Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

In vivo co-ordinated interactions between inhibitory systems to control glutamate mediated hippocampal excitability.

We present an overview of the long-term adaptation of hippocampal neurotransmission to cholinergic and GABAergic deafferentation caused by excitotoxic lesion of the medial septum. Two months after septal microinjection of 2.7 nmol a -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), a 220% increase of GABA A receptor labelling in the hippo- campal CA3 and the hilus was shown, and also changes in hippocampal neurotransmission characterised by in vivo microdialysis and HPLC. Basal amino acid and purine extra- cellular levels were studied in control and lesioned rats. In vivo effects of 100 m M KCl perfusion and adenosine A 1 receptor blockade with 1,3-dipropyl- 8-cyclopentylxanthine (DPCPX) on their release were also investigated. In lesioned animals GABA, glutamate and glutamine basal levels were decreased and taurine, adenosine and uric acid levels increased. A similar response to KCl infusion occurred in both groups except for GABA and glutamate, which release decreased in lesioned rats. Only in lesioned rats, DPCPX increased GABA basal level and KCl-induced glutamate release, and decreased glutamate turnover. Our results evidence that an excitotoxic septal lesion leads to increased hippocampal GABA A receptors and decreased glutamate neurotransmis- sion. In this situation, a co-ordinated response of hippocampal retaliatory systems takes place to control neuron excitability.

Multigram synthesis and in vivo efficacy studies of a novel multitarget anti-Alzheimer's compound

We describe the multigram synthesis and in vivo efficacy studies of a donepezil‒huprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer's disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aβ42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aβ42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aβ peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aβ lowering effect in vivo might be related to its lower in vitro potency toward Aβ aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

Effect of metal ions on the in vitro availability of enoxacin, its in vivo implications, kinetic and antibacterial studies

The present paper describes the effect of metals ions on the in vitro availability of enoxacin (a second generation quinolone antibiotic) owing to drug-metal interaction. These interaction studies were performed at 37 °C in different pH environments simulating human body compartments and were studied by UV spectroscopic technique. In order to determine the probability of these reactions different kinetic parameters (dissolution constants (K) and free energy change (ΔG)) for these reactions were also calculated. It is proposed that the structure of enoxacin contains various electron donating sites which facilitate its binding with metallic cations forming chelates. Hence taking food products, nutritional supplements or multivitamins containing multivalent cations at the same time as enoxacin, could reduce the absorption of the drug into the circulation and thus would decrease the effectiveness of the drug. In addition, the MIC of enoxacin for various microorganisms before and after interaction with metal ions was calculated which in most cases was increased which possibly could impair the clinical efficacy of the drug.

Efeito de solventes orgânicos usados como veículos de fungicidas no controle in vitro e in vivo da incidência e da transmissão de Bipolaris sorokiniana em sementes de cevada

O objetivo deste trabalho foi comparar e avaliar, in vitro e in vivo, o efeito de solventes orgânicos utilizados como veículos de fungicidas na erradicação do fungo em sementes de cevada. Foram empregados o monoetilenoglicol (MEG) e o propilenoglicol (PPG), ambos a 0,5, 1 e 2%. Os fungicidas testados foram a iminoctadina, a iprodiona, o triadimenol, o triticonazol, o flutriafol e o difenoconazol. A incidência foi determinada pela ocorrência de B. sorokiniana em sementes plaquadas em meio seletivo. A erradicação foi obtida nos tratamentos com iminoctadina + MEG; iminoctadina + PPG (a 1 e 2%) e iprodiona + PPG (2%). A eficiência dos demais fungicidas foi melhorada com o emprego dos solventes orgânicos, mas sem alcançar a erradicação do fungo. In vivo na testemunha foram registrados níveis de transmissão de 89,7% para o coleóptilo e de 12,3% para a plúmula. Diferentemente dos dados obtidos in vitro, o emprego do PPG influenciou muito pouco no controle da transmissão, pois os fungicidas comportaram-se satisfatoriamente quando misturados com água. Os fungicidas iminoctadina e difenoconazol foram 100% efetivos em evitar a transmissão do fungo das sementes para os coleóptilos. Os solventes orgânicos mostraram potencialidade para melhorar a eficiência da maioria dos fungicidas testados in vitro, aspecto que não foi corroborado in vivo.

Controle de Alternaria solani em tomateiro por extratos de Curcuma longa e curcumina: II. Avaliação in vivo

A pinta preta, causada por Alternaria solani, é uma das mais importantes doenças da cultura do tomateiro no Brasil. Várias alternativas aos fungicidas têm sido avaliadas nos últimos anos na busca de produtos que controlem satisfatoriamente as doenças, tenham pequeno impacto ambiental e baixa toxicidade aos seres vivos. A cúrcuma, Curcuma longa, apresenta em seus rizomas compostos com comprovada atividade antimicrobiana. Dessa forma, o objetivo deste trabalho foi avaliar o controle de pinta preta em tomateiro utilizando extratos de cúrcuma e curcumina em condições de casa de vegetação. Os tratamentos utilizados foram: extrato de cúrcuma (1 e 10%), curcumina (50 e 100 mg/L), acibenzolar-S-metil (ASM) (25 mg do i.a./L), oxicloreto de cobre (1.100 mg do i.a./L), azoxystrobin (80 mg do i.a./L) e testemunha (água). A curcumina e os extratos brutos de cúrcuma apresentaram níveis de controle de pinta preta similares ao tratamento com fungicida cúprico, mas inferior ao azoxystrobin. Não houve diferenças estatísticas na produção comercial de tomate entre tratamentos. Somente o tratamento de curcumina 50 mg/l apresentou maior porcentagem de frutos grandes em relação à testemunha. Esses resultados indicam o potencial de controle de pinta preta em tomateiro com cúrcuma e curcumina.

Avaliação de produtos químicos comerciais, in vitro e in vivo, no controle da doença foliar, mancha branca do milho, causada por Pantoea ananatis

Uma bactéria identificada como Pantoea ananatis foi recentemente isolada de lesões jovens da doença mancha branca do milho de plantas naturalmente infectadas. Esta bateria reproduziu sintomas semelhantes aos da doença quando inoculada em plantas de milho em casa de vegetação. Estudos anteriores realizados por outros autores demonstraram que o controle desta doença em condições de campo foi obtido pelo uso de fungicidas, principalmente o Mancozeb, nas fases iniciais de seu desenvolvimento. O objetivo deste estudo foi avaliar a freqüência de isolamento da bactéria P. ananatis a partir de plantas infectadas coletadas na região de Londrina, Estado do Paraná, e reproduzir sintomas da doença através de inoculações artificiais em plantas de milho em casa de vegetação. Utilizando os produtos químicos testados anteriormente por outros autores para o controle desta doença a campo, foi também objetivo deste trabalho avaliar o potencial destes produtos na inibição da bactéria tanto em condições de laboratório como em condições de infecção natural. Os resultados mostraram que P. ananatis foi isolada em 40% das lesões jovens coletadas a campo e quando inoculada em casa de vegetação sob condições controladas reproduziu sintomas semelhantes aos observados a campo. Entre os produtos químicos testados, o fungicida Mancozeb mostrou-se eficiente no controle da doença a campo, em concordância com os relatos anteriores. Este produto inibiu completamente o crescimento da bactéria em laboratório, explicando os resultados obtidos a campo. Os demais produtos não foram eficientes no controle a campo e eles também não inibiram a bactéria em laboratório. Estes resultados representam evidências adicionais de que a bactéria P. ananatis é o agente causal da doença mancha branca do milho.