Carboxyamidotriazole-orotate inhibits the growth of imatinib-resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML are often resistant. New therapeutic options are therefore needed for this fatal disease. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in hematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities. CTO, which has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound, was tested in this study for its ability to affect imatinib-resistant CML tumor growth in a xenograft model. The active cross talk between endothelial cells and leukemic cells in the bone marrow involving exosomes plays an important role in modulating the process of neovascularization in CML. We have thus investigated the effects of CTO on exosome-stimulated angiogenesis. Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients.     

Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies

Muscular dystrophies (MDs) are a heterogeneous group of inherited disorders characterized by progressive muscle wasting and weakness likely associated with exhaustion of muscle regeneration potential. At present, no cures or efficacious treatments are available for these diseases, but cell transplantation could be a potential therapeutic strategy. Transplantation of myoblasts using satellite cells or other myogenic cell populations has been attempted to promote muscle regeneration, based on the hypothesis that the donor cells repopulate the muscle and contribute to its regeneration. Embryonic stem cells (ESCs) and more recently induced pluripotent stem cells (iPSCs) could generate an unlimited source of differentiated cell types, including myogenic cells. Here we review the literature regarding the generation of myogenic cells considering the main techniques employed to date to elicit efficient differentiation of human and murine ESCs or iPSCs into skeletal muscle. We also critically analyse the possibility of using these cellular populations as an alternative source of myogenic cells for cell therapy of MDs.     

Recognition pliability is coupled to structural heterogeneity: a calmodulin intrinsically disordered binding region complex

Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP(145-165)) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP(145-165)-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.     

Exploitation of the semi-homothallic life cycle of Saccharomyces cerevisiae for the development of breeding strategies

A strain of Saccharomyces cerevisiae having desirable winemaking properties and high spore viability was bred from a semi-homothallic parent strain with similar winemaking properties but that produced sixfold fewer viable spores. Because the parent was homozygous for HO and for the MATa allele at both silent HMR and HML loci, it produced two MATa and two nonmating progeny per ascus. To obtain a segregant able to mate with the stable MATa progeny, a strain of the nonmating progeny, previously subjected to HO distruption with a KanMX4 cassette, was used. The resultant MATalphaho::KanMX4 transformant was mated to a MATa HO segregant and the diploid produced was sporulated to allow the isolation of a semi-homothallic diploid segregant designated 2D that lacked the KanMX4-disrupted HO allele as confirmed by sequence analysis. Genetic analysis indicated greater homozygosity in 2D than in the parent as assessed by PCR at five loci. The sugar consumption profiles of both 2D and the parent in grape juice fermentations were the same. Acetaldehyde levels and postfermentation biofilm formation were higher in 2D than in the parent. Because 2D has acceptable winemaking characteristics but produces significantly more viable spores than the parent strain, it will be useful in future breeding efforts.     

Fine scale dispersal in Banggai Cardinalfish, Pterapogon kauderni, a coral reef species lacking a pelagic larval phase

Dispersal in marine species results from complex interactions between biotic and abiotic factors. Importantly, the pelagic larval phase of most marine species adds a significant degree of complexity. Therefore, a growing body of work is focusing on those rare species that lack a pelagic larval phase (usually brooding species). For such species, large-scale gene flow has been shown to be very low, thus following the expectation of a relationship between realized dispersal and pelagic larval duration. Yet, little is known about the dispersal of those species at very small geographic scales. In this study, we focused on the Banggai Cardinalfish, Pterapogon kauderni, a mouthbrooding species that lacks a pelagic larval phase. Based on previously identified microsatellites, we scored 12 populations around the southern island of Bangkulu, in the Banggai Archipelago, Indonesia. While only 60 km in perimeter, we found that this island harbors very distinct populations of P. kauderni. Indeed, assignment tests self-assigned 10 out of those 12 populations. These results mirror the very high level of self-assignment at the level of the entire archipelago, where, out of 13 populations, 70% of the individuals were reassigned to their source population. Therefore, our data show consistency between small and large-scale dispersal. In addition, in light of the recent expansion in the harvesting of this species for the pet trade, our data have important conservation implications.     

Degradation and Transformability of DNA from Transgenic Leaves

The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.     

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg^-1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg^-1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034).

Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.