RESUMEN
In order to assess the impact of nanoplastics on marine species, polystyrene nanoparticles (PS NPs) have been largely used as model particles. Here we studied the effects of 50 nm amino-modified PS-NH2 on Mediterranean sea urchin Paracentrotus lividus immune system cells (coelomocytes) in the presence of celomic fluid (CF) and at different NP concentrations (1, 5, 10, and 25 µg mL-1) and experimental conditions (absence or presence of EDTA). PS-NH2 acquired a protein corona once incubated with CF, dominated by the toposome precursor protein (TPP). In short-term cultures, a significant concentration- and time-dependent decrease in lysosomal membrane stability and apoptotic-like nuclear alterations were observed in phagocytes upon exposure to PS-NH2 (10 and 25 µg mL-1) in CF but they resulted abolished in the presence of EDTA confirming the role of TPP in triggering PS-NH2-coelomocytes interaction and toxicity. PS-NH2 did not alter MXR phenotype but the observed dose-dependent decrease in calcein accumulation suggests the ability of PS-NH2 to affect pump's efflux activity. Overall results encourage additional studies on positively charged nanoplastics, since the observed effects on sea urchin coelomocytes as well as the TPP corona formation might represent a first step for addressing their impact on sensitive marine species.
Asunto(s)
Nanopartículas/toxicidad , Paracentrotus/efectos de los fármacos , Poliestirenos/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Cationes , Nanopartículas/química , Paracentrotus/inmunología , Poliestirenos/química , Contaminantes Químicos del Agua/químicaRESUMEN
Dried herbal preparations, based on "Zornia latifolia," are commonly sold on web, mainly for their supposed hallucinogenic properties. In this work, we demonstrate that these commercial products contain a different Fabacea, i.e., Stylosanthes guianensis, a cheaper plant, widely cultivated in tropical regions as a fodder legume. We were provided with plant samples of true Zornia latifolia from Brazil, and carried out a thorough comparison of the two species. The assignment of commercial samples was performed by means of micro-morphological analysis, DNA barcoding, and partial phytochemical investigation. We observed that Z. latifolia contains large amounts of flavonoid di-glycosides derived from luteolin, apigenin, and genistein, while in S. guianensis lesser amounts of flavonoids, mainly derived from quercetin, were found. It is likely that the spasmolytic and anxiolytic properties of Z. latifolia, as reported in traditional medicine, derive from its contents in apigenin and/or genistein.
Asunto(s)
Contaminación de Medicamentos , Fabaceae/química , Flavonoides/análisis , Extractos Vegetales/análisis , Apigenina , Brasil , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Luteolina , Plantas , QuercetinaRESUMEN
Creatine, a compound that is critical for energy metabolism of nervous cells, crosses the blood-brain barrier (BBB) and the neuronal plasma membrane with difficulty, and only using its specific transporter. In the hereditary condition where the creatine transporter is defective (creatine transporter deficiency) there is no creatine in the brain, and administration of creatine is useless lacking the transporter. The disease is severe and incurable. Creatine-derived molecules that could cross BBB and plasma membrane independently of the transporter might be useful to cure this condition. Moreover, such molecules could be useful also in stroke and other brain ischemic conditions. In this paper, we investigated three creatine salts, creatine ascorbate, creatine gluconate and creatine glucose. Of these, creatine glucose was ineffective after transporter block with guanidine acetic acid (GPA) administration. Creatine ascorbate was not superior to creatine in increasing tissue creatine and phosphocreatine content after transporter impairment, however even after such impairment it delayed synaptic failure during anoxia. Finally, creatine gluconate was superior to creatine in increasing tissue content of creatine after transporter block and slowed down PS disappearance during anoxia, an effect that creatine did not have. These findings suggest that coupling creatine to molecules having a specific transporter may be a useful strategy in creatine transporter deficiency. In particular, creatine ascorbate has effects comparable to those of creatine in normal conditions, while being superior to it under conditions of missing or impaired creatine transporter.
Asunto(s)
Ácido Ascórbico/farmacología , Creatina/farmacología , Gluconatos/farmacología , Glucosa/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Ácido Ascórbico/química , Creatina/química , Evaluación Preclínica de Medicamentos , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Gluconatos/química , Glucosa/química , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipoxia Encefálica/tratamiento farmacológico , Hipoxia Encefálica/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones Endogámicos ICR , Estructura Molecular , Fármacos Neuroprotectores/química , Técnicas de Cultivo de TejidosRESUMEN
Creatine, an ergogenic compound essential for brain function, is very hydrophilic and needs a transporter to cross lipid-rich cells' plasma membranes. Hereditary creatine transporter deficiency is a severe incurable neurological disease where creatine is missing from the brain. Creatine esters are more lipophylic than creatine and may not need the transporter to cross plasma membranes. Thus, they may represent a useful therapy for hereditary creatine transporter deficiency. Creatine ethyl ester (CEE) is commercially available and widely used as a nutritional supplement. It was reported that it enters the cells of patients lacking the transporter but was not useful when administered in vivo, by oral route, to affected patients. In this paper we investigated the effects of CEE in in vitro brain slices before and after biochemical block of the creatine transporter. We found that CEE is rapidly degraded in the aqueous incubation medium to creatinine, however it remains in solution long enough to cause an increase in tissue content of creatine and, more prominently, phosphocreatine. Both CEE and creatine delayed the anoxia-induced failure of synaptic transmission, and there was no difference between the two compounds. Contrary to what we expected, CEE did not increase tissue creatine content after the creatine transporter was blocked. We confirm that CEE is probably not an effective treatment for hereditary creatine transporter deficiency. Two factors seem to affect the possibility for creatine esters to be exploited in the therapy of creatine transporter deficiency. First, the size of their alcohol moiety should be increased since this would increase the lipophilicity of the compound and improve its ability to diffuse through biological membranes. Second, creatine esters should be further modified to slow their degradation to creatinine and increase their half-life in aqueous solutions. Moreover, we should not forget the possibility that they are degraded in vivo by plasma esterases.
Asunto(s)
Creatina/análogos & derivados , Hipocampo/metabolismo , Proteínas de Transporte de Membrana/deficiencia , Animales , Cromatografía Líquida de Alta Presión , Creatina/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Técnicas de Cultivo de Órganos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
In human Burkitt's Lymphoma (BL) BRG cells, a t(8;14) translocation, placing c-myc near the Emu enhancer of the H chain locus, causes tumor expansion. Earlier, we showed that a peptide nucleic acid complementary to the Emu sequence (PNAEmu), specifically inhibited the expression of translocated c-myc and impaired the growth of BRG cells-induced subcutaneous tumors in mice suffering from severe combined immunodeficiency (SCID). In this study, the therapeutic potential of PNAEmu was evaluated in a systemic mouse model. BRG-BL cells transfected with the luciferase gene were inoculated intravenously into SCID mice resulting in a preferential expansion, similar to the one of human adult patients, in the abdominal cavity, central nervous system and bone marrow. The mice were chronically injected intraperitoneally either with PNAEmu or with control PNA. The treatment was stopped when the control animals developed severe neurological symptoms. As detected both by inspection at necropsy and imaging, overall tumor growth in PNAEmu-treated mice decreased by >80%. Histological and immunohistochemical studies showed, only in PNAEmu-treated mice, a substantially reduced BL cell growth at the major sites of invasion and vast areas of necrosis in the lymphomatous tissues, with concomitant c-myc expression downregulation. Altogether, the data support the therapeutic potential of PNAEmu in human adult BL.
Asunto(s)
Linfoma de Burkitt/tratamiento farmacológico , Ácidos Nucleicos de Péptidos/farmacología , Animales , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Línea Celular Tumoral , Transformación Celular Viral , Femenino , Humanos , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Mediciones Luminiscentes , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/genética , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A new method for isolation and characterization of peptides presented in the context of the nonclassical human leukocytes antigen (HLA) class I molecule HLA-E was developed. A combination of different chromatographic steps coupled with electrospray mass spectrometry allowed us to detect the presence of small amounts of a naturally processed human Cytomegalovirus (HCMV)-derived peptide isolated from the HEK-293T/HLA-E+/UL40+ transfected cells of from HELA cell line. The peptide sequence was confirmed by tandem mass spectrometry (MS/MS). This approach provides a versatile and sensitive method for direct identification of MHC class I-binding peptides that might be derive from different pathogen or tumor-associated proteins.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Espectrometría de Masas/métodos , Péptidos/inmunología , Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Membrana Celular/química , Células Cultivadas , Citomegalovirus , Antígenos HLA/química , Células HeLa , Antígenos de Histocompatibilidad Clase I/química , Humanos , Datos de Secuencia Molecular , Péptidos/química , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem , Transfección , Antígenos HLA-ERESUMEN
Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.
Asunto(s)
Linfoma de Burkitt/genética , Elementos de Facilitación Genéticos , Cadenas mu de Inmunoglobulina/toxicidad , Mutágenos/toxicidad , Señales de Localización Nuclear/toxicidad , Ácidos Nucleicos de Péptidos/toxicidad , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Células CHO , Cricetinae , Cricetulus , Escherichia coli , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Linfocitos/efectos de los fármacos , Pruebas de Micronúcleos , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Salmonella typhimuriumRESUMEN
In Burkitt's lymphoma (BL) cells due to a t(8;14) chromosomal translocation c-myc is often placed in proximity to the Emu enhancer of the Ig locus and upregulated. We demonstrated that in BL cells a peptide nucleic acid (PNA), complementary to intronic Emu sequences (PNAEmuwt), specifically blocks the expression of the c-myc oncogene under the Emu enhancer control and inhibits BL cell growth in culture. Here, we investigated whether PNAEmuwt was also able to block tumor growth in SCID mice inoculated with human BL cell lines. After subcutaneous inoculum in mice BL cells reproducibly form tumors. Both pre-treatment of BL cells with PNAEmuwt before inoculum and chronic intravenous administration of PNAEmuwt to mice already inoculated with BL cells selectively caused increased latency of tumor appearance and decreased final tumor size. Tumors from PNAEmuwt-treated animals showed substantial areas of cell necrosis and of c-myc downregulation. Inhibition of tumor growth was specific and was not observed with PNAEmumut carrying sequence mutations and in BL cell lines where the translocated c-myc is not under the control of the Emu enhancer. These data confirm the potential therapeutic value of PNA targeted to regulatory non-coding regions.
Asunto(s)
Linfoma de Burkitt/patología , División Celular/efectos de los fármacos , Genes myc , Ácidos Nucleicos de Péptidos/farmacología , Animales , Secuencia de Bases , Inmunohistoquímica , Ratones , Ratones SCID , Trasplante de Neoplasias , Ácidos Nucleicos de Péptidos/química , Biosíntesis de Proteínas , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
BACKGROUND: We have recently demonstrated that low doses of Dexamethasone 21-P (Dex 21-P), loaded in autologous erythrocytes and administered at monthly intervals, have been able to maintain steroid-dependent patients with Crohn's disease (CD) and ulcerative colitis (UC) in clinical remission with a progressive and complete tapering of systemic steroids. AIM: Since multi-drug resistance 1 gene (MDR1) has a potential influence on Dexamethasone (Dex) bioavailability, we designed this study to investigate the correlation between MDR1 genotype and Dex pharmacokinetic after its delivery in patients with inflammatory bowel disease (IBD). MATERIALS AND METHODS: Seventeen steroid-dependent consecutive patients with IBD (10 UC mean age 36 +/- 12, and 7 Crohn's disease mean age 31 +/- 5) were consecutively recruited. The C3435T polymorphism of MDR1 gene was studied by Denaturing High Performance Liquid Chromatography (DHPLC). Serum level of Dex were determined at the end of the infusion and after 15 days by high performance liquid chromatography electrospray mass spectrometry. RESULTS: The mean dose of Dex 21-P administered was 9.9 mg +/- 4 (range 2.7-20.3), while the mean levels of Dex at the end of the infusion and after 15 days were 0.66 +/- 0.23 mM and 0.06 +/- 0.06 mM, respectively. Concerning the C3435T genotype, two patients were wild-type, eleven heterozygotes, and four homozygotes. No correlation between basal or 15-days plasma level of Dex and MDR1 genotype was found (r = 0.19 and r = 0.21, respectively). CONCLUSION: Our findings demonstrated that Dex plasma level, after infusion of autologous erythrocytes loaded with Dex 21-P are completely independent by the MDR 1 gene polymorphism. This could be another potential advantage of this modality of drug delivering.
Asunto(s)
Colitis Ulcerosa/genética , Enfermedad de Crohn/genética , Dexametasona/análogos & derivados , Genes MDR/genética , Profármacos/farmacocinética , Adulto , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/metabolismo , Dexametasona/administración & dosificación , Dexametasona/sangre , Dexametasona/farmacocinética , Portadores de Fármacos , Eritrocitos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Profármacos/administración & dosificaciónAsunto(s)
Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Dexametasona/análogos & derivados , Sistemas de Liberación de Medicamentos/métodos , Eritrocitos , Adulto , Niño , Colitis Ulcerosa/sangre , Enfermedad de Crohn/sangre , Preparaciones de Acción Retardada , Dexametasona/administración & dosificación , Dexametasona/sangre , Dexametasona/química , Dexametasona/farmacocinética , HumanosRESUMEN
Peptides corresponding to three alpha helices present in the C-terminal region of the human prion protein have been synthesized and their structural autonomy analyzed by circular dichroism (CD) and NMR spectroscopy. The results obtained indicate that the protein fragment corresponding to the alpha 3-helix, in contrast to alpha 1 and alpha 2 peptides, shows a complete structural autonomy. The chemical shifts values found for NH and CHalpha resonance of the isolated alpha 3 peptide, formed by 30 aminoacid residues, were markedly and surprisingly similar to the corresponding values of the alpha 3-helix in the protein. The structural autonomy of the alpha 3-helix is profoundly determined by the presence of the conserved capping box and, in part, by the ionic bond formed between Glu200 and Lys204. On the basis of these observations a novel PrP consensus pattern, centered on the alpha 3-helix region, has been defined. The data indicate that this autonomous and highly conserved region of the PrPc likely plays a critical role in folding and stability. This gives an explanation of why many of pathogenic mutations occur in this part of the molecule, sharing relevant effects on the overall protein conformation. In particular the D202N capping mutation almost completely destabilizes the isolated alpha 3 peptide. While it is well known that the D202N substitution is associated with a GSS disease, the possible structural basis of this fatal pathology has never been investigated. We propose that a lower alpha 3-helical propensity leading to a major destabilization of the PrPc molecule initiates the pathogenic process associated with D202N capping mutation.
Asunto(s)
Mutación/fisiología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Priones/síntesis química , Conformación ProteicaRESUMEN
Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).
Asunto(s)
Péptidos/farmacología , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-myc/química , Secuencia de Aminoácidos , Apoptosis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Neoplasias de la Mama , División Celular/efectos de los fármacos , Línea Celular Tumoral , Dicroismo Circular , Neoplasias del Colon , Dimerización , Estabilidad de Medicamentos , Fluoresceína , Polarización de Fluorescencia , Colorantes Fluorescentes , Calor , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Desnaturalización Proteica , Proteínas Proto-Oncogénicas c-myc/análisis , Rodaminas/química , Relación Estructura-ActividadRESUMEN
Prion diseases are neurodegenerative disorders of the central nervous system of humans and animals, characterized by spongiform degeneration of the central nervous system, astrogliosis, and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (prion protein, PrP(C)) into an altered isoform (PrP(Sc)) has been proposed to represent the causative event responsible for these diseases. The peptide corresponding to the residues 106-126 of PrP sequence (PrP106-126) is largely used to explore the neurotoxic mechanisms underlying the prion diseases. We investigated the intracellular signaling responsible for PrP106-126-dependent cell death in the SH-SY5Y human neuroblastoma cell line. In these cells, PrP106-126 treatment induced apoptotic cell death and the activation of caspase-3. The p38 MAP-kinase blockers (SB203580 and PD169316) prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 activation. However, whether the neuronal toxicity of PrP106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. In this study, we correlated the structural state of this peptide with its neurotoxicity. We show that the two conserved glycines in position 114 and 119 prevent the peptide to assume a structured conformation, favoring its aggregation in amyloid fibrils. The substitution of both glycines with alanine residues (PrP106-126AA) generates a soluble nonamyloidogenic peptide, that retained its toxic properties when incubated with neuroblastoma cells. These data show that the amyloid aggregation is not necessary for the induction of the toxic effects of PrP106-126.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Apoptosis/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/farmacología , Priones/farmacología , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Neuroblastoma/patología , Fragmentos de Péptidos/química , Priones/química , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.
Asunto(s)
Escherichia coli/genética , Priones/genética , Secuencia de Bases , Western Blotting , Cromatografía Liquida , Dicroismo Circular , Cartilla de ADN , Humanos , Espectrometría de Masas , Priones/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de FluorescenciaRESUMEN
Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly-Arg-Arg-Arg-Arg, corresponding to a region of the N-terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N-terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 microM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro-inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 degrees C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.
Asunto(s)
Núcleo Celular/metabolismo , Lactoferrina/metabolismo , Señales de Localización Nuclear/metabolismo , Ácidos Nucleicos de Péptidos/metabolismo , Secuencias de Aminoácidos , Nucléolo Celular/metabolismo , Endocitosis , Colorantes Fluorescentes/química , Humanos , Lactoferrina/química , Imitación Molecular , Ácidos Nucleicos de Péptidos/química , Rodaminas/química , Temperatura , Células Tumorales CultivadasRESUMEN
Systemic sclerosis is an autoimmune disease characterized by immunological and vascular abnormalities. Autoantibodies against intracellular antigens are associated with particular clinical features of the disease, whereas autoantibodies against cell surface antigens may be pathogenic by inducing endothelial cell damage, considered the primary event in the pathogenesis of the disease. Latent human cytomegalovirus infection may contribute to progression of systemic sclerosis through its ability to infect endothelial cells; however, direct links between human cytomegalovirus infection and systemic sclerosis are still lacking. Molecular mimicry is one of the mechanisms that account for the link between infection and autoimmunity. Here we have identified an immunodominant peptide using systemic sclerosis serum screening of a random peptide library; such peptide shares homology with autoantigens and with the human cytomegalovirus late protein UL94 (ref. 9). Immunoglobulin G antibodies against the peptide affinity-purified from the sera of patients with systemic sclerosis specifically recognized the viral product and autoantigens; moreover, such antibodies induced endothelial cell apoptosis through specific interaction with the cell surface integrin-NAG-2 protein complex. Our results provide evidence that antibodies against human cytomegalovirus cause apoptosis of endothelial cells, considered the initial pathogenic event of systemic sclerosis, and indicate a previously unknown mechanism for the etiological link between human cytomegalovirus infection and autoimmunity.
Asunto(s)
Autoanticuerpos/metabolismo , Proteínas de la Cápside , Cápside/metabolismo , Inmunoglobulina G/metabolismo , Esclerodermia Sistémica/inmunología , Apoptosis/inmunología , Estudios de Casos y Controles , Células Cultivadas , Reacciones Cruzadas , Citomegalovirus/química , Endotelio Vascular/inmunología , Epítopos , Femenino , Humanos , Masculino , Fragmentos de Péptidos/inmunología , Homología de Secuencia de AminoácidoRESUMEN
Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed to enter the cell, bind to the complementary polynucleotide sequence and inhibit DNA transcription and mRNA translation. Although PNAs have a very limited ability in penetrating nuclei of living cells, there are indications that covalent linkage of the PNA to appropriate vectors, e.g., a nuclear localization signal, permits access to the genome. Here we test the ability of dihydrotestosterone (T) covalently linked to PNA to act as a vector for targeting c-myc DNA to prostatic cancer cell nuclei. LNCaP cells, which express the androgen receptor gene, and DU145 cells, in which the androgen receptor gene is silent, offer a model to test this biologically active hormone as a cell-specific vector. T vector was covalently linked to the NH2-terminal position of a PNA complementary to a unique sequence of c-myc oncogene (PNAmyc-T). To localize PNAmyc-T and vector-free PNA within the cells, a rhodamine (R) group was attached at the COOH-terminal position (PNAmyc-R, PNAmyc-TR); cellular uptake was monitored by confocal fluorescence microscopy. PNAmyc-R was detected only in the cytoplasm of both prostatic cell lines, whereas PNAmyc-TR was localized in nuclei as well as in cytoplasm of LNCaP cells. In contrast, PNAmyc-TR uptake in DU145 cells was minimal and exclusively cytoplasmic. In LNCaP cells, MYC protein remained unchanged by exposure to vector-free PNAmyc, whereas a significant and persistent decrease was induced by PNAmyc-T. In DU145 cells, MYC expression was unaltered by PNAmyc with or without the T vector. Our data show that the T vector facilitates cell-selective nuclear localization of PNA and its consequent inhibition of c-myc expression. These findings suggest a strategy for targeting of cell-specific anti-gene therapy in prostatic carcinoma.
Asunto(s)
Núcleo Celular/metabolismo , Dihidrotestosterona/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ácidos Nucleicos de Péptidos/metabolismo , Ácidos Nucleicos de Péptidos/farmacología , Neoplasias de la Próstata/metabolismo , Elementos sin Sentido (Genética)/genética , Elementos sin Sentido (Genética)/metabolismo , Elementos sin Sentido (Genética)/farmacocinética , Elementos sin Sentido (Genética)/farmacología , Transporte Biológico , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoplasma/metabolismo , Genes myc/genética , Terapia Genética , Humanos , Masculino , Microscopía Fluorescente , Señales de Localización Nuclear , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/farmacocinética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Receptores Androgénicos/deficiencia , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Interferons upregulate the expression of HLA class I antigens on cancer cells. Nevertheless, little is known about the panel of HLA class I antigen-associated peptides presented by recombinant alpha-interferon (r(alpha)-IFN)-treated cells. For this reason, peptides were eluted from five cancer cell lines (four melanoma and one non-small cell lung cancer) following treatment with r(alpha)-IFN. High-performance liquid chromatography (HPLC) profiles of the peptide fractions were compared with those obtained from untreated cells. No significant differences in peptide characteristics (detectable on the basis of retention times) were observed, but significant differences in terms of peptide quantities were observed. Mass spectrometry performed on HPLC peaks allowed not only the detection of three different peptides (two derived from the MAGE family of genes and one from the mart-1) both in untreated and in treated cells, but also gave an indication of the number of peptides within one HPLC peak. This data demonstrates that r(alpha)-IFN-treated cells express a similar peptide pattern as untreated cells, with significant quantitative differences. Interestingly, this finding also explains the higher susceptibility to lysis (mediated by specific cytolytic lymphocytes, which recognize cancer cells in an HLA-restricted fashion) of r(alpha)-IFN-treated cells.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Interferón-alfa/farmacología , Péptidos/metabolismo , Antígenos de Neoplasias/metabolismo , Antígenos HLA-A/metabolismo , Antígenos HLA-B/metabolismo , Humanos , Células K562 , Antígeno MART-1 , Antígenos Específicos del Melanoma , Proteínas de Neoplasias/metabolismo , Células Tumorales CultivadasRESUMEN
Overexpression of inducible nitric oxide synthase causes the production of high levels of nitric oxide, which, under pathological conditions, leads to immunosuppression and tissue damage. The results recently obtained using peptide nucleic acids, rather than traditional oligonucleotides as antigen and antisense molecules, prompted us to test their efficacy in the regulation of nitric oxide production, thereby overcoming the obstacle of cellular internalization. The cellular permeability of four inducible nitric oxide synthase antisense peptide nucleic acids of different lengths was evaluated. These peptide nucleic acids were covalently linked to a hydrophobic peptide moiety to increase internalization and to a tyrosine to allow selective 125I radiolabelling. Internalization experiments showed a 3-25-fold increase in the membrane permeability of the modified peptide nucleic acids with respect to controls. Inducible nitric oxide synthase inhibition experiments on intact stimulated macrophages RAW 264.7 after passive permeation of the two antisense peptide nucleic acids 3 and 4 demonstrated a significant decrease (43-44%) in protein enzymatic activity with respect to the controls. These data offer a basis for developing a good alternative to conventional drugs directed against inducible nitric oxide synthase overexpression.
Asunto(s)
Macrófagos/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Ácidos Nucleicos de Péptidos/metabolismo , Ácidos Nucleicos de Péptidos/farmacología , Animales , Elementos sin Sentido (Genética)/genética , Elementos sin Sentido (Genética)/metabolismo , Elementos sin Sentido (Genética)/farmacología , Transporte Biológico , Línea Celular , Ratones , Óxido Nítrico Sintasa de Tipo II , Ácidos Nucleicos de Péptidos/genéticaRESUMEN
Disseminated infection with Mycobacterium avium complex (MAC) remains the most common serious bacterial infection in patients with advanced AIDS. The organisms that make up this complex are found ubiquitously in the environment, yet rarely cause disseminated disease in nonimmunocompromised human patients; on the contrary, up to 50% of patients with AIDS may ultimately develop the pathology. Hence, therapeutic strategies able to inhibit HIV and Mycobacterium replication are needed. Because of the rapid plasma elimination and toxicity of the most commonly used drugs, daily multiple-drug therapies must often be continued throughout life, frequently causing major side effects and, as a consequence, poor patient compliance. Therefore, alternative strategies that reduce the toxicity of the drugs and allow prolonged application intervals are sorely needed. Since erythrocytes (RBCs) can behave as bioreactors able to convert impermeant prodrugs to membrane-releasable active drugs, new compounds (AZTpEMB, AZTpEMBpAZT, and AZTp2EMB) consisting of both an antiretroviral and an antimicrobial drug were designed and synthesized. Among these, only AZTp2EMB was hydrolyzed by erythrocyte enzymes and could be encapsulated inside RBCs. AZTp2EMB-loaded RBCs slowly released AZT and EMB in culture medium, reducing its concentration by one-half about every 48 hr of incubation at 37 degrees C. Moreover, when AZTp2EMB-loaded erythrocytes were incubated for 6 days in the presence of human macrophages infected with Mycobacterium avium (M. avium) a marked bactericidal effect (>1 log) was observed. Thus, AZTp2EMB-loaded erythrocytes could be used as endogenous bioreactors for AZT and EMB delivery in the treatment of HIV and M. avium infection.