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Bidirectional dialogue between cellular and non-cellular components of the tumor microenvironment (TME) drives cancer survival. In the extracellular space, combinations of matrix molecules and soluble mediators provide external cues that dictate the behavior of TME resident cells. Often studied in isolation, integrated cues from complex tissue microenvironments likely function more cohesively. Here, we study the interplay between the matrix molecule tenascin-C (TNC) and chemokine CCL2, both elevated in and associated with the progression of breast cancer and playing key roles in myeloid immune responses. We uncover a correlation between TNC/CCL2 tissue levels in HER2+ breast cancer and examine the physical and functional interactions of these molecules in a murine disease model with tunable TNC levels and in in vitro cellular and cell-free models. TNC supported sustained CCL2 synthesis, with chemokine binding to TNC via two distinct domains. TNC dominated the behavior of tumor-resident myeloid cells; CCL2 did not impact macrophage survival/activation whilst TNC facilitated an immune suppressive macrophage phenotype that was not dependent on or altered by CCL2 co-expression. Together, these data map new binding partners within the TME and demonstrate that whilst the matrix exerts transcriptional control over the chemokine, each plays a distinct role in subverting anti-tumoral immunity.
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Neoplasias , Tenascina , Animales , Ratones , Quimiocinas/metabolismo , Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Tenascina/metabolismo , Quimiocina CCL2/metabolismoRESUMEN
The biomass from cereal side streams is rich in valuable components, such as hemicelluloses. Among the hemicelluloses, arabinoxylans and ß-glucans are the most acknowledged for potential health benefits. Numerous publications discuss the potential to use purified forms of these hemicelluloses for various applications. However, as the purification of hemicelluloses may not be economically feasible to upscale, sustainable and cost-effective methods are needed to make their valorization more realistic for industrial applications. Co-components present in hemicellulose-rich fractions may also provide added functionality, such as flavonoid content and antioxidant capacity. This review provides an overview on the feasibility of sustainably upscaling hemicellulose extraction processes, focusing on by-products from different cereal streams. We describe the hemicelluloses' physicochemical properties and provide various possible applications of pure and impure fractions from small scale to pilot and industrial scale. Furthermore, real case examples on the industrial utilization of cereal side streams are enclosed. This review provides pathways for future research for developing the hemicellulose extraction methods to obtain fractions with optimized purity, and offers suggestions to valorize them.
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Grano Comestible , Polisacáridos , Biomasa , Fraccionamiento QuímicoRESUMEN
Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease.
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Artritis/genética , Colitis/genética , Dermatitis/genética , Linfoma de Células B Grandes Difuso/genética , Quinasa Syk/genética , Adulto , Animales , Artritis/inmunología , Artritis/patología , Artritis/terapia , Secuencia de Bases , Trasplante de Médula Ósea , Colitis/inmunología , Colitis/patología , Colitis/terapia , Dermatitis/inmunología , Dermatitis/patología , Dermatitis/terapia , Familia , Femenino , Expresión Génica , Técnicas de Sustitución del Gen , Humanos , Lactante , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/terapia , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mutación , Linaje , Inhibidores de Proteínas Quinasas/farmacología , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/deficienciaRESUMEN
Human retromer, a heterotrimer of VPS26 (VPS26A or VPS26B), VPS35 and VPS29, orchestrates the endosomal retrieval of internalised cargo and promotes their cell surface recycling, a prototypical cargo being the glucose transporter GLUT1 (also known as SLC2A1). The role of retromer in the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR, also known as IGF2R) from endosomes back to the trans-Golgi network remains controversial. Here, by applying knocksideways technology, we develop a method for acute retromer inactivation. While retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1, we failed to observe a quantifiable defect in CI-MPR sorting. In contrast, knocksideways of the ESCPE-1 complex - a key regulator of retrograde CI-MPR sorting - revealed time-resolved defects in CI-MPR sorting. Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting.
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Nexinas de Clasificación , Proteínas de Transporte Vesicular , Endosomas/metabolismo , Células HeLa , Humanos , Transporte de Proteínas , Nexinas de Clasificación/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Red trans-Golgi/metabolismoRESUMEN
The interplay between cancer cells and immune cells is a key determinant of tumor survival. Here, we uncovered how tumors exploit the immunomodulatory properties of the extracellular matrix to create a microenvironment that enables their escape from immune surveillance. Using orthotopic grafting of mammary tumor cells in immunocompetent mice and autochthonous models of breast cancer, we discovered how tenascin-C, a matrix molecule absent from most healthy adult tissues but expressed at high levels and associated with poor patient prognosis in many solid cancers, controls the immune status of the tumor microenvironment. We found that, although host-derived tenascin-C promoted immunity via recruitment of proinflammatory, antitumoral macrophages, tumor-derived tenascin-C subverted host defense by polarizing tumor-associated macrophages toward a pathogenic, immune-suppressive phenotype. Therapeutic monoclonal antibodies that blocked tenascin-C activation of Toll-like receptor 4 reversed this phenotypic switch in vitro and reduced tumor growth and lung metastasis in vivo, providing enhanced benefit in combination with anti-PD-L1 over either treatment alone. Combined tenascin-C:macrophage gene-expression signatures delineated a significant survival benefit in people with breast cancer. These data revealed a new approach to targeting tumor-specific macrophage polarization that may be effective in controlling the growth and spread of breast tumors.
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Antineoplásicos Inmunológicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Macrófagos/inmunología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/inmunología , Femenino , Humanos , Vigilancia Inmunológica , Inmunoterapia/métodos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Ratones , Fenotipo , Tenascina/inmunología , Células Tumorales Cultivadas , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
BACKGROUND: As malaria transmission intensity approaches zero, measuring it becomes progressively more difficult and inefficient because parasite-positive individuals are hard to detect. This situation may arise shortly before achieving local elimination, or during surveillance post-elimination to prevent reintroduction. Antibody responses against the parasite last longer than the infections themselves. This "footprint" of infection may thus be used for assessing transmission intensity. A statistical approach is presented for measuring the seroconversion rate (SCR), a correlate of the force of infection, from individual-level longitudinal data on antibody titres in an area of low Plasmodium falciparum transmission. METHODS: Blood samples were collected from 160 Indonesian schoolchildren every month for six months. Titres of antibodies against AMA-1 and MSP-1(19) antigens of P. falciparum were measured using ELISA. The distribution of antibody titres among seronegative and -positive individuals, respectively, was estimated by comparing the titres from the study data (a mixture of both seropositive and -negative individuals) with titres from a (unexposed) negative control group of Indonesian individuals. Two Markov-Chain models for the transition of individuals between serological states were fitted to individual anti-PfAMA-1 or anti-PfMSP-1 titre time series using Bayesian Markov-Chain-Monte-Carlo (MCMC). This yielded estimates of SCR as well as of the duration of seropositivity. RESULTS: A posterior median SCR of 0.02 (Pf AMA-1) and 0.09 (PfMSP-1) person(-1) year(-1) was estimated, with credible intervals ranging from 1E-4 to 0.2 person(-1) year(-1). This level of transmission intensity is at the lower range of what can reliably be measured with the present study size. A Bayesian test for seroconversion of an individual between two observations is presented and used to identify the subjects who have most likely experienced an infection. Furthermore, the theoretical limits of measuring transmission intensity, and how these depend on duration and size of a study as well as on transmission intensity itself, is illustrated. CONCLUSIONS: This analysis shows that it is possible to measure SCR's from individual-level longitudinal data on antibody titres. In addition, individual seroconversion events can be identified, which can be useful in assessing interruption of transmission. Analyses of further serological datasets using the present method are required to improve and validate it. This includes measurement of the duration of antibody responses, how it depends on host age or cumulative exposure, or on the particular antigen used.
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Anticuerpos Antiprotozoarios/sangre , Malaria Falciparum/epidemiología , Malaria Falciparum/transmisión , Antígenos de Protozoos/inmunología , Niño , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Incidencia , Indonesia/epidemiología , Masculino , Proteínas de la Membrana/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Proteínas Protozoarias/inmunología , Estudios SeroepidemiológicosRESUMEN
The prediction of response or severe toxicity and therapy individualisation are extremely important in cancer chemotherapy. There are few tools to predict chemoresponse or toxicity in cancer patients. We investigated the correlation between the induction and repair of DNA double-strand breaks (DSBs) using constant-field gel electrophoresis (CFGE) and evaluating cell cycle progression and the sensitivity of four cancer cell lines to 5-fluorouracil (5FU). Using a sulphorhodamine-B assay, colon carcinoma cells (HCT116) were found to be the most sensitive to 5FU, followed by liver carcinoma cells (HepG2) and breast carcinoma cells (MCF-7). Cervical carcinoma cells (HeLa) were the most resistant. As measured by CFGE, DSB induction, but not residual DSBs, exhibited a significant correlation with the sensitivity of the cell lines to 5FU. Flow cytometric cell cycle analysis revealed that 14% of HCT116 or HepG2 cells and 2% of MCF-7 cells shifted to sub-G1 phase after a 96-h incubation with 5FU. Another 5FU-induced cell cycle change in HCT116, HepG2 and MCF-7 cells was the mild arrest of cells in G1 and/or G2/M phases of the cell cycle. In addition, 5FU treatment resulted in the accumulation of HeLa cells in the S and G2/M phases. Determination of Fas ligand (Fas-L) and caspase 9 as representative markers for the extrinsic and intrinsic pathways of apoptosis, respectively, revealed that 5FU-induced apoptosis in HCT116 and HepG2 results from the expression of Fas-L (extrinsic pathway). Therefore, the induction of DNA DSBs by 5FU, detected using CFGE, and the induction of apoptosis are candidate predictive markers that may distinguish cancer cells which are likely to benefit from 5FU treatment and the measurement of DSBs using CFGE may aid the prediction of clinical outcome.
RESUMEN
BACKGROUND: Acute neurogenic pulmonary edema, a common and underdiagnosed clinical entity, can occur after virtually any form of injury of the central nervous system and is a potential early contributor to pulmonary dysfunction in patients with head injuries. OBJECTIVE: To explore myocardial function in patients with evident neurogenic pulmonary edema after traumatic head injury. METHODS: During a 1-year period in a university hospital in Sfax, Tunisia, information was collected prospectively on patients admitted to the 22-bed intensive care unit because of isolated traumatic head injury who had neurogenic pulmonary edema. Data included demographic information, vital signs, neurological status, physiological status, and laboratory findings. All of the patients had computed tomography and plain radiography of the neck and determination of cardiac function. RESULTS: All 7 patients in the sample had cardiac dysfunction. Evidence of myocardial damage was confirmed by echocardiography in 3 patients, pulmonary artery catheterization in 3 patients, and/or postmortem myocardial biopsy in 4 patients. Echocardiography studies, repeated 7 days after the initial study in one patient and 90 days afterward in another, showed complete improvement in wall motion, with a left ventricular ejection fraction of 0.65. CONCLUSION: All patients who had neurogenic pulmonary edema due to traumatic head injury had myocardial dysfunction. The mechanisms of the dysfunction were multiple. The great improvement in wall motion seen in 2 patients indicated the presence of a stunned myocardium. Further studies are needed to understand the mechanisms of this cardiac dysfunction.
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Lesiones Encefálicas/complicaciones , Cardiopatías/etiología , Edema Pulmonar/etiología , Adolescente , Adulto , Cateterismo de Swan-Ganz , Niño , Ecocardiografía , Electrocardiografía , Cardiopatías/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Edema Pulmonar/diagnóstico , Radiografía Torácica , Tomografía Computarizada por Rayos XRESUMEN
An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K(D)) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC50 of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 microg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).
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Fluoroinmunoensayo/métodos , Narcóticos/orina , Anticuerpos Monoclonales , Autoanálisis , Unión Competitiva , Codeína/orina , Fluoresceína , Humanos , Microesferas , Morfina/inmunología , Derivados de la Morfina/orina , Oxicodona/orina , Sensibilidad y EspecificidadRESUMEN
A Protein C (PC) biosensor can be used to diagnose PC deficiency, to monitor the PC level in the blood of PC deficient patients, and to measure the PC concentration in other PC-containing samples, such as PC producing animal cell culture broth or transgenic animal milk. A fully functional biosensor requires extremely high sensitivity and specificity, and real-time measurement. To satisfy these requirements, it is proposed to develop an immuno-optical fiber biosensor that utilizes PC-specific biomolecules (PC probes) tagged with fluorophores. The method involves immobilizing monoclonal antibody against PC (anti-PC) on the surface of an optical fiber. When PC in a sample is adsorbed to the anti-PC on the fiber, it can be reached with the fluorophore tagged PC-probe. The intensity of light transported through the optical fiber, therefore, can be correlated with the concentration of PC in the sample. The sensor will be designed so it can be reused, following a simple elution step, thus reducing diagnostic expense. The preliminary study shows encouraging future for the real-time optical PC biosensor.
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Técnicas Biosensibles , Proteína C/análisis , Animales , Anticuerpos Monoclonales , Estudios de Evaluación como Asunto , Tecnología de Fibra Óptica , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Inmunoensayo/estadística & datos numéricos , Fibras Ópticas , Deficiencia de Proteína C , Sensibilidad y EspecificidadRESUMEN
A fiber-optic biosensor was developed for detection of cocaine, its metabolites, and other coca alkaloids, using a monoclonal antibody (mAb) against a derivatized benzoylecgonine (BE). The mAb was immobilized noncovalently on quartz fibers and a flow fluorometer was used to detect changes in evanescent wave fluorescence. A fluorescein (FL) conjugate of BE bound to the mAb specifically in a saturable manner and with high affinity (Kd = 7.6 nM). Cocaine or other test compounds competed with FL-BE for binding to the mAb in a concentration-dependent manner, thereby reducing the initial rate or steady-state fluorescence. Addition of cocaine to the flow buffer after reaching steady-state fluorescence enhanced the dissociation of bound FL-BE, and cocaine removal allowed fiber regeneration for multiple measurements. The detection limits for cocaine, cocaethylene, norcocaine, and BE were 5, 5, 29, and 30 ng/ml, respectively, but for ecgonine it was 4600 ng/ml and for methylecgonine it was 2000 ng/ml. Tropacocaine was detected at 10 ng/ml, but atropine was detected at 2900 ng/ml. The biosensor discriminated by 833-fold between cocaine and its stereoisomer pseudococaine. Structural features necessary for high-affinity recognition by this mAb are benzoate and 3 beta configuration, both of which are found in BE, cocaine, norcocaine, and cocaethylene.
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Técnicas Biosensibles , Cocaína/análogos & derivados , Cocaína/análisis , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Calibración , Cocaína/química , Cocaína/inmunología , Cocaína/aislamiento & purificación , Fluoresceína , Fluoresceínas/química , Inmunoconjugados/química , Inmunoconjugados/inmunología , Cinética , Sensibilidad y Especificidad , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diisopropylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.
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Acetilcolinesterasa/efectos de los fármacos , Colina/farmacología , Isoflurofato/toxicidad , Paraoxon/toxicidad , Fluoruro de Sodio/farmacología , Acetilcolinesterasa/metabolismo , Colina/metabolismo , Enzimas Inmovilizadas , FosforilaciónRESUMEN
Philanthotoxin (PhTX) is a neurotoxic constituent of the paralytic venom of the digger wasp, Philanthus triangulum. PhTX inhibits glutamate receptors of insect muscles mostly as a channel blocker, thereby producing muscle paralysis. Since glutamate receptor blockers may be of value as selective insect control agents, numerous derivatives of PhTX were synthesized and tested for their potencies as inhibitors of insect skeletal muscle glutamate receptors. Structure-activity relationship studies revealed that shortening the polyamine chain length reduced potency, and quaternarization of the nitrogen destroyed it. The potency was increased by a bulky anchoring group with moderate hydrophobicity at the end of the polyamine chain. The conversion of the tryosyl moiety to 3,5-diiodo-tyrosyl also increased potency and so did lengthening the butyryl chain from 4 to 10 carbons. Not only did PhTXs inhibit different subtypes of glutamate receptors, including the mammalian N-methyl-D-aspartate receptor, but also nicotinic receptors of insects and vertebrates. Because of this low selectively, and the hydrophilicity of the derivatives tested, which interferes with their penetration to the target receptor, these compounds cannot be used as insecticides. Nevertheless, the insect skeletal muscle glutamate receptor is a viable target for selective insecticides and major changes in PhTX structure may possibly produce derivatives that can be potential insecticides.
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Antagonistas de Aminoácidos Excitadores , Insecticidas/farmacología , Poliaminas , Venenos de Avispas/farmacología , Animales , Relación Estructura-Actividad , Venenos de Avispas/químicaRESUMEN
125I2-iodinated philanthotoxin-343 (PhTX-343), [125I2]PhTX-343-arginine, and [125I2]PhTX-343-lysine were synthesized and evaluated as probes for glutamate receptors in rat brain synaptic membranes. It was found that these probes were not specific for the glutamate receptors but may be useful for investigating the polyamine binding site. Filtration assays with Whatman GF/B fiber glass filters were unsuitable because the iodinated PhTX-343 analogues exhibited high nonspecific binding to the filters, thus hindering detection of specific binding to membranes. When binding was measured by a centrifugal assay, [125I2]PhTX-343-lysine bound with low affinity (KD = 11.4 +/- 2 microM) to a large number of sites (37.2 +/- 9.1 nmol/mg of protein). The binding of [125I2]PhTX-343-lysine was sensitive only to the polyamines spermine and spermidine, which displaced [125I2]PhTX-343-lysine with Ki values of (3.77 +/- 1.4) x 10(-5) M and (7.51 +/- 0.77) x 10(-5) M, respectively. The binding was insensitive to glutamate receptor agonists and antagonists. Binding results with [125I2]PhTX-343-arginine were similar to those of [125I2]-PhTX-343-lysine. Considering the high number of toxin binding sites (10000-fold more than glutamate) in these membranes and the insensitivity of the binding to almost all drugs that bind to glutamate receptors, it is evident that most of the binding observed is not to glutamate receptors. On the other hand, PhTX analogues with photoaffinity labels may be useful in the isolation/purification of various glutamate and nicotinic acetylcholine receptors; they could also be useful in structural studies of receptors and their binding sites.
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Encéfalo/metabolismo , Neurotoxinas/metabolismo , Receptores de Neurotransmisores/metabolismo , Venenos de Avispas/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Radioisótopos de Yodo , Cinética , Masculino , Neurotoxinas/síntesis química , Poliaminas/metabolismo , Ratas , Ratas Endogámicas , Receptores de Glutamato , Espermina/metabolismo , Membranas Sinápticas/metabolismo , Venenos de Avispas/síntesis químicaRESUMEN
1. The effect of electrophoretic ejection of philanthotoxin (the polyamine toxin, from the Egyptian digger wasp) was tested on responses of brainstem and spinal neurones in the pentobarbitone-anaesthetized rat to excitatory amino acids. 2. Philanthotoxin caused a dose-dependent reduction of responses to quisqualate, alpha-amino-3-hydroxy-5-phenyl-4-isoxazolepropionate (AMPA) and kainate with little effect on those to N-methyl-D-aspartate (NMDA). 3. The time-course of this antagonist action was slow. In particular the rate of recovery was dependent on frequency of ejection of the agonist. This agonist-dependent recovery suggests that philanthotoxin has a channel blocking mode of action on mammalian central neurones.
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Tronco Encefálico/citología , Ácido Iboténico/análogos & derivados , Ácido Kaínico/antagonistas & inhibidores , N-Metilaspartato/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Poliaminas , Ácido Quiscuálico/antagonistas & inhibidores , Venenos de Avispas/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Tronco Encefálico/efectos de los fármacos , Femenino , Ácido Iboténico/antagonistas & inhibidores , Ratas , Ratas Endogámicas , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
The effects of varying the structure of philanthotoxin (PhTX) were investigated on binding of the channel blockers: [3H]perhydrohistrionicotoxin (H12-HTX) to the nicotinic acetylcholine receptor (nACh-R) of Torpedo electric organ and [3H]MK-801 [( 3H]-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine maleate) to the N-methyl-D-aspartate receptor (NMDA-R) of rat brain cortex. The four moieties of PhTX (tyrosine, butyrate, spermine and the terminal amino group) were modified or conjugated resulting in 36 compounds. Although the potencies of the PhTX analogs on both receptors were higher with increasing lipophilicity and the polyamine chain length, there was considerable divergence between the two receptors' channels in the structural activity requirements for blockade by PhTX analogs. A major difference was the more critical role of the amine terminal for inhibition of the nACh-R than the NMDA-R, whereas the reverse might be true for the tyrosine moiety. The potency range of PhTX analogs on [3H]H12-HTX binding was 1070, but only 21 on [3H]MK-801 binding. Adding a lysine or arginine onto the spermine moiety increased the compound's potency on the nACh-R with little effect on the NMDA-R. Because spermine is a component of PhTX, the effects of five polyamines were also studied. Spermine and spermidine potentiated [3H]MK-801 binding, whereas putrescine, cadeverine and agmatine inhibited it. In presence of glutamate, higher concentrations of all polyamines inhibited [3H]MK-801 binding. On the nACh-R, spermine, spermidine and agmatine inhibited [125I]alpha-bungarotoxin and also [3H]H12-HTX binding in presence of carbamylcholine. The complex nature of PhTX interactions with the two receptors suggests that PhTX may bind to two sites: an external polyamine binding site and a channel binding site.
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Venenos de Abeja/farmacología , Poliaminas/farmacología , Receptores de Neurotransmisores/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Venenos de Avispas/farmacología , Animales , Anticonvulsivantes/metabolismo , Sitios de Unión , Dibenzocicloheptenos/metabolismo , Maleato de Dizocilpina , Ratas , Ratas Endogámicas , Receptores de N-Metil-D-Aspartato , Receptores de Neurotransmisores/metabolismo , Receptores Nicotínicos/metabolismo , Relación Estructura-Actividad , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/metabolismo , TorpedoRESUMEN
The actions of philanthotoxin (PhTX) were studied on the function of glutamate receptors expressed in Xenopus oocytes injected with rat brain mRNA and on binding of radioligands to rat brain glutamate receptors. PhTX reversibly inhibited the oocyte responses to quisqualate, N-methyl-D-aspartate (NMDA) and kainate in a dose-dependent manner. The NMDA receptor was the most sensitive to PhTX action (10-fold more than the kainate receptor) and the least sensitive was the smooth current component of the quisqualate response. Recovery from PhTX block differed among the three amino acids. NMDA responses recovered completely within a few minutes whereas responses to kainate and quisqualate recovered more slowly. PhTX had no effect on equilibrium binding of [3H]glutamate to rat brain cortical membranes studied in buffer treated to eliminate microorganisms. Based on the drug specificity of this [3H]glutamate binding, it is suggested to be mostly to the NMDA receptor. Low concentrations of PhTX (1-10 microM) potentiated binding of [3H] MK-801, a specific noncompetitive inhibitor of the NMDA receptor. However, higher PhTX concentrations inhibited this binding with an IC50 of 20 microM, similar to its inhibition of the oocyte-expressed NMDA receptor. Inhibition of [3H]MK-801 binding by PhTX was noncompetitive. It is suggested that PhTX, like the more potent MK-801, binds to an allosteric site on the NMDA receptor and inhibits its function but its binding site is not identical with the MK-801 binding site.