and a cascade of ring opening of cyclopropanol/1,5-hydrogen shift/desilylation-oxidation. The hidden yet undeniable problem of irreproducible specific rotation for this family of compounds was solved by sufficient acidification. The absolute configuration for the natural product was thus established as the mirror image of the synthetic sample.
Asunto(s)
Antibacterianos/síntesis química , Calcimicina/análogos & derivados , Antibacterianos/química , Calcimicina/síntesis química , Calcimicina/química , Hidrógeno/química , Oxidación-Reducción , EstereoisomerismoRESUMEN
The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.
Asunto(s)
Antibacterianos/biosíntesis , Calcimicina/biosíntesis , Furanos/metabolismo , Pirroles/metabolismo , Compuestos de Espiro/metabolismo , Streptomyces/metabolismo , ortoaminobenzoatos/metabolismo , Antibacterianos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Calcimicina/química , Furanos/química , Espectrometría de Masas , Estructura Molecular , Mutación , Pirroles/química , Compuestos de Espiro/química , Streptomyces/enzimología , Streptomyces/genética , ortoaminobenzoatos/químicaRESUMEN
To maintain their metal ion homeostasis, bacteria critically depend on membrane integrity and controlled ion translocation. Terrestrial Streptomyces species undermine the function of the cytoplasmic membrane as diffusion barrier for metal cations in competitors using ionophores. Although the properties of the divalent cation ionophores calcimycin and ionomycin have been characterized to some extent in vitro, their effects on bacterial ion homeostasis, the factors leading to bacterial cell death, and their ecological role are poorly understood. To gain insight into their antibacterial mechanism, we determined the metal ion composition of the soil bacterium Bacillus subtilis after treatment with calcimycin and ionomycin. Within 15 min the cells lost approximately half of their cellular iron and manganese content whereas calcium levels increased. The proteomic response of B. subtilis provided evidence that disturbance of metal cation homeostasis is accompanied by intracellular oxidative stress, which was confirmed with a ROS-specific fluorescent probe. B. subtilis showed enhanced sensitivity to the ionophores in medium lacking iron or manganese. Furthermore, in the presence of ionophores bacteria were sensitive to high calcium levels. These findings suggest that divalent cation ionophores are particularly effective against competing microorganisms in soils rich in available calcium and low in available iron and manganese.
Asunto(s)
Bacillus subtilis/metabolismo , Ionóforos/farmacología , Hierro/metabolismo , Manganeso/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/química , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Calcimicina/química , Calcio/metabolismo , Ionóforos de Calcio/farmacología , Ecología , Homeostasis , Ionomicina/química , Ionóforos/química , Hierro/aislamiento & purificación , Hierro/farmacología , Manganeso/aislamiento & purificación , Manganeso/farmacología , Micronutrientes/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteoma/efectos de los fármacos , Proteoma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Microbiología del SueloRESUMEN
Mass spectrometry techniques have enabled the identification of different lipid metabolites and mediators derived from omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFA) that are implicated in various biological processes. However, the broad-spectrum assessment of physiologically formed lipid metabolites and mediators in blood samples has not been presented so far. Here lipid mediators and metabolites of the n-6 PUFA arachidonic acid as well as the long-chain n-3 PUFA eicosapentaenoic acids (EPA) and docosahexaenoic acid (DHA) were measured in human blood samples as well as in mouse blood. There were detectable but mostly very low amounts of the assayed compounds in human native plasma samples, whereas in vitro activation of whole blood with the calcium ionophore A23187 led to highly significant increases of metabolite formation, with a predominance of the 12-lipoxygenase (12-LOX) products 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA). A23187 activation also led to significant increases in the formation of 5-LOX products including leukotriene B(4) (LTB(4)), leukotriene B(5) (LTB(5)) as well as of 15-LOX products and prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)). Levels were similar or even higher in A23187-activated mouse blood. The approach presented here thus provides a protocol for the comprehensive and concomitant assessment of the generation capacity of n-3 and n-6 PUFA-derived lipid metabolites as well as thromboxanes and prostaglandins in human and murine blood samples. Further studies will now have to evaluate lipid metabolite generation capacity in different physiological and pathophysiological contexts.
Asunto(s)
Ácidos Grasos Omega-3/sangre , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-6/sangre , Ácidos Grasos Omega-6/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/sangre , Araquidonato 12-Lipooxigenasa/metabolismo , Calcimicina/química , Cromatografía Liquida/métodos , Humanos , Metabolismo de los Lípidos , Ratones , Prostaglandinas/sangre , Prostaglandinas/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Tromboxanos/sangre , Tromboxanos/metabolismoRESUMEN
The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.
Asunto(s)
Antibacterianos/biosíntesis , Calcimicina/biosíntesis , Streptomyces/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Calcimicina/química , Datos de Secuencia Molecular , Estructura Molecular , Homología de Secuencia de Aminoácido , Streptomyces/genéticaRESUMEN
PURPOSE: A liposomal irinotecan formulation referred to as Irinophore C relies on the ability of copper to complex irinotecan within the liposome. It is currently being evaluated for critical drug-loading parameters. Studies presented here were designed to determine the optimum copper concentration required for the effective encapsulation and retention of irinotecan into liposomes. METHODS: Distearoylphosphatidylcholine/cholesterol liposomes were formulated using buffers containing various copper or manganese concentrations, and irinotecan loading was determined in the presence and absence of divalent metal ionophore A23187. The rate and extent of irinotecan encapsulation and the rate of irinotecan release from the liposomes were assessed. The amount of copper retained inside liposomes following irinotecan loading and the effect of copper on membrane permeability were determined. RESULTS: Efficient (>98%) irinotecan loading was achieved using encapsulated copper concentrations of 50 mM. However, irinotecan release was copper concentration dependent, with a minimum 300 mM concentration required for optimal drug retention. The presence of copper increased liposomal membrane permeability. CONCLUSION: Results explain why irinotecan loading rates are enhanced in the presence of formulations prepared with copper, and we speculate that the Irinophore C formulation exhibits improved drug retention, due to generation of a complex between copper and irinotecan.
Asunto(s)
Antineoplásicos/química , Calcimicina/química , Camptotecina/análogos & derivados , Química Farmacéutica/métodos , Cobre/química , Ionóforos/química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Camptotecina/administración & dosificación , Camptotecina/sangre , Camptotecina/química , Permeabilidad de la Membrana Celular , Colesterol/química , Cromatografía Líquida de Alta Presión , Femenino , Irinotecán , Liposomas , Ratones , Ratones Endogámicos BALB C , Fosfatidilcolinas/químicaRESUMEN
These studies describe the role of transition metal ions in the liposomal encapsulation of topotecan. Liposomes (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CH) (55:45, mole ratio)) were prepared with manganese (Mn), copper (Cu), zinc (Zn) or cobalt (Co) ion gradients (metal inside). Subsequently, topotecan was added to the liposome exterior (final drug-to-lipid ratio (mol/mol) of 0.2) and drug encapsulation was measured as a function of time and temperature. No drug loading was achieved with liposomes containing Co or Zn. Topotecan could be encapsulated into Mn-containing liposomes only in the presence of the ionophore, A23187 suggesting that a transmembrane pH gradient was necessary. However, Cu-containing liposomes, in the presence or absence of an imposed pH gradient, efficiently encapsulated topotecan. It has been reported that Cu(II) can form transition metal complexes with camptothecin; therefore, the Cu-topotecan interaction was characterized in solution as a function of pH. These investigations demonstrated that topotecan inhibited formation of an insoluble Cu hydroxide precipitate. Cryo-TEM analysis of the topotecan-loaded Cu liposomes showed electron-dense intravesicular precipitates. Further studies demonstrated that only the active lactone form of the drug was encapsulated and this form predominated in Cu-containing liposomes. Copper complexation reactions define a viable methodology to prepare liposomal camptothecin formulations.
Asunto(s)
Cobre/química , Liposomas/química , Topotecan/química , Tampones (Química) , Calcimicina/química , Cationes Bivalentes/química , Precipitación Química , Colesterol/química , Microscopía por Crioelectrón , Doxorrubicina/química , Composición de Medicamentos/métodos , Concentración de Iones de Hidrógeno , Lactonas/química , Compuestos de Manganeso/química , Estructura Molecular , Nigericina/química , Fosfatidilcolinas/química , Fuerza Protón-Motriz , Sulfatos/químicaRESUMEN
A comparative study of the loading and retention properties of three structurally very closely related vinca alkaloids (vincristine, vinorelbine and vinblastine) in liposomal formulations has been performed. All three vinca alkaloids showed high levels of encapsulation when accumulated into egg sphingomyelin/cholesterol vesicles in response to a transmembrane pH gradient generated by the use of the ionophore A23187 and encapsulated MgSO4. However, despite the close similarities of their structures the different vinca drugs exhibited very different release behavior, with vinblastine and vinorelbine being released faster than vincristine both in vitro and in vivo. The differences in loading and retention can be related to the lipophilicity of the drugs tested, where the more hydrophobic drugs are released more rapidly. It was also found that increasing the drug-to-lipid ratio significantly enhanced the retention of vinca alkaloids when the ionophore-based method was used for drug loading. In contrast, drug retention was not dependent on the initial drug-to-lipid ratio for vinca drugs loaded into liposomes containing an acidic citrate buffer. The differences in retention can be explained on the basis of differences in the physical state of the drug inside the liposomes. The drug-to-lipid ratio dependence of retention observed for liposomes loaded with the ionophore technique may provide a way to improve the retention characteristics of liposomal formulations of vinca drugs.
Asunto(s)
Vinblastina/análogos & derivados , Vinblastina/química , Vinblastina/farmacocinética , Vincristina/química , Vincristina/farmacocinética , Animales , Calcimicina/química , Femenino , Inyecciones Intravenosas , Ionóforos/química , Liposomas , Sulfato de Magnesio/química , Ratones , Ratones Endogámicos ICR , Solubilidad , Vinblastina/administración & dosificación , Vincristina/administración & dosificación , VinorelbinaRESUMEN
Vinorelbine (VRL) is a particularly lipophilic member of the vinca alkaloids which, as a class of drugs, exhibit improved cytotoxicity and therapeutic activity through increased duration of exposure. Here, we describe and optimize a sphingomyelin/cholesterol (SM/Chol) liposome formulation of VRL to maximize in vivo drug retention, plasma circulation time, and therapeutic activity. VRL was efficiently encapsulated (>90%) into 100 nm liposomes using an ionophore-mediated loading method. VRL retention in SM/Chol liposomes after intravenous injection in mice was dependent on drug-to-lipid ratio (D/L), with higher D/L ratios exhibiting increased drug retention (0.3 > 0.2 > 0.1, wt/wt) and improved pharmacokinetics. Cryo-electron microscopic examination of a high D/L ratio formulation indicated that the intravesicular regions of these liposomes were electron dense compared with empty liposomes. The optimized, high D/L ratio SM/Chol VRL formulation showed promising activity against subcutaneous B16 melanoma tumors compared with VRL or SM/Chol formulations of vincristine or vinblastine. Finally, the stability of the formulation was excellent (<5% drug leakage, >99% intact VRL, no changes in liposome size after 1 year at 2-8 degrees C). The optimized drug retention properties of the SM/Chol formulation of VRL, combined with its promising antitumor activity and pharmaceutical stability, make this formulation an excellent candidate for future clinical development.
Asunto(s)
Antineoplásicos Fitogénicos/química , Colesterol/química , Esfingomielinas/química , Vinblastina/análogos & derivados , Vinblastina/química , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacocinética , Calcimicina/química , Química Farmacéutica , Cromatografía Líquida de Alta Presión , Microscopía por Crioelectrón , Estabilidad de Medicamentos , Excipientes , Femenino , Concentración de Iones de Hidrógeno , Ionóforos/química , Liposomas , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Tamaño de la Partícula , Vinblastina/administración & dosificación , Vinblastina/farmacocinética , Alcaloides de la Vinca/administración & dosificación , Alcaloides de la Vinca/química , Alcaloides de la Vinca/farmacocinética , VinorelbinaRESUMEN
Topotecan can be encapsulated in liposomes, however little is known about the role encapsulated counter ions play in drug loading efficiency and drug release. Using 1,2-distearoyl-sn-glycero-3 phosphatidylcholine and cholesterol liposomes (55:45 mole ratio), encapsulation was achieved using manganese ion gradients (MnSO(4) or MnCl(2)), with the addition of A23187, a divalent cation/proton exchanger, to maintain a pH gradient. This methodology was compared to procedures where the pH gradient was generated by use of encapsulated (NH(4))(2)SO(4) or citrate (300 mM, pH 3.5). All methods facilitated topotecan encapsulation. Liposomes prepared in the presence of the citrate and MnCl(2) (+A23187) exhibited reduced loading capacities. Liposomes prepared in the presence of (NH(4))(2)SO(4) and MnSO(4) (+A23187) could be used to generate liposomes exhibiting a drug-to-lipid ratio of 0.3 (wt/wt) with an encapsulation efficiency of >90%. In vitro drug release data suggested that the (NH(4))(2)SO(4) and MnSO(4) (+A23187) formulations released drug at a reduced rate. For these formulations, the drug release rates decreased as the drug-to-lipid ratio (wt/wt) increased from 0.1 to 0.2. Cryo-electron micrographs indicated that encapsulated topotecan precipitated as linear particles within liposomes. The stability of topotecan loaded liposomes appeared to be dependent on the presence of both a pH gradient and encapsulated sulfate.
Asunto(s)
Antineoplásicos/administración & dosificación , Topotecan/administración & dosificación , Sulfato de Amonio/química , Antibacterianos/administración & dosificación , Antibacterianos/química , Calcimicina/administración & dosificación , Calcimicina/química , Química Farmacéutica , Citratos/química , Microscopía por Crioelectrón , Composición de Medicamentos , Concentración de Iones de Hidrógeno , Lípidos/química , Liposomas , Manganeso/química , Membranas Artificiales , Tamaño de la Partícula , Solventes , Sulfatos/químicaRESUMEN
Waves of elevated intracellular free calcium that propagate between neighboring astrocytes are important for the intercellular communication between astrocytes as well as between neurons and astrocytes. However, the mechanisms responsible for the initiation and propagation of astrocytic calcium waves remain unclear. In this study, intercellular calcium waves were evoked by focal photolysis of a caged calcium ionophore (DMNPE-caged Br A23187) in cultured astrocytes from newborn rats. The focal photolysis of the caged compound resulted in the increase in intracellular calcium in a single astrocyte, and this increase then propagated to neighboring astrocytes. We also analyzed the spatiotemporal characteristics of the intercellular calcium waves, and estimated the propagation pathways for them. The method using a caged calcium ionophore described in this study provides a new in vitro model for the analysis of intercellular calcium waves.
Asunto(s)
Astrocitos/fisiología , Calcimicina/análogos & derivados , Calcimicina/farmacología , Señalización del Calcio/fisiología , Calcio/fisiología , Ionóforos/farmacología , Animales , Animales Recién Nacidos , Calcimicina/química , Células Cultivadas , Difusión , Espacio Extracelular/fisiología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/fisiología , Inmunohistoquímica , Neuroglía/efectos de los fármacos , Neuroglía/fisiología , Nitrobencenos/química , Estimulación Luminosa , Fotólisis , RatasRESUMEN
This study is an investigation of the temporal relationship between transmembrane Ca(2+) fluxes, and glycogen phosphorylase activation in dispersed trophocytes from the fat body of the cockroach, Periplaneta americana. Phosphorylase is maximally activated within 5 min after treating the trophocytes with either of the hypertrehalosemic hormones, Pea-HTH-I and Pea-HTH-II. Activation caused by Pea-HTH-II is sustained for a longer period than that produced by Pea-HTH-I. Chelation of extracellular Ca(2+) with EGTA blocks the activation of phosphorylase by HTH. Similarly, chelation of intracellular Ca(2+) with Quin 2 greatly diminishes the phosphorylase activating effect of both HTHs. The data support the view that an increase in the intracellular Ca(2+ )concentration is required for the activation of phosphorylase and that extracellular Ca(2+) is an essential, although not necessarily sole, source of Ca(2+) for this purpose. Using (45)Ca(2+) to trace the movement of Ca(2+) following a challenge with either Pea-HTH-I or -II, it was shown that (45)Ca(2+)influx nearly doubled during the first 30 s. At this time, the trophocytes begin to expel Ca(2+) at a rate higher than that of untreated cells and this state persists for approximately 4 min. The Ca(2+) fluxes are consistent with its postulated role in the activation of phosphorylase. Arch.
Asunto(s)
Calcio/metabolismo , Cuerpo Adiposo/enzimología , Hormonas de Insectos/fisiología , Periplaneta/enzimología , Fosforilasas/metabolismo , Aminoquinolinas/química , Animales , Calcimicina/química , Radioisótopos de Calcio , Quelantes/química , Ácido Egtácico/química , Activación Enzimática , Colorantes Fluorescentes/química , Ionóforos/química , Masculino , Fosforilasas/análisis , Conteo por Cintilación , Factores de TiempoRESUMEN
Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.
Asunto(s)
Bovinos/fisiología , Criopreservación/veterinaria , Técnicas de Transferencia Nuclear , Resultado del Embarazo/veterinaria , Animales , Animales Recién Nacidos , Calcimicina/química , Bovinos/embriología , Criopreservación/métodos , Cicloheximida/química , ADN/química , ADN/aislamiento & purificación , Femenino , Fertilización In Vitro/veterinaria , Masculino , Repeticiones de Microsatélite/genética , Donación de Oocito/veterinaria , Oocitos/química , Oocitos/fisiología , Reacción en Cadena de la Polimerasa/veterinaria , Embarazo , Ultrasonografía PrenatalRESUMEN
The processes of capacitation and acrosomal exocytosis of dog spermatozoa in vitro have yet to be fully investigated. Firstly, we investigated the effectiveness of a technique for staining dog spermatozoa with the fluorescent labels Hoechst 33258 and chlortetracycline. A modified fluorescence microscopy staining method was shown to be effective for the assessment of both viability and functional status in this species. Secondly, the presence of the ionophore A23187 in culture medium was shown to promote capacitation and the acrosome reaction of dog spermatozoa. We have therefore established that this dual fluorescent staining method can be used for monitoring these events in the dog, and it may be useful in future studies of optimal in vitro culture conditions.
Asunto(s)
Acrosoma/fisiología , Perros/fisiología , Colorantes Fluorescentes/química , Capacitación Espermática/fisiología , Espermatozoides/fisiología , Compuestos de Anilina/química , Animales , Bisbenzimidazol/química , Calcimicina/química , Clortetraciclina/química , Ionóforos/química , Masculino , Microscopía Fluorescente/veterinariaRESUMEN
The effects of two polyunsaturated fatty acids, 18:4n-3 and 16:4n-3 purified from the marine algae, Undaria pinnatifida and Ulva pertusa, on icosanoid production in MC/9 mouse mast cells were assessed. Both fatty acids suppressed the production of leukotriene B4 (LTB4), leukotriene C4 (LTC4), and 5-hydroxyeicosatetraenoic acid (5-HETE). The order of the suppressive activity for the two marine algae-derived fatty acids and three other common polyunsaturated fatty acids was as follows; 22:6n-3 = 18:4n-3 = 18:3n-3 > 20:5n-3 = 16:4n-3 for LTB4; 22:6n-3 = 18:4n-3 = 18:3n-3 > 16:4n-3 > 20:5n-3 (no suppression) for LTC4; 22:6n-3 = 18:4n-3 > 18:3n-3 > 20:5n-3 = 16:4n-3 for 5-HETE.