Effect of 3,4-ethylenedioxy-extension of thiophene core on the DNA/RNA binding properties and biological activity of bisbenzimidazole amidines

Novel bisbenzimidazoles (4-6), characterized by 3,4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. They interact within ds-DNA grooves as dimmers or even oligomers and agglomerate along ds-RNA. Compounds 4-6 have shown moderate to strong antiproliferative effect toward panel of eight carcinoma cell lines. Compound 5 displayed the best inhibitory potential and in equitoxic concentration (IC(50) = 1 x 10 (6) M) induced accumulation of cells in G2/M phase after 48 h of incubation. Fluorescence microscopy showed that 5 entered into live HeLa cells within 30 min, but did not accumulate in nuclei even after 2.5 h. Compound 5 inhibited the growth of Trypanosome cruzi epimastigotes (IC(50) = 4.3 x 10 (6) M). (C) 2009 Elsevier Ltd. All rights reserved.

Sample stacking in CZE using dynamic thermal junctions II: Analytes with high dpK(a)/dT crossing a single thermal junction in a BGE with low dpH/dT

In a previous work [M. Mandaji, et al., this issue] a sample stacking method was theoretically modeled and experimentally demonstrated for analytes with low dpK(a)/dT (analytes carrying carboxylic groups) and BGEs with high dpH/dT (high pH-temperature-coefficients). In that work, buffer pH was modulated with temperature, inducing electrophoretic mobility changes in the analytes. In the present work, the opposite conditions are studied and tested, i.e. analytes with high dpK(a)/dT and BGEs that exhibit low dpH/dT. It is well known that organic bases such as amines, imidazoles, and benzimidazoles exhibit high dpK(a)/dT. Temperature variations induce instantaneous changes on the basicity of these and other basic groups. Therefore, the electrophoretic velocity of some analytes changes abruptly when temperature variations are applied along the capillary. This is true only if BGE pH remains constant or if it changes in the opposite direction of pK(a) of the analyte. The presence of hot and cold sections along the capillary also affects local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band stacking efficacy was also taken into account in the theoretical model presented. Finally, this stacking method is demonstrated for lysine partially derivatized with naphthalene-2,3-dicarboxaldehyde. In this case, the amino group of the lateral chain was left underivatized and only the alpha amino group was derivatized. Therefore, the basicity of the lateral amino group, and consequently the electrophoretic mobility, was modulated with temperature while the pH of the buffer used remained unchanged.

Conformational analysis: A new approach by means of chemometrics

In conformational analysis, the systematic search method completely maps the space but suffers from the combinatorial explosion problem because the number of conformations increases exponentially with the number of free rotation angles. This study introduces a new methodology of conformational analysis that controls the combinatorial explosion. It is based on a dimensional reduction of the system through the use of principal component analysis. The results are exactly the same as those obtained for the complete search but, in this case, the number of conformations increases only quadratically with the number of free rotation angles. The method is applied to a series of three drugs: omeprazole. pantoprazole, lansoprazole-benzimidazoles that suppress gastric-acid secretion by means of H(+), K(+)-ATPase enzyme inhibition. (C) 2002 John Wiley Sons. Inc.

Desempenho de fungicidas no controle da mancha-de-macrospora na cultura do milho

Fungicide application is one of the control strategies of fungal diseases in corn leaves. In Brazil, there are no fungicides recorded for the control of corn macrospora leaf spot (MLS). The aim of this study was to assess the efficacy of 16 fungicides on MLS control in a protective, curative and eradicant form. Fungicides of the chemical groups of benzimidazoles, strobilurins and triazoles were used alone or in mixture, in completely randomized block design, with six replicates of five plants, totaling 30 plants per treatment. The experiments were carried out in a greenhouse with the single-cross hybrid AS 1565 in phenological stage of two to six expanded leaves, using an isolate of S. macrospsora from the same hybrid. The inoculum was deposited into the cartridge of plants at 48 hours after, 48 hours before and 10 days before fungicide applications for preventive, curative and eradicant action, respectively. The experiment was repeated twice. The data underwent analysis of variance (p<0.05), and the means of treatments were compared by using the Scott-Knott test (p<0.05). Severity was estimated at 21 days after inoculations. All fungicides significantly differed from the control treatment in the preventive, curative and eradicant action. For the preventive action, mean disease control was 85%. The mixture of triazoles plus strobilurins controlled, on average, 75% of the disease severity, while the isolated products such as strobilurins reduced it by 62%, benzimidazoles by 55% and triazoles by 38% for the curative action. The lowest control was obtained for the eradicant action, with mean reduction of 40.1% in the disease severity and no significant difference among fungicides.

Haemonchus contortus: A multiple-resistant Brazilian isolate and the costs for its characterization and maintenance for research use

The aim of this work was to determine the resistance level of Haemonchus contortus isolated from the Santa Inês flock of the Embrapa (Brazilian government's Agricultural Research Company), Southeast Livestock Unit (CPPSE), as well as to determine costs of characterizing and maintaining this isolate in host donors. Forty-two male Santa Inês lambs were experimentally infected with 4000 H. contortus infective larvae of the field isolate of CPPSE, called Embrapa2010, and divided into six treatment groups, which received triclorfon, albendazol plus cobalt sulfate, ivermectin, moxidectin, closantel and levamisole phosphate, as well as a negative control group (water). Egg per gram (EPG) counts were performed at 0, 3, 7, 10 and 14. days post treatment when the animals were slaughtered for parasite count. The data were analyzed using the RESO statistical program, considering anthelmintic resistance under 95% of efficacy. EPG and worm count presented a linear and significant relation with 94% determination coefficient. The susceptibility results obtained by RESO through both criteria (EPG and worm count) were equal, except for closantel, showing that the isolate Embrapa2010 is resistant to benzimidazoles, macrocyclic lactones and imidazothiazoles. The need of a control group did not appear to be essential since the result for susceptibility in the analyses with or without this group was the same. Suppression in egg production after treatment did not occur in the ivermectin and moxidectin groups. In the control group, the establishment percentage was just 12.5 because of the low number of third-stage larvae, resistance (innate and infection immunity) of the animals studied plus good nutrition. Drug classes presented similar efficacy between adults and immature stages. The costs for isolate characterization were calculated for 42 animals during 60. days. The total cost based on local market rates was approximately US$ 8000. The precise identification of Brazilian isolates and their establishment in host donors would be useful for laboratorial anthelmintic resistance diagnoses through in vitro tests, which has an annual cost of approximately US$ 2500 for maintenance in host donors. © 2012 Elsevier B.V.

Sensibilidade de stenocarpella macrospora a fungicidas

The application of fungicides in the aerial organs is control strategy to macrospora spot caused by fungus Stenocarpella macrospora. The objective of this study was to determine the sensitivity of S. macrospora to fungicides by inhibition of mycelial growth (MG) and conidial germination (CG). It was eval uated 12 fungicides belonging to the chemical groups of the benzimidazoles, triazoles and strobilurins, six concentrations and two isolates of the fungus (SC and MT). The fungicides were diluted in sterile distilled water and added to the culture medium of potato dextrose agar (mycelium) and water-agar (spore) after sterilization. The percentage of inhibition of MC and CG was calculed in comparison with control, estimating of 50% inhibitory concentration (IC50). The fungicides tested were effective in inhibiting the MC. The IC50 was less than 1 ppm for all fungicides. There was no difference between isolates. The inhibition of CG had higher fungitoxicity strobilurins, and the IC50 was between 0.0035 and 0.03 ppm, and the isolated SC showed the higher sensitivity to the fungicides. The IC50 values obtained for fungicides and specific S. macrospora will be useful in monitoring the sensitivity of the fungus, especially in regions with intense demand for fungicides in corn.

Resistência anti-helmíntica em equinos na Região Oeste do Estado de São Paulo

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Podridão floral dos citros: controle preventivo mediante fungicidas, biorreguladores e fertilizantes químicos

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

In vitro efficacy of dicationic compounds and mefloquine enantiomers against Echinococcus multilocularis metacestodes

The current chemotherapy of alveolar echinococcosis (AE) is based on benzimidazoles such as albendazole and has been shown to be parasitostatic rather than parasiticidal, requiring lifelong duration. Thus, new and more efficient treatment options are urgently needed. By employing a recently validated assay based on the release of functional phosphoglucose isomerase (PGI) from dying parasites, the activities of 26 dicationic compounds and of the (+)- and (-)-erythro-enantiomers of mefloquine were investigated. Initial screening of compounds was performed at 40 muM, and those compounds exhibiting considerable antiparasitic activities were also assessed at lower concentrations. Of the dicationic drugs, DB1127 (a diguanidino compound) with activities comparable to nitazoxanide was further studied. The activity of DB1127 was dose dependent and led to severe structural alterations, as visualized by electron microscopy. The (+)- and (-)-erythro-enantiomers of mefloquine showed similar dose-dependent effects, although higher concentrations of these compounds than of DB1127 were required for metacestode damage. In conclusion, of the drugs investigated here, the diguanidino compound DB1127 represents the most promising compound for further study in appropriate in vivo models for Echinococcus multilocularis infection.

Application of an in vitro drug screening assay based on the release of phosphoglucose isomerase to determine the structure-activity relationship of thiazolides against Echinococcus multilocularis metacestodes

OBJECTIVES: The disease alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is fatal if treatment is unsuccessful. Current treatment options are, at best, parasitostatic, and involve taking benzimidazoles (albendazole, mebendazole) for the whole of a patient's life. In conjunction with the recent development of optimized procedures for E. multilocularis metacestode cultivation, we aimed to develop a rapid and reliable drug screening test, which enables efficient screening of a large number of compounds in a relatively short time frame. METHODS: Metacestodes were treated in vitro with albendazole, the nitro-thiazole nitazoxanide and 29 nitazoxanide derivatives. The resulting leakage of phosphoglucose isomerase (PGI) activity into the medium supernatant was measured and provided an indication of compound efficacy. RESULTS: We show that upon in vitro culture of E. multilocularis metacestodes in the presence of active drugs such as albendazole, the nitro-thiazole nitazoxanide and 30 different nitazoxanide derivatives, the activity of PGI in culture supernatants increased. The increase in PGI activity correlated with the progressive degeneration and destruction of metacestode tissue in a time- and concentration-dependent manner, which allowed us to perform a structure-activity relationship analysis on the thiazolide compounds used in this study. CONCLUSIONS: The assay presented here is inexpensive, rapid, can be used in 24- and 96-well formats and will serve as an ideal tool for first-round in vitro tests on the efficacy of large numbers of antiparasitic compounds.

Innovative chemotherapeutical treatment options for alveolar and cystic echinococcosis

Echinococcus granulosus and Echinococcus multilocularis are cestode parasites, of which the metacestode (larval) stages cause the diseases cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. Albendazole and mebendazole are presently used for chemotherapeutical treatment. However, these benzimidazoles do not appear to be parasiticidal in vivo against AE. In addition, failures in drug treatments as well as the occurrence of side-effects have been reported. New drugs are needed to cure AE and CE, which are considered to be neglected diseases. Strategies currently being implemented to identify novel chemotherapeutical treatment options include (i) conventional primary in vitro testing of broad-spectrum anti-infective drugs, either in parallel with, or followed by, animal experimentation; (ii) studies of drugs which interfere with the proliferation of cancer cells and of Echinococcus metacestodes; (iii) exploitation of the similarities between the parasite and mammalian signalling machineries, with a special focus on targeting specific signalling receptors; (iv) in silico approaches, employing the current Echinococcus genomic database information to search for suitable targets for compounds with known modes of action. In the present article, we review the efforts toward obtaining better anti-parasitic compounds which have been undertaken to improve chemotherapeutical treatment of echinococcosis, and summarize the achievements in the field of host-parasite interactions which may also lead to new immuno-therapeutical options.

In vitro selection of Haemonchus contortus for benzimidazole resistance reveals a mutation at amino acid 198 of beta-tubulin

Benzimidazoles were the first broad-spectrum anthelmintics and are still in use today against gastro-intestinal nematodes of ruminants such as Haemonchus contortus. Benzimidazoles block the polymerization of nematode microtubules. However, their efficacy is jeopardized by the spread of drug-resistant parasites that carry point mutations in beta-tubulin. Here we use a novel in vitro selection-in vivo propagation protocol to breed drug-resistant H. contortus. After 8 generations of selection with thiabendazole an in vitro resistance factor of 1000 was reached that was also relevant in vivo in infected sheep. The same procedure carried out with ivermectin produced only a moderate resistance phenotype that was not apparent in sheep. Cloning and sequencing of the beta-tubulin genes from the thiabendazole-resistant H. contortus mutants revealed all of the isotype 1 alleles, and part of the isotype 2 alleles, to carry the mutation glutamate(198) to alanine (E198A). An allele-specific PCR was developed, which may be helpful in monitoring the prevalence of alanine(198) encoding alleles in the beta-tubulin isotype 1 gene pool of H. contortus in the field.

Alveolar and cystic echinococcosis: towards novel chemotherapeutical treatment options

Echinococcus granulosus and Echinococcus multilocularis are cestode parasites, of which the metacestode (larval) stages cause the neglected diseases cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. The benzimidazoles albendazole and mebendazole are presently used for the chemotherapeutical treatment, alone or prior to and after surgery. However, in AE these benzimidazoles do not appear to be parasiticidal in vivo. In addition, failures in drug treatments as well as the occurrence of side-effects have been reported, leading to discontinuation of treatment or to progressive disease. Therefore, new drugs are needed to cure AE and CE. Strategies that are currently employed in order to identify novel chemotherapeutical treatment options include in vitro and in vivo testing of broad-spectrum anti-infective drugs or drugs that interfere with unlimited proliferation of cancer cells. The fact that the genome of E. multilocularis has recently been sequenced has opened other avenues, such as the selection of novel drugs that interfere with the parasite signalling machinery, and the application of in silico approaches by employing the Echinococcus genome information to search for suitable targets for compounds of known mode of action.

In vitro and in vivo activities of dicationic diguanidino compounds against Echinococcus multilocularis metacestodes.

Alveolar echinococcosis (AE) is a disease predominantly affecting the liver, with metacestodes (larvae) of the tapeworm Echinococcus multilocularis proliferating and exhibiting tumor-like infiltrative growth. For many years, chemotherapeutical treatment against alveolar echinococcosis has relied on the benzimidazoles albendazole and mebendazole, which require long treatment durations and exhibit parasitostatic rather than parasiticidal efficacy. Although benzimidazoles have been and still are beneficial for the patients, there is clearly a demand for alternative and more efficient treatment options. Aromatic dications, more precisely a small panel of di-N-aryl-diguanidino compounds, were screened for efficacy against E. multilocularis metacestodes in vitro. Only those with a thiophene core group were active against metacestodes, while furans were not. The most active compound, DB1127, was further investigated in terms of in vivo efficacy in mice experimentally infected with E. multilocularis metacestodes. This diguanidino compound was effective against AE when administered intraperitoneally but not when applied orally. Thus, thiophene-diguanidino derivatives with improved bioavailability when administered orally could lead to treatment options against AE.

Transcriptional profiles of cytokine/chemokine factors of immune cell-homing to the parasitic lesions: a comprehensive one-year course study in the liver of E. multilocularis-infected mice.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.