Investigation of wild-type and mycolactone-negative mutant Mycobacterium ulcerans on skeletal muscle: IGF-1 protects against mycolactone-induced muscle catabolism.

Buruli ulcer (BU), which is caused by Mycobacterium ulcerans (MU), is an endemic and neglected tropical disease that affects mostly subcutaneous tissues. Skeletal muscle under infected skin is also subject to serious dysfunctions and contractures. The goal of this study was to investigate the effects of an infection with the wild-type M. ulcerans (WT-MU) or the mycolactone-negative Mycobacterium ulcerans (M(neg)-MU) mutant strains on myotubes or fully differentiated skeletal muscles. WT-MU infection decreased by 22% and 29% the maximal muscle force at days 7 and 42 postinfection, respectively, while M(neg)-MU induced no decrease at day 7 postinfection and a small but significant 13% decrease in muscle force at day 42. A 13.2-fold and 4.3-fold increase in neutrophil and macrophage concentrations, respectively, was observed on day 42 following the injection of WT-MU. However, the increases in neutrophil and macrophage concentrations were 2.4-fold and 5.5-fold in M(neg)-MU. Myoblast proliferation decreased by 20%, myotube diameter by 45%, MyHC levels by 32%, while MuRF-1 levels increased by 22.8% when C2C12 cells and WT-MU were cocultured for 48 h at a multiplicity of infection of 5:1. In contrast, M(neg)-MU had no significant effect. Interestingly, the addition of 1,000 ng/ml of IGF-1 to the WT-MU/C2C12 coculture significantly improved all of these biological parameters. The present investigation clearly established that muscle dysfunction and chronic inflammation in the presence of WT-MU are largely caused by the release of mycolactone, and the addition of recombinant IGF-1 was sufficient to alleviate some of the antiproliferative and atrophic effects of mycolactone.

Noncovalent DNA binding drives DNA alkylation by leinamycin: evidence that the Z,E-5-(thiazol-4-yl)-penta-2,4-dienone moiety of the natural product serves as an atypical DNA intercalator.

Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product leinamycin. Previous studies suggested that alkylation of duplex DNA by activated leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because leinamycin does not contain a classical noncovalent DNA-binding motif, such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by leinamycin provided evidence that activated leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 10(3) M(-1). Activated leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated leinamycin with palindromic duplexes containing 5'-CG and 5'-GC target sites, bulge-containing duplexes, and 5-methylcytosine-containing duplexes provided evidence regarding the orientation of leinamycin with respect to target guanine residues. The data allow construction of a model for the leinamycin-DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA.

ABC transporters influence sensitivity of Brugia malayi to moxidectin and have potential roles in drug resistance.

Some ABC transporters play a significant role in human health and illness because they confer multidrug resistance (MDR) through their overexpression. Compounds that inhibit the drug efflux mechanism can improve efficacy or reverse resistance. Of the eight described ABC transporter subfamilies, those proteins conferring MDR in humans are in subfamilies A, B, C, and G. In nematodes, transporters in subfamilies B and C are suggested to confer resistance to ivermectin. The Brugia malayi ABC transporter superfamily was examined to assess their potential to influence sensitivity to moxidectin. There was an increase in expression of ABC transporters in subfamilies A, B, C, and G following treatment. Co-administration of moxidectin with inhibitors of ABC transporter function did not enhance sensitivity to moxidectin in males; however, sensitivity was significantly enhanced in females and microfilariae. The work suggests that ABC transporters influence sensitivity to moxidectin and have a potential role in drug resistance.

Polyketides from the ascomycete fungus Leptosphaeria sp.

The new polyketides leptosphaerins A-G (1-7) and the known compounds monodictysin B (8), 2-(2,6-dihydroxybenzoyl)-3-hydroxy-5-methylbenzoic acid (9), and 2-(2,6-dihydroxy-4-methylbenzoyl)-6-hydroxybenzoic acid (10) have been isolated from solid cultures of the ascomycete fungus Leptosphaeria sp. The absolute configurations of compounds 1, 2, and 6 were assigned using the modified Mosher method, whereas that of C-2 in 3 was determined via the circular dichroism data of the [Rh(2)(OCOCF(3))(4)] complex. Compound 7 showed antifungal activity against Aspergillus flavus.

Vacuolar H+-ATPase inhibitors overcome Bcl-xL-mediated chemoresistance through restoration of a caspase-independent apoptotic pathway.

The anti-apoptotic oncoproteins Bcl-2 and Bcl-xL play crucial roles in tumorigenesis and chemoresistance, and are thus therapeutic cancer targets. We searched for small molecules that disturbed the anti-apoptotic function of Bcl-2 or Bcl-xL, and found vacuolar H(+)-ATPase (V-ATPase) inhibitors, such as bafilomycin A1 (BMA), that showed such activity. Bcl-xL-overexpressing Ms-1 cells displayed resistance to anticancer drugs, but underwent apoptosis following treatment with a combination of V-ATPase inhibitors at doses similar to those that caused inhibitory activities of V-ATPase. We investigated the apoptosis mechanism induced by cotreatment of Bcl-xL-overexpressing Ms-1 cells with BMA as a V-ATPase inhibitor and taxol (TXL) as an anticancer drug. With BMA, TXL triggered mitochondrial membrane potential loss and cytochrome c release, whereas downstream caspase activation was not observed. In contrast, pronounced nuclear translocation of mitochondrial apoptosis-inducing factor and endonuclease G, known as effectors of caspase-independent apoptosis, was observed with BMA and TXL cotreatment. Moreover, depletion of apoptosis-inducing factor and endonuclease G using each siRNA significantly rescued cells from BMA- and TXL-induced apoptosis. Hence, the apoptosis-inducing factor- and endonuclease G-dependent pathway was critical for apoptosis induction by BMA and TXL cotreatment. Our data suggest that V-ATPase inhibitors could not only suppress anti-apoptotic Bcl-2 nor Bcl-xL but could also facilitate the caspase-independent apoptotic pathway. V-ATPase inhibition will be a promising therapeutic approach for Bcl-2- or Bcl-xL-overexpressing malignancies.

Enhanced CML stem cell elimination in vitro by bryostatin priming with imatinib mesylate.

OBJECTIVE: In chronic myeloid leukemia (CML), imatinib mesylate (IM; Gleevec, Glivec) induces a G0/G1 cell-cycle block in total CD34(+) cells without causing significant apoptosis. Bryostatin-1 (bryo), a protein kinase C (PKC) modulator, was investigated for its ability to increase IM-mediated apoptosis either through induction of cycling of G0/G1 Ph(+) cells or antagonism of the IM-induced cell-cycle block. METHODS: The Ph(+) K562 cell line and primary CD34(+) CML cells were studied for cell-cycle progression (PI staining), proliferation ((3)H thymidine uptake), and survival (dye exclusion). RESULTS: Following 48 hours exposure to IM, on average more than 80% of surviving K562 cells were in G0/G1 as compared to approximately 50% for untreated control cultures (p < 0.001). After accounting for IM-induced cell kill, the absolute number of viable G0/G1 cells was significantly increased, confirming its anti-proliferative effect. However, pretreatment for 24 hours with bryo both increased K562 total cell kill and normalized the percentage of cells recovered in G0/G1, thus reducing their absolute number. For primary CML CD34(+) cells, pretreatment with bryo prior to IM significantly enhanced cell death of both total and, critically, G0/G1 populations. CONCLUSION: These results suggest that carefully scheduled drug combinations that include an agent to antagonize the anti-proliferative effect of IM may prove more efficacious within the Ph(+) stem cell compartment than IM monotherapy.

A triazole derivative elicits autophagic clearance of polyglutamine aggregation in neuronal cells.

Trinucleotide CAG repeat expansion in the coding region of genes has a propensity to form polyglutamine (polyQ) aggregates that contribute to neuronal disorders. Strategies in elevating autophagy to disintegrate the insoluble aggregates without injuring cells have become a major goal for therapy. In this work, a triazole derivative, OC-13, was found accelerating autophagic clearance of polyQ aggregation in human neuroblastoma cells following induction of the enhanced green fluorescence-conjugated chimeric protein that enclosed 79 polyQ repeats (Q79-EGFP). OC-13 accelerated autophagy development and removed nuclear Q79-EGFP aggregates. The increase of Beclin-1, turnover of LC3-I to LC3-II and degradation of p62 supported autophagy activation. Pretreatment of autophagy inhibitor, bafilomycin A1, not only suppressed autophagolysome fusion, but also impeded aggregate eradication. The study also showed that c-Jun N-terminal kinase/Beclin-1 pathway was activated during OC-13 treatment and c-Jun N-terminal kinase inhibitor impaired autophagy and final breakdown. Autophagic clearance of the insoluble aggregates demonstrated the feasibility of OC-13 in alleviating neuronal disorders because of expanded glutamine stretches.

Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

In vitro antagonism between beta-lactam and macrolide in Streptococcus pneumoniae: how important is the antibiotic order?

We found that the in vitro interaction between penicillin or cefotaxime and erythromycin against Streptococcus pneumoniae varies depending on the order of antibiotic exposure. Time-kill experiments were performed with penicillin, cefotaxime, erythromycin and different order combinations of both beta-lactams with erythromycin. The mean difference between the colony count at 0 and 6h for penicillin, cefotaxime and erythromycin tested separately was 3.5 log cfu/mL, 2.4 and 1.5 respectively for susceptible strains. The mean difference for the combination of beta-lactam and erythromycin studied simultaneously was 1.8 log cfu/mL for these strains. The association of penicillin or cefotaxime with erythromycin added two hours later showed an activity similar to those of beta-lactam alone (mean difference was 3.0 for this association with penicillin and 2.5 with cefotaxime). Therefore, the antagonistic effect of macrolide activity could be less important if erythromycin was administrated after beta-lactam.

Biofilm formation by Rhodococcus equi and putative association with macrolide resistance / Formação de biofilme por Rhodococcus equi e possível associação com resistência a macrolídeos

Rhodococcus equi is a facultative intracellular pathogen, which cause severe pyogranulomatous pneumonia in foals and tuberculosis-like lesions in humans. Its ability to form biofilm was described in strains isolated from chronic diseases associated to treatment failures in humans. This study aimed to verify the biofilm formation by 113 R. equi isolated from equine samples (clinical and fecal) using two different methods (biofilm-culturing with and without additional glucose and epifluorescence microscopy). We also aimed to determine the efficacy of azithromycin, clarithromycin and erythromycin on R. equi in established biofilm. We found 80.5% (26/41) and 63% (58/72) biofilm-positive isolates, in fecal and clinical samples, respectively. The additional glucose increased the biofilm formation by R. equi fecal samples, but not by clinical samples. The antimicrobials tested herein were not able to eradicate R. equi in biofilm even at higher concentrations. This is the first study showing the biofilm formation by R. equi isolated from equine samples. Our findings indicate that R. equi biofilm-producers may be more resistant to the antimicrobials evaluated. Further studies are warranted to test this hypothesis...

Rhodococcus equi é um patógeno intracelular facultativo, o qual causa pneumonia piogranulosa severa em potros e lesões semelhantes à tuberculose em humanos. A sua capacidade de formar biofilme foi descrita em cepas humanas, isoladas a partir de doenças crônicas associadas a falhas de tratamento. Este estudo teve como objetivo verificar a formação de biofilme por 113 cepas de R. equi, isoladas a partir de amostras de equinos (clínicas e fecais), utilizando-se dois diferentes métodos (biofilme em cultura - com e sem adição de glicose - e microscopia de epifluorescência). Além disso, buscou-se determinar a eficácia da azitromicina, claritromicina e eritromicina sobre biofilme consolidado de R. equi. Verificou-se 80,5% (26/41) e 63% dos isolados (58/72) positivos para formação de biofilme, em amostras fecais e clínicas, respectivamente. A adição de glicose amentou a formação de biofilme em amostras fecais, mas não em amostras clínicas. Os antimicrobianos aqui testados não foram capazes de erradicar R. equi em biofilme consolidado, mesmo em concentrações elevadas. Este é o primeiro estudo a demonstrar a formação de biofilme por cepas de R. equi isoladas a partir de amostras de equinos. Os resultados indicam que os isolados de R. equi produtores de biofilme podem ser mais resistentes aos antimicrobianos avaliados. Estudos adicionais são necessários para testar essa hipótese...

In vitro effects of VD-99-11 on Angiostrongylus cantonensis and isolated frog rectus.

In vitro effects of VD-99-11 were examined using adult Angiostrongylus cantonensis and isolated frog rectus. In A. cantonensis, paralysis was elicited by VD-99-11 at 10(-9)-10(-6) g/ml. The paralysis caused by VD-99-11 (10(-8) g/ml) was antagonized by picrotoxin or bicuculline but not by phentolamine. A relationship between VD-99-11 and gabergic antagonists was observed in worm preparations contracted by eserine or pyrantel: VD-99-11 at higher concentrations (3x10(-6) g/ml) caused a marked contraction. In worm preparations contracted with eserine or pyrantel, the only additional contraction induced by VD-99-11 (5x10(-6) g/ml) was antagonized by strychnine. In experiments on the guanidine (2.5x10(-3) M)-induced twitch responses in isolated frog rectus, marked stimulation was caused by VD-99-11 (3-5x10(-6) g/ml). The stimulated responses induced by VD-99-11 were antagonized by tetrodotoxin, D-tubocurarine, strychnine, and hemicholinium-3, respectively. These results suggest that VD-99-11 seems superior to milbemycin D, milbemycin oxime, and ivermectin in some aspects, such as in vitro potency, though this new substance is similar to these drugs in having two different actions on the gabergic mechanism at lower concentrations and on the cholinergic mechanism at higher concentrations.