Anticonvulsant and Antioxidant Effects of Cyano-carvone and Its Action on Acetylcholinesterase Activity in Mice Hippocampus

The anticonvulsant effect of cyano-carvone, a monoterpene monocyclic, was investigated in epilepsy model induced by pilocarpine. Cyano-carvone at doses of 25, 50 or 75 mg/kg promoted a reduction of 16.7, 33 and 66.7%, respectively, against pilocarpine-induced seizures, and it was efficacious in increasing both the latency to first seizures and the survival percentage, resulting in 33.3, 67 and 91.7% of protection against death induced by seizures, respectively (P < 0.05). The reference drug atropine (25 mg/kg) also produced a significant protection (100%). Its monoterpene, at 25, 50 and 75 mg/kg, was also capable to increase the latency for installation of status epilepticus induced by pilocarpine, and presented a significant protection against lipid peroxidation and nitrite formation in mice hippocampus (P < 0.05). In addition, it was observed that the cyano-carvone pretreatment increased the acetylcholinesterase activity in mice hippocampus after pilocarpine-induced seizures. The present results clearly indicate the anticonvulsant ability of cyano-carvone, which can be, at least in part, explained by the increased activity of the acetylcholinesterase enzyme. Our data suggest that the action mechanism can also be due to a direct activation of the antioxidant enzymes that could be associated with a reduction observed in oxidative stress in mice hippocampus, probably involving an inhibition of free radical production.     

Diversity of cloacal microbial community in migratory shorebirds that use the Tagus estuary as stopover habitat and their potential to harbor and disperse pathogenic microorganisms

The diversity of the cloacal microbial community in migratory shorebirds, caught at the Tagus estuary, Portugal, was assessed by cultivation (R2A and Nutrient Agar media) and denaturing gradient gel electrophoresis profiling (DGGE) to provide a better understanding of the birds' potential to harbor and disperse pathogens. Three different bird species belonging to four different populations were studied: common redshank (Tringa totanus), black-winged stilt (Himantopus himantopus) and nominate and Icelandic populations of black-tailed godwit (Limosa limosa). DGGE profiling and partial 16S RNA gene sequences of 240 isolates, and 26 DGGE bands resulting in 58 clones, were analyzed. Most isolates were members of the phylum Firmicutes and Actinobacteria and only a small portion belonged to the Proteobacteria and Deinococcus-Thermus phyla. Potentially pathogenic strains carried by the birds were found such as Helicobacter and Staphylococcus in all bird species, and Clostridium, Mycobacterium, Rhodococcus, Legionella and Corynebacterium in black-winged stilts. Unexpectedly, bacteria from the phylum Deinococcus-Thermus were isolated in shorebirds and were present in all the bird species studied.     

Pol kappa partially rescues MMR-dependent cytotoxicity of O6-methylguanine

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.     

Label-free MS(E) proteomic analysis of chronic myeloid leukemia bone marrow plasma: disclosing new insights from therapy resistance

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic disorder that is currently considered incurable. The tyrosine kinase product of the Philadelphia chromosome, P210 BCR-ABL, provided a pathogenetic explanation for the initiation of the CML chronic phase and is the molecular therapeutic target for the disease. Imatinib mesylate, an orally available BCR-ABL kinase inhibitor, can induce haematologic and cytogenetic remission of CML. However, imatinib resistance occurs frequently, resulting in relapse. New treatment strategies are focusing on resistant CML stem cells and the bone marrow stroma. The identification of novel pathways and mechanisms in the bone marrow microenvironment could significantly contribute to the development of such strategies. In this work, we used a high-resolution label-free MS(E) proteomic approach to identify differential protein expression in the CML bone marrow plasma of responsive and resistant patients. Oxidative lipid metabolism and regulation of the switch from canonical to noncanonical WNT signaling may contribute to CML resistance in the bone marrow compartment.     

A rat model of diabetic wound infection for the evaluation of topical antimicrobial therapies

Diabetes mellitus is an epidemic multisystemic chronic disease that frequently is complicated by complex wound infections. Innovative topical antimicrobial therapy agents are potentially useful for multimodal treatment of these infections. However, an appropriately standardized in vivo model is currently not available to facilitate the screening of these emerging products and their effect on wound healing. To develop such a model, we analyzed, tested, and modified published models of wound healing. We optimized various aspects of the model, including animal species, diabetes induction method, hair removal technique, splint and dressing methods, the control of unintentional bacterial infection, sampling methods for the evaluation of bacterial burden, and aspects of the microscopic and macroscopic assessment of wound healing, all while taking into consideration animal welfare and the '3Rs' principle. We thus developed a new wound infection model in rats that is optimized for testing topical antimicrobial therapy agents. This model accurately reproduces the pathophysiology of infected diabetic wound healing and includes the current standard treatment (that is, debridement). The numerous benefits of this model include the ready availability of necessary materials, simple techniques, high reproducibility, and practicality for experiments with large sample sizes. Furthermore, given its similarities to infected-wound healing and treatment in humans, our new model can serve as a valid alternative for applied research.     

Cytotoxicity Analysis of Three Bacillus thuringiensis Subsp. israelensis δ-Endotoxins towards Insect and Mammalian Cells

Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL.     

A High-throughput Platform for the Production and Analysis of Transgenic Cassava (Manihot esculenta) Plants

A platform for high-throughput production and analysis of transgenic cassava (Manihot esculenta) has been developed for the variety 60444 and implemented to generate plants expressing traits for nutritional enhancement, modified metabolism, promoter analysis and disease resistance. Over a three and a half year period this system has been utilized to produce more than 3500 independent transgenic plant lines from 50 different genetic constructs within a single laboratory. Plants recovered through this system have proven robust and efficacious for engineered traits under greenhouse conditions and within the first confined field trials of transgenic cassava carried out in Uganda, Kenya, Nigeria and Puerto Rico. Detailed procedures are described for the operation of this platform, including all steps in tissue culture, genetic transformation, copy number estimation, greenhouse establishment for shoot and storage root formation and systems for centralized quality control, transgenic plant tracking and regulatory compliance. In addition to providing reliable transgenic plant production for proof of concept studies in the variety 60444, the systems implemented and described here form the structure for high throughput production of transgenic farmer-preferred cultivars of cassava.     

A case study: the effect of visitors on two captive pumas with respect to the time of the day

This study analyzed the effects of noise levels and number of visitors on the behaviors of a mother puma and her daughter in a zoo environment with respect to the time of day. The study monitored visitation (noise and number) over two 1-week periods (4 weeks between periods) and frequency of various puma behaviors (videorecorded). The study analyzed videotaped behavior of the pumas based on the time of day and visitors' number and noise levels. There was a direct association of puma behaviors with visitors' number and noise levels. The daughter puma was the most affected and behavioral changes of both pumas correlated more strongly with the noise level than the number of visitors. The noise level and number of visitors affected the behavior of the mother and her daughter only in the morning. The results of this study indicate that both noise level and number of visitors affected these pumas but revealed the noise as predominant. Moreover, the response of these pumas to visitors depended on the time of the day.     

Nitric oxide at the CVLM is involved in the attenuation of the reflex bradycardia in renovascular hypertensive rats

Hypertension is associated to an increase in central oxidative stress and an attenuation of the baroreflex control of arterial pressure. The present study evaluated the effect of alterations in the levels of nitric oxide (NO) and superoxide anion in the caudal ventrolateral medulla (CVLM), a key area of the brainstem for the baroreflex control of arterial pressure, in renovascular hypertensive rats (2K1C). Baseline mean arterial pressure (MAP), heart rate (HR), and reflex bradycardia were evaluated 30 days after renal artery occlusion in anesthetized (urethane, 1.2 g/kg, i.p.) 2K1C or normotensive (SHAM) rats. The MAP, HR, and baroreflex control of HR were evaluated before and after CVLM microinjections of the non-selective NOS inhibitor L-NAME (10 nmol), the NO precursor L-ARG (50 nmol), or the antioxidant ascorbic acid, Vit C (10 nmol). In both 2K1C and SHAM animals, CVLM microinjection of L-NAME produced a decrease in MAP, whereas L-ARG induced a significant increase in MAP. However, microinjection of Vit C into the CVLM produced a decrease in MAP and HR only in 2K1C and not in SHAM rats. Cardiovascular effects produced by microinjection of l-ARG into the CVLM were abolished by prior microinjection of L-NAME in the CVLM of 2K1C and SHAM rats. Microinjection of L-NAME into the CVLM increased the sensitivity of reflex bradycardia in 2K1C animals. In contrast, the CVLM microinjection of L-ARG reduced reflex bradycardia only in SHAM rats. Vit C in the CVLM did not change reflex bradycardia in either 2K1C or in SHAM rats. These results suggest that increased oxidative stress in the CVLM during hypertension contributes to the reduced baroreflex sensitivity and to maintain hypertension in the 2K1C model.