RESUMEN
BACKGROUND: The oligomeriztion of α-synuclein (α-syn) into ordered assemblies is associated with the symptoms of Parkinson's Disease (PD). Yet, it is still debatable whether oligomers are formed as part of a multistep process towards amyloid fibril formation or alternatively as "off-pathway" aggregates. METHODS: 100µM α-syn was incubated with decreasing amounts of cinnamon extract precipitation (CEppt). The fibril formation was measured using spectroscopy and microscopy analyses and oligomers were detected using western blot analysis. The secondary structure of the protein was analyzed using CD. Drosophila brains were studied using immunostaining and confocal microscopy. RESULTS: Here we probed the inhibition pattern of oligomeric and fibrillar forms of α-syn, using a natural substance, CEppt which was previously shown to effectively inhibit aggregation of ß-amyloid polypeptide. We demonstrated that CEppt has a differential inhibitory effect on the formation of soluble and insoluble aggregates of α-synuclein in vitro. This inhibition pattern revokes the possibility of redirection to "off-pathway" oligomers. When administering to Drosophila fly model expressing mutant A53T α-syn in the nervous system, a significant curative effect on the behavioral symptoms of the flies and on α-syn aggregation in their brain was observed. CONCLUSIONS: We conclude that CEppt affects the process of aggregation of α-syn without changing its secondary structure and suggest that increasing amounts of CEppt slow this process by stabilizing the soluble oligomeric phase. When administered to Drosophila fly model, CEppt appears to have a curative effect on the defective flies. GENERAL SIGNIFICANCE: Our results indicate that CEppt can be a potential therapeutic agent for PD.
Asunto(s)
Amiloide/antagonistas & inhibidores , Cinnamomum zeylanicum , Drosophila , Enfermedad de Parkinson/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/metabolismo , Amiloide/genética , Amiloide/metabolismo , Animales , Animales Modificados Genéticamente , Células CHO , Cinnamomum zeylanicum/química , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Drosophila/genética , Femenino , Humanos , Masculino , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Fitoterapia/métodos , Multimerización de Proteína/genética , alfa-Sinucleína/genéticaRESUMEN
Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.
Asunto(s)
Neoplasias Encefálicas/terapia , Encéfalo/metabolismo , Terapia Genética , Glioma/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/farmacocinética , Adenoviridae/genética , Animales , Barrera Hematoencefálica , Encéfalo/efectos de los fármacos , Encéfalo/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Ensayos Clínicos Fase I como Asunto , ADN Viral/metabolismo , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Multimerización de Proteína/genética , Ratas , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.
Asunto(s)
Animales , Humanos , Ratas , Adenoviridae/genética , Barrera Hematoencefálica , Encéfalo/efectos de los fármacos , Neoplasias Encefálicas/genética , Ensayos Clínicos Fase I como Asunto , ADN Viral/metabolismo , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Terapia Genética , Glioma/genética , Hígado/efectos de los fármacos , Multimerización de Proteína/genética , Bazo/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.
Asunto(s)
Animales , Humanos , Ratas , Adenoviridae/genética , Barrera Hematoencefálica , Encéfalo/efectos de los fármacos , Neoplasias Encefálicas/genética , Ensayos Clínicos Fase I como Asunto , ADN Viral/metabolismo , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Terapia Genética , Glioma/genética , Hígado/efectos de los fármacos , Multimerización de Proteína/genética , Bazo/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
The pollen-specific receptor-like kinases (PRKs) from Solanum lycopersicum, LePRK1 and LePRK2, are believed to be involved in the regulation of pollen germination and pollen tube growth. They appear to be part of a multimeric complex in which the transmembranic LePRKs presumably have a key position in transducing exogenous signals through the plasma membrane. Here, we focused on extra- and intracellular interactions involving the LePRKs. We show in yeast two-hybrid experiments a cross-interaction of putative PRK-ligands, the oligomerization of LePRK2 and a direct contact of LePRKs to activated Rho proteins of plants (ROPs). Moreover, we observed that pollen-specific RopGEFs, which catalyze ROP activation and may be regulated by PRK interaction, are active in vitro while autoinhibition seems to occur in vivo. We suggest that activation of RopGEFs as a checkpoint in PRK signal transduction is a more complex event including further components in planta. Our findings point to some new aspects in PRK-mediated signal transduction implying a LePRK2 complex with different signaling activity and a further direct control of LePRKs by activated ROP.
Asunto(s)
Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Solanum lycopersicum/enzimología , Proteínas de Plantas/genética , Polen/genética , Polen/metabolismo , Tubo Polínico/genética , Tubo Polínico/metabolismo , Proteínas Quinasas/genética , Multimerización de Proteína/genética , Multimerización de Proteína/fisiología , Proteínas Recombinantes/genética , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 microM CoCl(2). Ten- to 100-fold less CoCl(2) sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mm phosphate, pH 7.0, 25 degrees C). Divalent metal ions including Co(2+), Cu(2+), Zn(2+), and Ni(2+) stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co(2+) with K(d) of 0.12 +/- 0.06 nM and 110 +/- 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn(2+) (K(d) = 11 +/- 2 nM) and Ni(2+) (K(d) = 49 +/- 17 nM) and did not detectably bind Cu(2+). Substitution of the sole histidine residue did not affect Co(2+) binding. Holo-AnhE had a weak absorption band at 490 nM (epsilon = 9.7 +/- 0.1 m(-1) cm(-1)), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.