Desenvolvimento de inibidores duais de histona desacetilase 6 (HDAC6) e tirosina quinase (TQ) com vistas ao tratamento do câncer / Design of histone deacetylase 6 (HDAC6) and tyrosine kinase (TK) dual inhibitors for cancer treatment

Câncer é a denominação atribuída a um conjunto de doenças que são responsáveis pela segunda maior causa de morte no Brasil e no mundo. A quimioterapia figura entre uma das estratégias utilizadas para o tratamento e cura do câncer, sendo amplamente empregada em estratégias terapêuticas isoladas, ou em associação à radioterapia e cirurgia. A enzima histona desacetilase 6 (HDAC6) é responsável por desacetilar a cadeia lateral de N-acetillisinas em -tubulinas, desempanhando papel crítico na dinâmica do citoesqueleto celular, estando superexpressa em uma série de neoplasias. Neste sentido, na última década os receptores tirosina quinase (TQ) foram os principais alvos de fármacos aprovados para o tratamento do câncer e de doenças autoimunes e continuam atraindo a atenção de grupos de pesquisa dada a exorbitante diversidade do quinoma humano. É sabido que a monoterapia seja com inibidores de HDAC, seja com inibidores TQ, apresenta problemas de toxicidade, reações adversas, ineficácia, resistência e/ou recidiva. Diversos estudos relatam o desenvolvimento de inibidores duais de HDAC-TQ, almejando tanto a simplificação do tratamento, quanto sinergismo terapêutico e redução de efeitos adversos. Assim, o presente trabalho apresenta o planejamento, síntese e avaliação da citotoxicidade de inibidores duais, potencialmente seletivos para HDAC6 e receptores TQ. No total, 23 compostos foram sintetizados entre 2 a 4 etapas. Todos os compostos finais foram caracterizados por RMN (1H e 13C) e espectrometria de massas de alta resolução (HRMS). A citotoxicidade foi determinada pelo ensaio de MTT, em linhagens derivadas de tumores sólidos (HCT116 e MCF-7) e hematológicos (Jurkat e Namalwa). Os compostos apresentaram citotoxicidade em concentrações micro e nanomolares em todas as linhagens testadas, sendo que a linhagem MCF-7 foi a mais resistente à ação dos compostos, e as linhagens hematológicas foram as mais sensíveis. Os inibidores 4d-f foram os mais ativos na triagem por MTT, com IC50 iguais a 20, 30 e 50 nM, respectivamente, em células Jurkat. Estudos mecanísticos do efeito citotóxico indicaram que os compostos 4d-f exercem atividade de forma tempo-dependente, e majoritariamente por ação antiproliferativa, embora estímulos apoptóticos também tenham sido observados nos estudos. Simulações de ancoramento molecular (docking) e de relação entre as estruturas químicas dos compostos e suas respectivas atividades biológicas (REA) permitiram identificar padrões moleculares, propriedades físico-químicas e eletrônicas que potencialmente possuem relação com a atividade biológica dos compostos, permitindo futuras otimizações do arcabouço molecular desta série de compostos. Tomados em conjunto, os resultados deste trabalho revelam o potencial terapêutico de inibidores duais de HDAC6-TQ. Notadamente, os compostos apresentados aqui podem ser os primeiros potenciais inibidores duais de HDAC6-TQ a serem reportados na literatura

Cancer is the name of a series of diseases that are the second main cause of death in Brazil and worldwide. Chemotherapy is one of the main strategies to treat and cure cancer, and has been widely applied as a single therapeutic agent, and in association with radiotherapy and surgery. Histone deacetylase 6 (HDAC6) deacetylates N-acetyllysine side chains of tubulin, playing crucial role on cytoskeletal dynamics, and could be overexpressed in several cancers. Tyrosine kinase receptors (TK) have been the main targets of FDA-approved drugs through the last decade for both cancer and autoimmune diseases, and have been attracting special attention of research groups due to the exorbitant diversity of the human kinome. It is known that either HDAC or TK single therapy have toxicity issues, adverse effects, inefficacy, resistance and/or recidive. Therefore, many studies report the design of HDAC-TK dual inhibitors aiming simpler treatments, synergism of action and side effects reduction. Herein, the design, synthesis and cytotoxic evaluation of dual and selective HDAC6-TK inhibitors are presented. A total of 23 compounds were designed and synthesized through 2 to 4 steps. All final compounds were characterized by 1H/13C NMR and high-resolution mass spectrometry (HRMS). The cytotoxicity of compounds was determined by MTT assay for both solid (HCT116 and MCF-7 cells) and hematological cancers (Jurkat and Namalwa cells). Compounds exhibited micro and nanomolar ranges of cytotoxicity for all cell lines tested. MCF-7 cells were the most resistant against the treatment, and hematological cells were more susceptible to the cytotoxic effect of the compounds. Compounds 4d-f were the most actives in the MTT screening against Jurkat cells (IC50 = 20, 30 and 50 nM, respectively). Mechanistic studies regarding the cytotoxic effects of 4d-f indicated that the compounds induced cell death in a time-dependent manner mainly via cytostatic activity even though apoptotic stimuli were observed also. Molecular docking and structure-activity relationships (SARs) allowed the identification of molecular patterns, and physicochemical and electronic properties that potentially modulate the biological activity of these compounds, allowing further optimizations of the molecular scaffold for these series of compounds. Taken together, the results of this study reveal the therapeutic potential of HDAC6-TK dual inhibitors. Noteworthy, the compounds reported herein could be the first HDAC6-TK dual inhibitors ever reported in literature

Clinical features and tubulin folding cofactor E gene analysis in Iranian patients with Sanjad-Sakati syndrome / Características clínicas e análise genética e de cofatores de dobramento da tubulinaem pacientes iranianos com síndrome de Sanjad-Sakati

Abstract Objective Permanent hypoparathyroidism can be presented as part of genetic disorders such as Sanjad-Sakati syndrome (also known as hypoparathyroidism—intellectual disability-dysmorphism), which is a rare autosomal recessive disorder. Our aim was to confirm the diagnosis of a group of patients with dysmorphism, poor growth, and hypoparathyroidism clinically labeled as Sanjad-Sakati syndrome and to identify for the first time the genetic variations on Iranian patients with the same ethnic origin. Methods In this study, 29 cases from 23 unrelated Arab kindreds with permanent hypoparathyroidism and dysmorphism indicating Sanjad-Sakati syndrome were enrolled for 10 years in the southwest of Iran. The mutational analysis by direct sequencing of the tubulin folding cofactor E gene was performed for the patients and their families, as well as their fetuses using genomic DNA. Results Twenty-eight out of 29 cases had parental consanguinity. Twenty-seven cases presented with hypocalcemia seizure and two were referred because of poor weight gain and were found to have asymptomatic hypocalcemia. The dysmorphic features, hypocalcemia in the setting of low to normal parathyroid hormone levels and high phosphorus led to the diagnosis of these cases. Sequencing analysis of the tubulin folding cofactor E gene revealed a homozygous 12-bp deletion (c.155-166del) for all patients. Following that, prenatal diagnosis was performed for eight families, and two fetuses with a homozygous 12-bp deletion were identified. Conclusion These results make it much easier and faster to diagnose this syndrome from other similar dysmorphisms and also help to detect carriers, as well as prenatal diagnosis of Sanjad-Sakati syndrome in high-risk families in this population.

Resumo Objetivo O hipoparatireoidismo permanente pode estar presente como parte das doenças genéticas como na síndrome de Sanjad-Sakati (também chamada de síndrome de hipoparatireoidismo, retardo e dismorfismo), que é um distúrbio autossômico recessivo raro. Nosso objetivo foi confirmar o diagnóstico de um grupo de pacientes com dismorfismo, crescimento deficiente e hipoparatireoidismo clinicamente identificado como síndrome de Sanjad-Sakati e identificar as variações genéticas, pela primeira vez, em pacientes iranianos com a mesma origem étnica. Métodos Neste estudo, foram inscritos 29 casos de 23 famílias árabes sem parentesco com hipoparatireoidismo e dismorfismo indicando síndrome de Sanjad-Sakati, durante 10 anos no sudoeste do Irã. Foi feita a análise mutacional por sequenciamento direto do gene do cofator E de dobramento da tubulina dos pacientes e de suas famílias e também de seus fetos com o DNA genômico. Resultados Apresentaram consanguinidade parental 28 dos 29 casos. Desses, 27 casos apresentaram convulsão por hipocalcemia e dois foram encaminhados devido ao baixo ganho de peso, considerando diagnóstico de hipocalcemia assintomática. As características dismórficas, hipocalcemia na configuração de níveis de hormônio da paratireoide baixos a normais e alto nível de fósforo levaram ao diagnóstico dos casos. A análise de sequenciamento do gene do cofator E de dobramento da tubulina revelou deleção homozigótica de 12 pares de base (pb) (c.155-166del) em todos os pacientes. Após isso, foi feito o diagnóstico pré-natal em oito famílias e dois fetos foram identificados com deleção homozigótica de 12 pb. Conclusão Esses resultados tornam o diagnóstico dessa síndrome muito mais fácil e rápido do que outros dismorfismos semelhantes e também ajudam a detectar portadores, bem como o diagnóstico pré-natal da síndrome de Sanjad-Sakati em famílias de alto risco nessa população.

Absence of mutations associated with resistance to benzimidazole in the beta-tubulin gene of Ascaris suum

Abstract INTRODUCTION: Benzimidazoles are commonly used for the control of veterinary nematodes. Resistance to benzimidazoles has been associated with three single nucleotide polymorphisms in the β-tubulin gene of common nematodes. However, these mutations are infrequent in the genus Ascaris spp. METHODS: In order to determine mutations associated with benzimidazole resistance in Ascaris suum, worms were collected from slaughtered pigs and a partial region of the β-tubulin gene was sequenced. RESULTS: All parasites showed the wildtype genotype for codons 167, 198, and 200 of the β-tubulin gene. CONCLUSIONS: This is the first report of genetic sequences associated with benzimidazole resistance in A. suum.

Modelamiento proteico de la Tubulina de G. lambliapor homología de secuencias / Protein modeling of G. Lamblia Tubulin through sequence homology

La Giardiasis ocasionada por Giardia intestinalis (conocida como Giardia lamblia), es una de las infecciones parasíticas más comunes en todo el mundo y con mayor prevalencia en países en desarrollo como el nuestro. Existen muchas drogas para el tratamiento de la giardiasis, de diferente eficacia y efectos adversos como la curcumina que inhibe la polimerización de los microtubulos por un mecanismo distinto al de la colchicina. Sin embargo, la estructura cristalográfica de la Tubulina de G. lamblia (cadenas α y ß) permanece desconocida. El análisis de alineamiento de secuencias (PBLAST) indica una identidad del 86,98 y 88,32 % entre las cadenas α y ß de la Tubulina de G. lamblia y B. taurus (PDB:5NQT). El Modelamiento por Homología de la estructura proteica de la Tubulina de G. lamblia utilizando como molde a la Tubulina de B. taurus mediante el servidor SWISS-MODEL, generó una estructura proteica con los siguientes parámetros: z-core = -1,16 y -1,41, QMEANscore6 = 0,71 y 0,70, % de confiabilidad (de los diagramas de Ramachandran) = 96,08 y 96,95 %, RMS (Root Mean Square) = 0,081 y MOLPROBITYscore = 1,26. Estos parámetros indican que la estructura proteica de la Tubulina de G. lamblia obtenida a partir del Modelamiento por Homología es de buena calidad, por tanto, esta estructura podría ser utilizada en futuras evaluaciones, como el análisis in silico de compuestos antiGiardia.

Giardiasis caused by Giardia intestinalis (known as Giardia lamblia), is one of the most common parasitic infections in the world and with a higher prevalence in developing countries like ours. There are many drugs for the treatment of giardiasis, of different efficacy and adverse effects such as curcumin that inhibits the polymerization of microtubules by a mechanism other than colchicine. However, the crystallographic structure of G. lamblia Tubulin (α and ß chains) remains unknown. The sequence alignment analysis (PBLAST) indicates an identity of 86,98 and 88,32 % between the α and ß chains of the Tubulin of G. lamblia and B. taurus (PDB: 5NQT). Homologous Modeling of the protein structure of G. lamblia Tubulin using the B. taurus Tubulin as a template employing the SWISS-MODEL server, generated a protein structure with the following parameters: z-core = -1,16 and -1,41, QMEANscore6 = 0,71 and 0,70, % of reliability (from Ramachandran plots) = 96,08 y 96,95 %, RMS (Root Mean Square) = 0,081 and MOLPROBITYscore = 1,26. These parameters indicate that the protein structure of G. lamblia Tubulin obtained from Homology Modeling is of good quality, therefore, this structure could be used in further evaluations, such as the in silico analysis of antiGiardia compounds.

Homology modeling and epitope prediction of Der f 33

Dermatophagoides farinae (Der f), one of the main species of house dust mites, produces more than 30 allergens. A recently identified allergen belonging to the alpha-tubulin protein family, Der f 33, has not been characterized in detail. In this study, we used bioinformatics tools to construct the secondary and tertiary structures and predict the B and T cell epitopes of Der f 33. First, protein attribution, protein patterns, and physicochemical properties were predicted. Then, a reasonable tertiary structure was constructed by homology modeling. In addition, six B cell epitopes (amino acid positions 34-45, 63-67, 103-108, 224-230, 308-316, and 365-377) and four T cell epitopes (positions 178-186, 241-249, 335-343, and 402-410) were predicted. These results established a theoretical basis for further studies and eventual epitope-based vaccine design against Der f 33.

JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.

Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México

The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.

Biodegradation of phenol in static cultures by Penicillium chrysogenum ERK1: catalytic abilities and residual phototoxicity / Biodegradación de fenol en cultivos estáticos por Penicillium chrysogenum ERK1: habilidades catalíticas y fitotoxicidad residual

A phenol-degrading fungus was isolated from crop soils. Molecular characterization (using internal transcribed spacer, translation elongation factor and beta-tubulin gene sequences) and biochemical characterization allowed to identify the fungal strain as Penicillium chrysogenum Thorn ERK1. Phenol degradation was tested at 25 °C under resting mycelium conditions at 6, 30, 60, 200, 350 and 400 mg/l of phenol as the only source of carbon and energy. The time required for complete phenol degradation increased at different initial phenol concentrations. Maximum specific degradation rate (0.89978 mg of phenol/day/mg of dry weight) was obtained at 200 mg/l. Biomass yield decreased at initial phenol concentrations above 60 mg/l. Catechol was identified as an intermediate metabolite by HPLC analysis and catechol dioxygenase activity was detected in plate assays, suggesting that phenol metabolism could occur via ortho fission of catechol. Wheat seeds were used as phototoxicity indicators of phenol degradation products. It was found that these products were not phytotoxic for wheat but highly phytotoxic for phenol. The high specific degradation rates obtained under resting mycelium conditions are considered relevant for practical applications of this fungus in soil decontamination processes.

Un aislamiento fúngico capaz de degradar fenol como única fuente de carbono y energía fue aislado de suelos agrícolas. La caracterización molecular (basada en el empleo de secuencias de espaciadores de transcriptos internos, de factores de la elongación de la traducción y del gen de la beta-tubulina) y la caracterización bioquímica permitieron identificar a esta cepa como Penicillium chrysogenum Thom ERK1. Se estudió la degradación de fenol a 25 °C en cultivos estáticos con 6, 30, 60, 200, 350 y 400 mg/l de fenol inicial. El tiempo requerido para completar la degradación de fenol aumentó al elevarse las concentraciones iniciales de dicho compuesto. La máxima tasa de degradación específica (0,89978 mg de fenol/día/mg de peso seco) se obtuvo con 200 mg/l. El rendimiento en biomasa disminuyó con concentraciones Iniciales de fenol mayores de 60 mg/l. Se identificó al catecol como intermediarlo metabolico por HPLC y se observó actividad de catecol dioxigenasa en placa, lo que sugiere que el metabolismo de degradación del fenol ocurre vía orto fisión del catecol. Se utilizaron semillas de trigo como indicadores de fitotoxicidad de los productos de degradación. Estos productos no fueron fitotóxicos para trigo, mientras que el fenol mostró una alta fitotoxicidad. La alta tasa de degradación específica obtenida en condiciones estáticas resulta de gran interés para la aplicación de este hongo en procesos de descontaminación de suelos.

Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: An in vitro model of HTLV-I-induced disease

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could beinvolved in this disease, configuring an axonal transport disease. We measured tubulin and its posttranslational modified forms (acetylated and tyrosinated) in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulinbetween patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levels were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes theviral Tax protein) we observed neurite retraction and an increase in tau phosphorylation at T181. A disruptionof normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK2.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain

Dysmyelination, demyelination and reactive astrogliosis in the optic nerve of the taiep rat

Taiep is an autosomal recessive mutant rat that shows a highly hypomyelinated central nervous system (CNS). Oligodendrocytes accumulate microtubules (MTs) in association with endoplasmic reticulum (ER) membranes forming MT-ER complexes. The microtubular defect in oligodendrocytes, the abnormal formation of CNS myelin and the astrocytic reaction were characterized by immunocytochemical and ultrastructural methods during the first year of life. Optic nerves of both control and taiep rats were processed by the immunoperoxidase method using antibodies against tubulin, myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). Taiep oligodendrocytes are strongly immunoreactive against tubulin, indicative of a significant accumulation of microtubules. Early differentiated oligodendrocytes observed with electron microscopy show that MT-ER complexes are mainly present in the cell body. This defect increases during the first year of life; oligodendrocytes show large MT-ER complexes projected within oligodendrocyte processes. Using anti-MBP, there was a progressive reduction of immunolabeling in the myelin sheaths as taiep rats grew older. Ultrastructural analysis revealed severely dysmyelinated axons with a frequently collapsed periaxonal collar. However, through age the myelin sheath became gradually infiltrated by MTs, suggesting their contribution to premature loss of myelin in the taiep rat. Axons of one-year-old taiep rats were severely demyelinated. Modifications in astrocytes revealed by the GFAP antibody showed a strong hypertrophy with increased immunostaining in their processes. As demyelination of axons progressed, taiep rats developed a strong astrogliosis. The present findings suggest that in taiep rats the early abnormal myelination of axons affects the adequate maintenance of myelin, leading to a progressive loss of myelin components and severe astrogliosis, features that should be considered in the pathogenesis of dysmyelinating diseases

The anthelmintic albendazole affects in vivo the dynamics and the detyrosination-tyrosination cycle of rat brain microtubules

Albendazole (ABZ) is an anthelmintic benzimidazole drug widely used in human and veterinary medicine. ABZ has binding affinity to both mammalian and helminth parasite tubulin. In the current work, we have performed in vitro assays and in vivo experiments in which rats were given ABZ orally to better characterize the action of the drug on the polymerization of rat brain microtubules and on the detyrosination/tyrosination cycle that occurs on the COOH-terminal end of alpha-tubulin. The results showed that ABZ inhibits brain microtubule polymerization in vitro, and significantly delayed microtubule assembly in vivo. The tyrosination reaction cycle was not affected in vitro; however, in rats to which the drug was administered orally, the levels of in vitro tyrosination were reduced when compared to the controls with mock treatment. These results suggest that this apparent inhibition would be due to a decrease in the amount of substrate caused by the depolymerizing effect of ABZ and the subsequent tyrosination in the intact brain with endogenous tyrosine. In conclusion, ABZ strongly affects tubulin dynamics both in vivo and in vitro. The outcome of these experiments is a contribution to the understanding of the molecular mechanisms involved in the antimicrotubular action of benzimidazole compounds.

Cloning and sequence analysis of a trypanosoma cruzi alpha-butulin cDNA

A cDNA clone derived from the Trypanosoma cruzi alpha-tubulin gene was isolated and sequenced (Tc alpha tub; L37345). Tc alpha tub revealed an 87.79 percent and an 85.36 percent identity with the DNA sequence of T. brucei and Leishmania, respectively. This clone was used to study, by Northern blots, alpha-tubulin gene expression in epimastigotes, cell-cultured derived trypomastigotes and extracellular amastigotes. alpha-tubulin MRNA levels were the same in epimastigotes and trypomastigotes, however, there was a drastic decrease in amastigotes. This clone could be useful to elucidate the regulatory mechanisms of alpha-tubulin gene expression during the differentiation of T. cruzi

Tubulin aggregates induced by Ni2+ present microtubular characteristics

Rat brain tubulin in a proper buffered solution became insoluble in the presence of 10 mM NiCl2, and sedimented at centrifugal forces as low as 500 x g for 30 min. Both nickel-sedimented and microtubular tubulin conserved 65 of colchicine binding activity after 25 days of storage at -20 degrees C. However in brain cytosol, only 9 of the initial binding activity was conserved. The electrophoretic mobility of tubulin recovered from aggregates also remained unaltered. Therefore the aggregates formed with Ni2+ share important physicochemical properties with microtubules.

Organization and structure of an Onchocerca ß-tubulin gene

In filarial worms, as in other eukaryotes, microtubules are essential multifunctional components. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides, Ó e ß.Tubulin is particulary important in helminthic parasites as a target for anthelminthic benzimidazoles, wich bind to it and inhibit microtubule assembly. Two genomic Onchocerca gibsoni libraries were constructed in NM1149(EcoRi and HindIII). Three clones accounted for the entire gene: one from the EcoRi library (using a Plasmodium falciparum probe) containing the central part of the gene, and two from the HindIII library (using as probes PCR amplified fragments from the ends of the EcoRI clone) which, respectively, contained the 5'- and 3' -ends of the gene. The sequencing procedure for the EcoRI clone relied on the construction of a double-digested DraI/HindIII shotgun library. A number of recombinants were sequenced and aligned with each other for comparison. The sequencing of the overlapping 5' - and 3'-end clones was done by ...

Acetylated alpha-tubulin in Trypanosoma cruzi: immunocytochemical localization

We have used monoclonal antibodies specific for acetylated and non-acetylated alpha-tubulin to localize microtubules containing acetylated alpha-tubulin in all developmental forms of the life cycle of Trypanosoma cruzi. This was demonstrated using immunofluorescence and by transmission electron microscopy of thin sections, negative stained cells, and replicas of whole Triton X-100 extracted cells immunolabeled with antibody-gold complex. The antibody specific for acetylated alpha-tubulin (6-11B-1) binds to the flagellar, as well as to the sub-pellicular microtubules. The extent of labeling of the sub-pellicular microtubules with the monoclonal antibody recognized alpha-acetylated tubulin was smaller than that observed with the antibody which recognizes all tubulin isoforms. In relation to the developmental forms, the extent of labeling of the microtubules with antibody 6-11B-1 was larger in epimastigote and trypomastigote than in amastigote forms. Incubation of the parasites for 1 h at 0 degrees C or in the presence of either colchicine or vinblastine did not interfere with the sub-pellicular microtubules. These observations, in agreement with those reported for Trypanosoma brucei brucei (Schneider et al., 1987; Schulze et al., 1987; Sasse per cent Gull, 1988) indicate that the sub-pellicular microtubules of trypanosomatids represent stable microtubules containing acetylated alpha-tubulin (or the alpha 3-tubulin isotype)

Identification of guanine nucleotide binding proteins from Trypanosoma cruzi

Guanine nucleotide binding proteins (GTP-binding proteins) function as transducers of signals in different cellular processes. We have identified several GTP-binding proteins in Trypanosoma cruzi by Western blot analyses. Six polypeptide bands, p20, p25, p28, p31, p37 and p38, were specifically detected in epimastigote crude extracts, using polyclonal antibodies directed against transducin (T) or the alpha-subunit of transducin (T alpha). Four of these bands, p28, p31, p37 and p38, were found in both the soluble and the particulate epimastigote fractions. On the other hand, two of the polypeptides, p20 and p25, were observed only in the particulate fraction, and were not solubilized using 0.2 percent Triton X-100 and 0.2 percent Nonidet P-40. A rat monoclonal antibody directed against the ras oncogene, immunorecognized a band with molecular mass of 20,000 daltons, in epimastigote homogenates. In view of their identical apparent molecular weight and solubilization properties, p20, recognized by anti-T or anti-T alpha antibodies, and the 20 KDa band, recognized by anti-ras antibodies, seem to correspond to the same polypeptide. [3H] GDP and [3H] GMP-PNP binding experiments revealed the presence of guanine nucleotide binding proteins in total epimastigote crude extracts, as well as, in the soluble, detergent soluble, and particulate fractions. A primary screening of a T. cruzi cDNA library with anti-T alpha antibodies, followed by secondary and tertiary screenings with anti-ras antibodies yielded six positive clones. One of these clones (Tc-ras1) contains a 600 bp insert which we believe encodes for the ras protein from T. cruzi. On a Northern blot, this cDNA hybridizes to a unique mRNA band of 2.0 Kilobases in epimastigotes

Differential turn-over of beta-tubulin during the cell differentiation of Trypanosoma cruzi

We investigated the expression of beta-tubulin during the differentiation of non-infective epimastigotes to infective metacyclics of Trypanosoma cruzi to underlay some of the regulatory mechanisms of the gene expression in this pathogenic parasite. Given the strong evolutionary conservation of tubulin, it was possible to study its translational and transcriptional products with heterologous probes. Quantitative Western blotting with specific monoclonal antibodies against beta-tubulin revealed an increase in the relative amounts of this protein in metacyclics with respect to epimastigotes. Pulse-chase experiments with radioactive methionine followed by immunoprecipitation and polyacrylamide gel electrophoresis showed that beta-tubulin has a slower degradation in metacyclics, which may contribute to its relative higher abundance in these parasite forms. In contrast with these results, both in vitro translation of poly (A+) mRNA in a wheat germ system and Northern blots of total and poly (A+) mRNA with a heterologous DNA probe from Leishmania enriettii, revealed a significant decrease (5 fold) in the specific transcripts of beta-tubulin in the metacyclics with respect to epimastigotes. It thus appeared that after differentiation of T. cruzi the translational machinery for a key protein such as beta-tubulin is shut off by a decrease in its specific message. The protein levels of this protein are maintained, however, by a compensatory mechanism that involves a slower turn-over of the synthesized protein