Detection of lipid peroxidation in dog spermatozoa with the fluorescent dye C11-BODIPY581/591.

C11-BODIPY581/591 is a fluorescent probe that has been successfully used to evaluate lipid peroxidation in different species, but it has not been completely studied in the dog. Thus, the aim of the present study was to assess lipid peroxidation of dog spermatozoa using C11-BODIPY581/591 and compare different positive controls of the technique. Twenty-four ejaculates were collected from 8 adult male dogs. Routine seminal characteristics were evaluated in raw semen. Lipid peroxidation evaluation was performed as described in other species. Samples were divided in three aliquots, exposed to UV radiation, incubated with hydrogen peroxide or left without treatment (control). Lipid peroxidation was significantly greater only in UV-exposed samples than in the control ones (91 ± 6% vs. 8.3 ± 3.5%, p Ë‚ .01). In conclusion, C11-BODIPY581/591 is useful to evaluate lipid peroxidation of dog spermatozoa and UV radiation is a good promoter of membrane oxidation, so irradiated samples can be used as a positive control of this technique.

Electroporation optimizes the uptake of boron-10 by tumor for boron neutron capture therapy (BNCT) mediated by GB-10: a boron biodistribution study in the hamster cheek pouch oral cancer model.

Boron neutron capture therapy (BNCT) is a promising cancer binary therapy modality that utilizes the nuclear capture reaction of thermal neutrons by boron-10 resulting in a localized release of high- and low-linear energy transfer (LET) radiation. Electrochemotherapy (ECT) is based on electroporation (EP) that induces opening of pores in cell membranes, allowing the entry of compounds. Because EP is applied locally to a tumor, the compound is incorporated preferentially by tumor cells. Based on the knowledge that the therapeutic success of BNCT depends centrally on the boron content in tumor and normal tissues and that EP has proven to be an excellent facilitator of tumor biodistribution of an anti-tumor agent, the aim of this study was to evaluate if EP can optimize the delivery of boronated compounds. We performed biodistribution studies and qualitative microdistribution analyses of boron employing the boron compound sodium decahydrodecaborate (GB-10) + EP in the hamster cheek pouch oral cancer model. Syrian hamsters with chemically induced exophytic squamous cell carcinomas were used. A typical EP treatment was applied to each tumor, varying the moment of application with respect to the administration of GB-10 (early or late). The results of this study showed a significant increase in the absolute and relative tumor boron concentration and optimization of the qualitative microdistribution of boron by the use of early EP + GB-10 versus GB-10 without EP. This strategy could be a tool to improve the therapeutic efficacy of BNCT/GB-10 in vivo.

Deletion of liaR Reverses Daptomycin Resistance in Enterococcus faecium Independent of the Genetic Background.

We have shown previously that changes in LiaFSR, a three-component regulatory system predicted to orchestrate the cell membrane stress response, are important mediators of daptomycin (DAP) resistance in enterococci. Indeed, deletion of the gene encoding the response regulator LiaR in a clinical strain of Enterococcus faecalis reversed DAP resistance (DAP-R) and produced a strain hypersusceptible to antimicrobial peptides. Since LiaFSR is conserved in Enterococcus faecium, we investigated the role of LiaR in a variety of clinical E. faecium strains representing the most common DAP-R genetic backgrounds. Deletion of liaR in DAP-R E. faecium R446F (DAP MIC of 16 µg/ml) and R497F (MIC of 24 µg/ml; harboring changes in LiaRS) strains fully reversed resistance (DAP MICs decreasing to 0.25 and 0.094 µg/ml, respectively). Moreover, DAP at concentrations of 13 µg/ml (achieved with human doses of 12 mg/kg body weight) retained bactericidal activity against the mutants. Furthermore, the liaR deletion derivatives of these two DAP-R strains exhibited increased binding of boron-dipyrromethene difluoride (BODIPY)-daptomycin, suggesting that high-level DAP-R mediated by LiaR in E. faecium involves repulsion of the calcium-DAP complex from the cell surface. In DAP-tolerant strains HOU503F and HOU515F (DAP MICs within the susceptible range but bacteria not killed by DAP concentrations of 5× the MIC), deletion of liaR not only markedly decreased the DAP MICs (0.064 and 0.047 µg/ml, respectively) but also restored the bactericidal activity of DAP at concentrations as low as 4 µg/ml (achieved with human doses of 4 mg/kg). Our results suggest that LiaR plays a relevant role in the enterococcal cell membrane adaptive response to antimicrobial peptides independent of the genetic background and emerges as an attractive target to restore the activity of DAP against multidrug-resistant strains.

Assessment of biological effectiveness of boron neutron capture therapy in primary and metastatic melanoma cell lines.

PURPOSE: In order to optimize the effectiveness of Boron Neutron Capture Therapy (BNCT), Relative Biological Effectiveness (RBE) and Compound Biological Effectiveness (CBE) were determined in two human melanoma cell lines, M8 and Mel-J cells, using the amino acid p-boronophenylalanine (BPA) as boron carrier. MATERIALS AND METHODS: The effects of BNCT on the primary amelanotic cell line M8 and on the metastatic pigmented melanoma cell line Mel-J were studied using colony formation assay. The RBE values were determined using both a gamma ray source, and the neutron beam from the Nuclear Reactor of the National Atomic Energy Commission (RA-3). For the determination of the RBE, cells were irradiated with increasing doses of both sources, between 1 and 8 Gy; and for the determination of CBE factors, the cells were pre-incubated with BPA before irradiation. Afterwards, the cell surviving fraction (SF) was determined for each treatment. RESULTS: Marked differences were observed between both cell lines. Mel-J cells were more radioresistant than the M8 cell line. The clonogenic assays showed that for a SF of 1%, the RBE values were 1.3 for M8 cells and 1.5 for Mel-J cells. Similarly, the CBE values for a 1% SF were 2.1 for M8 and 3 for Mel-J cell lines. For the endpoint of 0.1% of SF the RBE values obtained were 1.2 for M8 and 1.4 for Mel-J cells. Finally, CBE values calculated for a 0.1% were 2 and 2.6 for M8 and Mel-J cell lines respectively. In order to estimate the uptake of the non-radioactive isotope Boron 10 ((10)B), a neutron induced autoradiographic technique was performed showing discrepancies in (10)B uptake between both cell lines. CONCLUSIONS: These obtained in vitro results are the first effectiveness factors determined for human melanoma at the RA-3 nuclear reactor and show that BNCT dosimetry planning for patients could be successfully performed using these new factors.

Isolation and characterization of indigenous Streptomyces and Lentzea strains from soils containing boron compounds in Argentina.

The Salta Province - in the northwest of Argentina - is the main worldwide producer of hydroboracite and leads in exports of boron mineral and its derivatives in Latin America. In addition to the natural presence of boron compounds in the soils, there are others contaminated due to the boron mining industry. Although some bacteria are known to require boron for their growth or to be capable of storing boron, no studies have been published about Streptomyces or Lentzea genera's capacity to tolerate high boron concentrations, or about their metabolic capacities in boron contaminated environments. The results of this research show the isolation and molecular characterization of eight strains belonging to the actinobacteria phylum collected from different soils contaminated with high boron concentration in Salta state. The boron tolerance assays, which show that three of the strains were able to tolerate up 60-80 mM boron, demonstrate the potential capability of this group of bacteria to grow and maybe to remove boron from the environment. They appear to be promising, considering that these microorganisms are infrequent pathogens, are metabolically versatile and many Streptomyces can synthesize boron containing metabolites.

Studies for the application of boron neutron capture therapy to the treatment of differentiated thyroid cancer.

The aim of these studies was to evaluate the possibility of treating differentiated thyroid cancer by BNCT. These carcinomas are well controlled with surgery followed by therapy with (131)I; however, some patients do not respond to this treatment. BPA uptake was analyzed both in vitro and in nude mice implanted with cell lines of differentiated thyroid carcinoma. The boron intracellular concentration in the different cell lines and the biodistribution studies showed the selectivity of the BPA uptake by this kind of tumor.

Tolerance to wood preservatives by copper-tolerant wood-rot fungi native to south-central Chile.

Understanding the effect of heavy metals and wood preservatives on the growth of wood-rot fungi native to a certain region may improve reliability in determining the effectiveness of antifungal products, particularly when dealing with new formulations. In this investigation, strains of copper-tolerant wood-rot fungi native to south-central Chile were evaluated against two preservatives: commercial chromated copper arsenate type C (CCA-C) and a new formulation with boron and silicon (BS). Thirteen native strains, mainly white-rot fungi, were selected for their high growth rates in solid medium containing 3 mM of copper. A short-term test was then carried out, consisting of adding cellulose disks impregnated with different concentrations of preservatives to solid culture media inoculated with selected copper tolerant strains. There was a great variability in interspecific and intraspecific responses to the presence of copper and preservatives in culture media. Among the native and commercial strains evaluated, the white-rot fungi Trametes versicolor 38 and mainly Ganoderma australe 100 were notable for their tolerance to all the CCA-C and BS concentrations. The brown-rot fungus Wolfiporia cocos, used as reference strain, showed a high tolerance to CCA-C, but not to BS preservative. T. versicolor 38 and G. australe 100 were selected for subsequent studies on preserved wood degradation.

A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca(2+) signaling.

The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis.

A novel methodology for the investigation of intracellular proteolytic processing in intact cells.

Taking advantage of the unique spectral properties of the fluorescent probe FL-Bodipy, we have developed a new methodology to study processing of exogenous proteins in intact cells. FL-Bodipy was conjugated to bovine serum albumin (BSA) at a molar ratio of 29 probe molecules to 1 albumin equivalent. The resulting conjugate was 98% self-quenched due to fluorescence resonance energy transfer (homotransfer) between neighboring Bodipy molecules. In vitro proteolytic cleavage of the conjugate led to relaxation of self-quenching and to a significant increase in fluorescence. Flow cytometry and fluorescence microscopy indicated that Bodipy-labeled BSA was readily internalized by J774 macrophages and accumulated in intracellular compartments. The kinetics of intracellular degradation of Bodipy-BSA was linear for up to 2 hours and was completely inhibited by a combination of protease inhibitors. Future applications of the methodology reported here may comprise studies of antigen processing and presentation, as well as the investigation of cellular events related to processing and disassembly of intracellular pathogens such as parasites, bacteria and viruses.