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1.
J Ethnopharmacol ; 271: 113884, 2021 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-33529639

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Piper capense is a medicinal spice whose fruits are traditionally used as aqueous decoction to heal several ailments such as trypanosomiasis, helminthic infections, and cancer. AIM OF THE STUDY: (1) To perform phytochemical investigation of the methanol extract of Piper capense; (2) to evaluate the cytotoxicity of botanicals (PCF, fractions PCFa-e), isolated phytochemicals on a broad panel of animal and human cancer cell lines; (3) to evaluate the induction of apoptosis of the most active samples. MATERIAL AND METHODS: Resazurin reduction assay (RRA) was used to determine the cytotoxicity of the studied samples. Cell cycle distribution (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP; JC-1) and reactive oxygen species (ROS; H2DCFH-DA) were measured by flow cytometry. Column chromatography (CC) was used for the purification of PCF, whilst nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) analyses were applied for structural elucidation. RESULTS: The phytochemical investigation of PCF led to the isolation of 11 compounds: licarin B (1), licarin A (2), 7-(1,3-benzodioxol-5-yl)-7,8-dihydro-8-methyl-5-(2-propenyl)-furo[3,2-e]-1,3-benzodioxole (3), nitidine isocyanate (4), 5-hydroxy-7,4'-dimethoxyflavone (5), cardamomin (6), sitosterol (7) and stigmasterol (8), ß-sitosterol 3-O-ß-D-glucopyranoside (9), oleanolic acid (10) and lupeol (11). Fraction PCFb, compound 2 and doxorubicin (as positive control drug) revealed cytotoxic effects towards the 18 tested cancer cell lines. The IC50 values ranged from 6.1 µg/mL (against CCRF-CEM cells) to 44.2 µg/mL (against BRAF-V600E homozygous mutant melanoma cells) for PSCb; from 4.3 µM (against CCRF-CEM cells) to 21.8 µM (against HCT116 p53-/-) for compound 2 and from 0.02 µM (against CCRF-CEM cells) to 123.0 µM (against CEM/ADR5000 cells) for doxorubicin. PCFb and compound 2 induced apoptosis in CCRF-CEM cells mediated by activation of caspase 3/7, 8 and 9, MMP alteration and increased ROS production. CONCLUSION: Piper capense is a source of potent cytotoxic botanicals and phytochemicals that could help to fight various types of cancer including multidrug resistance phenotypes. PCFb and compound 2 should further be explored to develop new drugs to fight malignancies.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Frutas/química , Fitoquímicos/farmacología , Piper/química , Extractos Vegetales/farmacología , Animales , Antineoplásicos Fitogénicos/química , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Lignanos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Fitoquímicos/química , Extractos Vegetales/química , Especies Reactivas de Oxígeno/metabolismo , Valinomicina/farmacología
2.
Artículo en Inglés | MEDLINE | ID: mdl-32284379

RESUMEN

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families, including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (coxsackievirus, rhinovirus), flavirivuses (Zika virus), and coronaviruses (human coronavirus 229E [HCoV-229E] and Middle East respiratory syndrome CoV [MERS-CoV]). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication.


Asunto(s)
Antivirales/farmacología , Encefalitis de California/tratamiento farmacológico , Ionóforos/farmacología , Virus La Crosse/efectos de los fármacos , Potasio/metabolismo , Valinomicina/farmacología , Replicación Viral/efectos de los fármacos , Coronavirus/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Encefalitis de California/virología , Enterovirus/efectos de los fármacos , Flavivirus/efectos de los fármacos , Humanos , Orthobunyavirus/efectos de los fármacos , Estados Unidos
3.
Anal Biochem ; 567: 8-13, 2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30503709

RESUMEN

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular and extracellular concentrations of the measured ion must be made equal. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two potassium probes, APG-4 and APG-2. APG-4 shows good sensitivity to potassium but its fluorescence is sensitive to cell volume. Because ionophores cause cell swelling, calibration buffers had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured at 132 mM and 118 mM, respectively. The other tested probe, APG-2, is nonselective for cations; this is, however, a potentially useful property because the sum [K+] + [Na+] determines the amount of intracellular water.


Asunto(s)
Colorantes Fluorescentes/química , Calibración , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Citometría de Flujo/normas , Colorantes Fluorescentes/farmacología , Humanos , Modelos Teóricos , Valinomicina/farmacología
4.
Biochim Biophys Acta Biomembr ; 1859(12): 2373-2380, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28888365

RESUMEN

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active Kv1.1 and Kv1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv1.1 and Kv1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.


Asunto(s)
Canal de Potasio Kv.1.1/metabolismo , Canal de Potasio Kv1.3/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Proteolípidos/efectos de los fármacos , Venenos Elapídicos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Isoxazoles/química , Canal de Potasio Kv.1.1/genética , Canal de Potasio Kv1.3/genética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/metabolismo , Valinomicina/farmacología
5.
J Surg Res ; 188(2): 473-9, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24582214

RESUMEN

BACKGROUND: Mitochondrial dysfunction has been closely related to many pathologic processes, such as cellular apoptosis. Alterations in organelle membrane potential are associated with mitochondrial dysfunction. A fluorine-18 labeled phosphonium compound: (18)F-triphenylphosphonium ((18)F-TPP) was prepared to determine its potential use as a mitochondria-targeting radiopharmaceutical to evaluate cellular apoptosis. METHODS: Studies were conducted in both ex vivo cell lines and in vivo using a burned animal model. Uptake of (18)F-TPP was assessed in PC-3 cells by gamma counting under the following conditions: graded levels of extracellular potassium concentrations, incubation with carbonyl cyanide m-chlorophenylhydrazone and staurosporine. Apoptosis was studied in a burn animal model using terminal deoxynucleotidyl transferase dUTP nick end labeling staining and simultaneous assessment of (18)F-TPP uptake by biodistribution. RESULTS: We found that stepwise membrane depolarization by potassium (K) resulted in a linear decrease in (18)F-TPP uptake, with a slope of 0.62 ± 0.08 and a correlation coefficient of 0.936 ± 0.11. Gradually increased concentrations of m-chlorophenylhydrazone lead to decreased uptake of (18)F-TPP. Staurosporine significantly decreased the uptake of (18)F-TPP in PC-3 cells from 14.2 ± 3.8% to 5.6 ± 1.3% (P < 0.001). Burn-induced significant apoptosis (sham: 4.4 ± 1.8% versus burn: 24.6 ± 6.7 %; P < 0.005) and a reduced uptake of tracer in the spleens of burn-injured animals as compared with sham burn controls (burn: 1.13 ± 0.24% versus sham: 3.28 ± 0.67%; P < 0.005). Biodistribution studies demonstrated that burn-induced significant reduction in (18)F-TPP uptake in spleen, heart, lung, and liver, which were associated with significantly increased apoptosis. CONCLUSIONS: (18)F-TPP is a promising new voltage sensor for detecting mitochondrial dysfunction and apoptosis in various tissues.


Asunto(s)
Apoptosis , Quemaduras/diagnóstico por imagen , Radioisótopos de Flúor , Potencial de la Membrana Mitocondrial , Compuestos Organofosforados/uso terapéutico , Animales , Carbonil Cianuro m-Clorofenil Hidrazona , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Ratones Endogámicos C57BL , Tomografía de Emisión de Positrones , Potasio , Bazo/diagnóstico por imagen , Estaurosporina , Valinomicina
6.
PLoS One ; 8(7): e67079, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23861753

RESUMEN

In vitro testing can contribute to reduce the risk that the use of genetically modified (GM) crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2) as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment.


Asunto(s)
Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Células Epiteliales/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Proteínas Hemolisinas/farmacología , Mucosa Intestinal/efectos de los fármacos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Línea Celular , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Impedancia Eléctrica , Endotoxinas/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas Hemolisinas/genética , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ionóforos/farmacología , L-Lactato Deshidrogenasa/metabolismo , Plantas Modificadas Genéticamente , Porcinos , Valinomicina/farmacología , Zea mays/química , Zea mays/genética
7.
Appl Environ Microbiol ; 76(23): 7683-90, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20889782

RESUMEN

An oxalate-fermenting brown rot fungus, Fomitopsis palustris, secretes large amounts of oxalic acid during wood decay. Secretion of oxalic acid is indispensable for the degradation of wood cell walls, but almost nothing is known about the transport mechanism by which oxalic acid is secreted from F. palustris hyphal cells. We characterized the mechanism for oxalate transport using membrane vesicles of F. palustris. Oxalate transport in F. palustris was ATP dependent and was strongly inhibited by several inhibitors, such as valinomycin and NH(4)(+), suggesting the presence of a secondary oxalate transporter in this fungus. We then isolated a cDNA, FpOAR (Fomitopsis palustris oxalic acid resistance), from F. palustris by functional screening of yeast transformants with cDNAs grown on oxalic acid-containing plates. FpOAR is predicted to be a membrane protein that possesses six transmembrane domains but shows no similarity with known oxalate transporters. The yeast transformant possessing FpOAR (FpOAR-transformant) acquired resistance to oxalic acid and contained less oxalate than the control transformant. Biochemical analyses using membrane vesicles of the FpOAR-transformant showed that the oxalate transport property of FpOAR was consistent with that observed in membrane vesicles of F. palustris. The quantity of FpOAR transcripts was correlated with increasing oxalic acid accumulation in the culture medium and was induced when exogenous oxalate was added to the medium. These results strongly suggest that FpOAR plays an important role in wood decay by acting as a secondary transporter responsible for secretion of oxalate by F. palustris.


Asunto(s)
Coriolaceae/enzimología , Coriolaceae/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Oxalatos/metabolismo , Adenosina Trifosfato/metabolismo , Análisis por Conglomerados , Coriolaceae/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/genética , Inhibidores Enzimáticos/metabolismo , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , Compuestos de Amonio Cuaternario/metabolismo , Vesículas Secretoras/enzimología , Análisis de Secuencia de ADN , Homología de Secuencia , Valinomicina/metabolismo , Madera/metabolismo , Madera/microbiología
8.
Chem Phys Lipids ; 160(2): 109-13, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19446541

RESUMEN

Nano-black lipid membranes (nano-BLMs) were obtained by functionalization of highly ordered porous alumina substrates with an average pore diameter of 60nm based on a self-assembled alkanethiol submonolayer followed by spreading of 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane on the hydrophobic substrate. By means of impedance spectroscopy, we analyzed the influence of the self-assembled alkanethiol submonolayer on the electrical properties of the nano-BLMs as well as their long-term stability. We were able to stably integrate nano-BLMs into a flow through system, which allowed us to readily exchange buffer solutions several times and accounts for mass transport phenomena. The ionophore valinomycin was successfully inserted into nano-BLMs and its transport activity monitored as a function of different potassium and sodium ion concentrations reflecting the specificity of valinomycin for potassium ions.


Asunto(s)
Técnicas Electroquímicas/métodos , Ionóforos/química , Membranas Artificiales , Valinomicina/química , Óxido de Aluminio/química , Impedancia Eléctrica , Transporte Iónico , Ionóforos/metabolismo , Éteres Fosfolípidos/química , Potasio/química , Potasio/metabolismo , Sodio/química , Sodio/metabolismo , Valinomicina/metabolismo
9.
Chemphyschem ; 9(13): 1920-4, 2008 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-18704903

RESUMEN

Tethered bilayer lipid membranes are established as well-suited model membrane systems adaptable to different surfaces, for example, gold and silicon. These solid supported membranes are highly flexible in their tethering and lipid parts and can thus be optimized for functional incorporation of membrane proteins. The excellent sealing properties of the tethered membranes allow incorporated ion-channel proteins to be investigated. Preparation of ultrasmooth aluminum oxide by sputtering and synthesis of new tethering lipids with phosphonic acid anchor groups enable formation of an electrically sealing membrane on this surface. This process is monitored by electrochemical impedance spectroscopy and by surface plasmon resonance spectroscopy. High sealing performance of the membrane and functional incorporation of the ion carrier valinomycin are demonstrated.


Asunto(s)
Óxido de Aluminio/química , Membrana Dobles de Lípidos/química , Electrones , Éteres de Glicerilo/química , Microscopía de Fuerza Atómica , Estructura Molecular , Valinomicina/química
10.
FEMS Microbiol Lett ; 283(1): 15-22, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18397288

RESUMEN

Crithidia deanei, a monoxenic trypanosomatid, presents an endosymbiotic bacterium in its cytoplasm. Both the protozoan and the bacterium maintain intensive metabolic exchange, resulting in an interesting model to study the coevolution of metabolisms. The relevance of l-proline for the growth of C. deanei and its transport into these cells was studied. Both the endosymbiont-containing (wild) and the endosymbiont-free protozoa (aposymbiont or cured) strains, when grown in medium supplemented with l-proline, reached higher cell densities than those grown in unsupplemented media. We biochemically characterized the uptake of l-proline in both the wild (K(m)=0.153+/-0.022 mM, V(max)=0.239+/-0.011 nmol min(-1) per 4 x 10(7) cells) and the aposymbiont strains (K(m)=0.177+/-0.049 mM, V(max)=0.132+/-0.012 nmol min(-1) per 4 x 10(7) cells). These data suggest a single type of proline transporter whose activity is upregulated by the presence of the symbiotic bacterium. Proline transport was further characterized and was found to be insensitive to the extracellular concentration of Na+, but sensitive to K+ and pH. The abolition of proline uptake by respiratory chain inhibitors and valinomycin indicates that the proline transport in C. deanei is dependent on the plasma membrane K+ gradient.


Asunto(s)
Crithidia/metabolismo , Crithidia/microbiología , Prolina/metabolismo , Simbiosis , Animales , Antimicina A/análogos & derivados , Antimicina A/farmacología , Bacterias/metabolismo , Medios de Cultivo , ADN Bacteriano/análisis , Depresión Química , Concentración de Iones de Hidrógeno , Monensina/farmacología , Potasio/metabolismo , ARN Ribosómico 16S/análisis , Rotenona/farmacología , Sodio/metabolismo , Temperatura , Factores de Tiempo , Regulación hacia Arriba , Valinomicina/farmacología
11.
Food Chem Toxicol ; 46(6): 1976-84, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18325653

RESUMEN

The growing use of genetically modified crops necessitates viable screening methods for safety evaluation of recombinant feed, particularly for ruminants. A new sheep rumen epithelial cell culture is introduced as an in vitro cell system for safety evaluation especially focussing on feed and food compounds. We used lactate dehydrogenase (LDH) release, WST-1 conversion, ATP content and caspase 3/7 activity to evaluate cytotoxicity of Cry1Ab, one of the newly expressed Bt-proteins in transgene maize. The results were compared to the effects of valinomycin, a potassium ionophore known to induce cytotoxic effects on a wide range of cells. Whereas no toxicity of Cry1Ab was observed in short as well as in long term experiments, even at non-physiological high concentrations, exposure to valinomycin induced apoptosis and a significant response of all viability parameters after a number of hours. The ATP content and the WST-1 conversion reflecting the energy metabolism of the cells appear to be more sensitive indicators of valinomycin toxicity than the LDH release, a parameter which reflects the membrane integrity. This study presents an in vitro model system, that may be useful as a supplementary tool in toxicity screening before testing substances on animals in vivo.


Asunto(s)
Bacillus thuringiensis/química , Proteínas Bacterianas/toxicidad , Endotoxinas/toxicidad , Células Epiteliales/efectos de los fármacos , Alimentos Modificados Genéticamente/toxicidad , Proteínas Hemolisinas/toxicidad , Rumen/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Antibacterianos/toxicidad , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/aislamiento & purificación , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Supervivencia Celular/efectos de los fármacos , Endotoxinas/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Células Epiteliales/patología , Proteínas Hemolisinas/aislamiento & purificación , L-Lactato Deshidrogenasa/metabolismo , Rumen/patología , Ovinos , Estaurosporina/farmacología , Sales de Tetrazolio/metabolismo , Valinomicina/toxicidad
12.
Biochim Biophys Acta ; 1554(1-2): 94-100, 2002 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-12034474

RESUMEN

Upon sudden exposure of plants to an actinic light of saturating intensity, the yield of chlorophyll fluorescence increases typically by 200-400% of the initial O-level. At least three distinct phases of these O-J-I-P transients can be resolved: O-J (0.05-5 ms), J-I (5-50 ms), and I-P (50-1000 ms). In thylakoid membranes, the J-I increase accounts for approximately 30% of the total fluorescence increase; in Photosystem II membranes, the J-I phase is always lacking. In the presence of the ionophore valinomycin, which is known to inhibit specifically the formation of membrane voltages, the magnitude of the J-I phase is clearly diminished; in the presence of valinomycin supplemented by potassium, the J-I phase is fully suppressed. We conclude that the light-driven formation of the thylakoid-membrane voltage results in an increase of the chlorophyll excited-state lifetime, a phenomenon explainable by the electric-field-induced shift of the free-energy level of the primary radical pair [Dau and Sauer, Biochim. Biophys. Acta 1102 (1992) 91]. The assignment of the J-I increase in the fluorescence yield enhances the potential of using O-J-I-P fluorescence transients for investigations on photosynthesis in intact organisms. A putative role of thylakoid voltages in protection of PSII against photoinhibitory damage is discussed.


Asunto(s)
Clorofila , Tilacoides/fisiología , Valinomicina/farmacología , Antibacterianos/farmacología , Electrofisiología , Fluorescencia , Luz , Spinacia oleracea , Tilacoides/efectos de los fármacos
13.
Biochem J ; 323 ( Pt 3): 777-83, 1997 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-9169612

RESUMEN

A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531-543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1-drug interactions directly.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bloqueadores de los Canales de Calcio/farmacología , Secuencia de Consenso , ADN Complementario/genética , Resistencia a Múltiples Medicamentos/genética , Fluoresceínas/farmacología , Vectores Genéticos , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Nucleopoliedrovirus/genética , Proteínas Recombinantes de Fusión/metabolismo , Rodamina 123 , Rodaminas/farmacología , Spodoptera/citología , Relación Estructura-Actividad , Valinomicina/farmacología , Verapamilo/farmacología
14.
Biochem J ; 316 ( Pt 1): 143-7, 1996 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-8645197

RESUMEN

Vacuolar proton-pyrophosphatase (H(+)-PPase) of mung bean seedlings contains a single kind of polypeptide with a molecular mass of approx. 73 kDa. However, in this study, a molecular mass of approx. 140 kDa was obtained for the purified vacuolar H(+)-PPase by size-exclusion gel-filtration chromatography, suggesting that the solubilized form of this enzyme is a dimer. Radiation inactivation analysis of tonoplast vesicles yielded functional masses of 141.5 +/- 10.8 and 158.4 +/- 19.5 kDa for PP1 hydrolysis activity and its supported proton translocation respectively. These results confirmed the in situ dimeric structure of the membrane-bound H(+)-PPase of plant vacuoles. Further target-size analysis showed that the functional unit of purified vacuolar H(+)-PPase was 71.1 +/- 6.7 kDa, indicating that only one subunit of the purified dimeric complex would sufficiently display its enzymic reaction. Moreover, in the presence of valinomycin and KCl, the functional size of membrane-bound H(+)-PPase was decreased to approx. 63.4 +/- 6.3 kDa. A working model was proposed to elucidate the structure of native H(+)-PPase on vacuolar membrane as a functional dimer. Factors that would disturb the membrane, e.g. membrane solubilization and the addition of valinomycin and KCl, may induce an alteration in its enzyme structure, subsequently resulting in a different functional size.


Asunto(s)
Fabaceae/enzimología , Plantas Medicinales , Pirofosfatasas/química , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Cromatografía en Gel , Radioisótopos de Cobalto , Relación Dosis-Respuesta en la Radiación , Gramicidina/farmacología , Pirofosfatasa Inorgánica , Cinética , Sustancias Macromoleculares , Modelos Estructurales , Pirofosfatasas/antagonistas & inhibidores , Pirofosfatasas/efectos de la radiación , Vacuolas/enzimología , Valinomicina/farmacología
15.
FEBS Lett ; 318(2): 113-7, 1993 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-8440367

RESUMEN

Pea leaf mitochondria had a high ATP hydrolase activity following the collapse of the membrane potential by addition of valinomycin in state 4. In mitochondria isolated from potato tubers such ATP hydrolase activity was not observed. Pea leaf mitochondria also had a delta pH, in contrast to what was previously found for potato tuber mitochondria. This delta pH could, however, not explain the different results on ATP hydrolysis since this activity was also observed in the presence of nigericin. The results suggest a tissue-specific regulation of ATP hydrolysis in resting organs (potato tubers) as compared to active organs (leaves).


Asunto(s)
Adenosina Trifosfato/metabolismo , Mitocondrias/fisiología , ATPasas de Translocación de Protón/metabolismo , Cianuros/farmacología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Nigericina/farmacología , Solanum tuberosum , Valinomicina/farmacología
16.
EMBO J ; 10(11): 3263-72, 1991 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-1915293

RESUMEN

The alternative secretion pathway which exports hemolysin across both Escherichia coli membranes into the surrounding medium is directed by an uncleaved C-terminal targeting signal and the membrane translocator proteins HlyD and HlyB. In order to identify stages and intermediates in this unconventional secretion process we have examined the effect of inhibition of the total proton motive force (delta P) and its components during the in vivo HlyB/HlyD-dependent export of a 22.4 kDa secretion competent HlyA C-terminal peptide (Actp). Secretion of Actp was severely inhibited by the proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses simultaneously membrane potential delta psi and the proton gradient delta pH, and also by valinomycin/K+, a potassium ionophore which disrupts delta psi. The inhibition of secretion by valinomycin/K+ was ameliorated by imposition of a pH gradient, the second component of the delta P, and selective depletion of delta pH by nigericin also blocked secretion. This indicates that, as in the secretion of beta-lactamase to the periplasm, HlyB/D-directed secretion requires delta P itself and not specifically one of its components. However, inhibition of HlyB/D-dependent secretion was only marked when CCCP, valinomycin/K+ or nigericin were present during the early stage of Actp secretion; at a later stage the secretion was not significantly inhibited. HlyB/D-dependent secretion appears therefore to share with conventional secretion across the cytoplasmic membrane an early requirement for delta P, but comprises in addition a late stage which does not require delta P, delta psi or delta pH. The translocation intermediate identified in the delta P-independent late stage of secretion was associated with the membrane fraction. Analysis of the protease accessibility of this intermediate in whole cells and spheroplasts showed that it was not in the periplasm, nor was it exposed on the cell surface or on the periplasmic faces of either the inner or outer membranes. This may reflect its close association with the inner membrane or a membrane translocation complex.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Proteínas Portadoras/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/efectos de los fármacos , Expresión Génica , Proteínas Hemolisinas/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Nigericina/farmacología , Potasio/farmacología , Valinomicina/farmacología
17.
Plant Cell ; 3(7): 709-17, 1991 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-1841725

RESUMEN

The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.


Asunto(s)
Cloroplastos/metabolismo , Fabaceae/genética , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Plantas/metabolismo , Plantas Medicinales , Adenosina Trifosfato/farmacología , Secuencia de Bases , Transporte Biológico/efectos de los fármacos , Compartimento Celular , Fraccionamiento Celular , Clonación Molecular , ADN de Cadena Simple/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Nigericina/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estructura Secundaria de Proteína , Valinomicina/farmacología
18.
J Biol Chem ; 265(36): 22167-73, 1990 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-2176205

RESUMEN

Scopadulcic acid B (SA-B), a novel diterpenoid, is a main ingredient of the Paraguayan traditional medicinal herb "Typychá kuratú (Scoparia dulcis L.). SA-B and its debenzoyl derivative, diacetyl scopadol (DAS), specifically inhibit ATP hydrolysis of gastric H+,K(+)-ATPase. Both compounds inhibit the K(+)-dependent dephosphorylation step of the enzyme without any effect on the phosphorylation step. SA-B is a mixed-type inhibitor with respect to the activating cation, K+. SA-B lowers the affinity of H+,K(+)-ATPase to K+ and decreases the maximal velocity of ATP hydrolysis, whereas DAS is an uncompetitive inhibitor with respect to K+. Furthermore, the effects of SA-B and DAS on conformational states of the ATPase were studied by measuring the changes in the fluorescence intensity of the fluorescein isothiocyanate-labeled enzyme. The fluorescence study shows that SA-B primarily binds to the E2K form in the presence of Mg2+ and stabilizes the form and that DAS stabilizes the E2PK form. Therefore, the chemical modification of SA-B, debenzoylation, induced the changes in the pattern of inhibition of H+,K(+)-ATPase. Furthermore, the inhibition mechanisms of SA-B and DAS were different from those of omeprazole, which is an irreversible inhibitor, and SCH 28080, which is a reversible, competitive inhibitor with respect to K+. DAS also inhibited the K(+)-dependent p-nitrophenyl phosphatase activity, and the inhibition was competitive with respect to K+, indicating that the K(+)-dependent p-nitrophenylphosphatase activity does not represent the partial reaction step of H+,K(+)-ATPase.


Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Diterpenos/farmacología , Mucosa Gástrica/enzimología , Animales , Proteínas de Transporte de Catión , ATPasa Intercambiadora de Hidrógeno-Potásio , Riñón/enzimología , Cinética , Medicina Tradicional , Modelos Teóricos , Estructura Molecular , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Porcinos , Valinomicina/farmacología
20.
Biochim Biophys Acta ; 1018(1): 77-90, 1990 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-2165420

RESUMEN

(1) The hydrophobic pH indicator Bromthymol blue and the hydrophilic pH indicator Phenol red have been used to follow the redox-pump-linked proton flows during transition from anaerobiosis to static head. The domains monitored by the pH indicators, whether external or internal, and the localization of the dye, whether free or membrane bound, have been identified by recording the absorbance changes following addition of nigericin or valinomycin to anaerobic or aerobic mitochondria and the effects of permeant and impermeant buffers. (2) After addition of the H+/K+ exchanger, nigericin, to anaerobic mitochondria. Phenol red and Bromthymol blue record an alkalinization and an acidification, respectively, indicating that while the hydrophilic pH indicator faces an external domain, the hydrophobic pH indicator faces, at least partly, an internal domain. The latter effect is sensitive to phosphate and to phosphate carrier inhibitors. On the other hand, addition of nigericin to aerobic mitochondria leads to an increased Bromthymol blue absorbance, which reflects an alkalinization, indicating that the pH indicator faces an external domain. The reorientation of the dye from the internal to the external domain is a function of the uncoupler concentration and thus of the membrane potential (cf. Mitchell et al. (1968) Eur. J. Biochem. 4, 9-19). (3) The amount of oxygen required for the transition from anaerobiosis to static head has been determined by following in parallel the extent of oxidation of cytochrome aa3 and the rise of delta mu H+. With succinate as substrate, 50% levels of cytochrome oxidation are obtained at 0.125 ngatom oxygen/mg and 50% of Safranine response at about 0.2 ngatom oxygen/mg. These amounts of oxygen correspond to an H+ displacement of about 0.8-1.2 ngatom/mg on the basis of the H+/O stoichiometry. It is concluded that mitochondria are in presteady state below, and in static head above, displacement of 2-3 ngatom H+/mg. This figure is very close to the original calculation of Mitchell (Mitchell, P. (1966) Biol. Rev. 41, 445-502). (4) Transition, by oxygen pulses, of EGTA-supplemented mitochondria from anaerobiosis to either presteady state or static head state results in a response of the hydrophilic pH indicator, Phenol red, which is negligible in amount and/or kinetically unrelated to the delta mu H+ rise. The fact that H+ extrusion in the bulk aqueous phase is negligible also in presteady state excludes proton cycling as an explanation. Addition of oxygen pulses to Sr2(+)-supplemented anaerobic mitochondria results in an H+ extrusion whose amount and rate is proportional to the Sr2+ concentration.(ABSTRACT TRUNCATED AT 400 WORDS)


Asunto(s)
Mitocondrias Hepáticas/metabolismo , Oxígeno/farmacología , Protones , Anaerobiosis , Animales , Azul de Bromotimol , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Ácido Egtácico/farmacología , Complejo IV de Transporte de Electrones/metabolismo , Etilmaleimida/farmacología , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Nigericina/farmacología , Oxidación-Reducción , Fenazinas/metabolismo , Fenolsulfonftaleína , Potasio/metabolismo , Ratas , Valinomicina/farmacología
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