Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity

Imipramine Is an Orally Active Drug against Both Antimony Sensitive and Resistant Leishmania donovani Clinical Isolates in Experimental Infection

Background: In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported. Methodology/Principal Findings: Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug wasfound to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD) promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF a, IFN c and iNOS activity increased with the concomitant decrease in IL 10 and TGF b level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity. Conclusions: This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar

Evaluation of apical deviation in root canals instrumented with K3 and ProTaper systems Avaliação do desvio apical em canais instrumentados pelos sistemas K3 e ProTaper

OBJECTIVES: this study evaluated the apical deviation of curved root canals instrumented with K3 and ProTaper systems. MATERIAL AND METHODS: twenty root canals of human maxillary and mandibular first molars were employed, which were divided into 2 groups: group A (10 teeth) was instrumented with the K3 system, and group B (10 teeth) with the ProTaper system. Evaluation of deviation was performed by double radiographic exposure. Radiographs were achieved before and after instrumentation, with 0.3-second, thus allowing superimposition of images. Three-dimensional computerized tomograph was performed in 3 specimens in each group, as an additional means to evaluate the apical deviation. RESULTS: were evaluated by the parametric test Student-Newman-Keuls at 5%, which did not reveal significance between groups concerning the apical deviation. The results of computerized tomograph images demonstrated that the larger deviation of the root canal occurred at the distolingual area for both systems. CONCLUSIONS: both techniques produced a mild apical deviation. Computerized microtomography was shown to be accurate for experimental endodontics studies.<br>OBJETIVOS: avaliar o desvio apical de canais radiculares curvos instrumentados pelos sistemas K3 e ProTaper. MATERIAIS E MÉTODOS: foram utilizados 20 canais radiculares de primeiros molares superiores e inferiores humanos, os quais foram divididos em 2 grupos, o grupo A (10 dentes) foi instrumentado pelo sistema K3 e o grupo B (10 dentes) com o sistema ProTaper, a forma de avaliação do desvio foi a dupla exposição radiográfica. As tomadas radiográficas foram feitas antes e após a instrumentação, com um tempo de 0,3 segundos em cada exposição, ocorrendo assim, sobreposição das imagens. Em 3 espécimes, de cada grupo, foi realizada a tomografia computadorizada tridimensional, como forma adicional de avaliar o desvio apical . Os resultados obtidos foram avaliados pelo teste paramétrico Student-Newman-Keuls 5% o qual denotou não haver significância em relação ao desvio apical entre os grupos estudados. Os resultados das imagens de tomografia computadorizada mostraram que o maior desvio do canal radicular ocorreu na área disto-lingual, para ambos os sistemas estudados. CONCLUSÃO: ambas as técnicas promoveram desvio apical. A tomografia computadorizada revelou ser um método de análise promissor em estudos in vitro na área de endodontia

Estrogen receptor beta activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma