Antitumoral synergism between a copper(II) complex and cisplatin improves in vitro and in vivo anticancer activity against melanoma, lung and breast cancer cells.

BACKGROUND: Intrinsic resistance of cancer cells is a major concern for the success of chemotherapy, and this undesirable feature stimulates further research into the design of new compounds and/or alternative multiple drug chemotherapy protocols. METHODS: In this study, we investigated the antitumoral potential of the coordination compounds [Cu(HPClNOL)Cl]Cl (1), [Fe(HPClNOL)Cl2]NO3(2) and [Mn(HPClNOL)Cl2] (3). Using the human, MCF-7 and A549, and the murine melanoma, B16-F10, cell lines, we determined the cytotoxicity, DCFH oxidation, disruption of mitochondrial membrane potential (ΔΨm), Sub-G1 and TUNEL positive cells, and caspase 8 and 9 activities. Fractional inhibitory concentration (FIC) and xenograft models were also assessed to evaluate the efficacy of antitumoral potential. RESULTS: We observed that only complex 1 was cytotoxic. The treatment of cancer cells with complex 1 triggered ROS generation and promoted the disruption of ΔΨm. Complex 1 increased the number of Sub-G1 and TUNEL positive cells, and the measurement of caspase 8 and 9 activity confirmed that apoptosis was triggered by the intrinsic pathway. FIC demonstrated that the combination of complex 1 with cisplatin was additive for the A549 cells whilst it was synergic for MCF-7 and B16-F10. Treatment with complex 1, either alone or combined with cisplatin, reduced tumor growth on xenograft models. CONCLUSIONS: The present study brings new clues regarding the mechanism of action of [Cu(HPClNOL)Cl]Cl, either alone or in combination with cisplatin. GENERAL SIGNIFICANCE: These results indicate that complex 1, administered either singly or in combination with current drugs, has real potential for use in cancer therapy.

Epigenetic variation at the SLC6A4 gene promoter in mother-child pairs with major depressive disorder.

BACKGROUND: Genetic and epigenetic variations of the serotonin transporter gene (SLC6A4) have been related to the etiology of depression. The 5-HTTLPR polymorphism at the SLC6A4 promoter region has two variants, a short allele (S) and a long allele (L), in which the S allele results in lower gene transcription and has been associated with depression. The short S-allele of 5-HTTLPR polymorphism of this gene has been associated with depression. In addition to molecular mechanisms, exposure to early life risk factors such as maternal depression seems to affect the development of depression in postnatal life. The present study investigated the association of 5-HTTLPR polymorphism and CpG DNA methylation (5mC) levels of an AluJb repeat element at the SLC6A4 promoter region in mother-child pairs exposed to maternal depression. METHODS: We analyzed DNA samples from 60 subjects (30 mother-child pairs) split into three groups, with and without major depression disorder (DSM-IV) among children and mothers. The genotyping of 5-HTTLPR polymorphism and quantification of 5mC levels was performed by qualitative PCR and methylation-sensitive restriction enzyme digestion, and real-time quantitative PCR (MSRED-qPCR), respectively. RESULTS: The sample analyzed presented a higher frequency of S allele of 5-HTTLPR (67.5%). Despite the high frequency of this allele, we did not find statistically significant differences between individuals carrying at least one S allele between the depression and healthy control subjects, or among the mother-child pair groups with different patterns of occurrence of depression. In the group where the mother and child were both diagnosed with depression, we found a statistically significant decrease of the 5mC level at the SLC6A4 promoter region. LIMITATIONS: The limitations are the relatively small sample size and lack of gene expression data available for comparison with methylation data. CONCLUSION: In this study, we demonstrated a repeat element specific 5mC level reduction in mother-child pairs, concordant for the diagnosis of depression.

Impacts of selective logging on inbreeding and gene flow in two Amazonian timber species with contrasting ecological and reproductive characteristics.

Selective logging in Brazil allows for the removal of up to 90% of trees above 50 cm diameter of a given timber species, independent of a species' life history characteristics or how quickly it will recover. The genetic and demographic effects of selective logging on two Amazonian timber species (Dipteryx odorata Leguminosae, Jacaranda copaia Bignoniaceae) with contrasting ecological and reproductive characteristics were assessed in the same forest. Genetic diversity and gene flow were characterized by genotyping adults and seed sampled before and after logging, using hypervariable microsatellite markers. Overall, there were no short-term genetic impacts on the J. copaia population, with commercial application of current Brazilian forest management regulations. In contrast, for D. Odorata, selective logging showed a range of genetic impacts, with a 10% loss of alleles, and reductions in siring by pollen from trees within the 546-ha study area (23-11%) and in the number of pollen donors per progeny array (2.8-1.6), illustrating the importance of the surrounding landscape. Asynchrony in flowering between D. odorata trees led to trees with no breeding partners, which could limit the species reproduction and regeneration under current regulations. The results are summarized with other published studies from the same site and the implications for forest management discussed. The different types and levels of impacts associated with each species support the idea that ecological and genetic information by species, ecological guild or reproductive group is essential in helping to derive sustainable logging guidelines for tropical forests.

Long-term impacts of selective logging on two Amazonian tree species with contrasting ecological and reproductive characteristics: inferences from Eco-gene model simulations.

The impact of logging and subsequent recovery after logging is predicted to vary depending on specific life history traits of the logged species. The Eco-gene simulation model was used to evaluate the long-term impacts of selective logging over 300 years on two contrasting Brazilian Amazon tree species, Dipteryx odorata and Jacaranda copaia. D. odorata (Leguminosae), a slow growing climax tree, occurs at very low densities, whereas J. copaia (Bignoniaceae) is a fast growing pioneer tree that occurs at high densities. Microsatellite multilocus genotypes of the pre-logging populations were used as data inputs for the Eco-gene model and post-logging genetic data was used to verify the output from the simulations. Overall, under current Brazilian forest management regulations, there were neither short nor long-term impacts on J. copaia. By contrast, D. odorata cannot be sustainably logged under current regulations, a sustainable scenario was achieved by increasing the minimum cutting diameter at breast height from 50 to 100 cm over 30-year logging cycles. Genetic parameters were only slightly affected by selective logging, with reductions in the numbers of alleles and single genotypes. In the short term, the loss of alleles seen in J. copaia simulations was the same as in real data, whereas fewer alleles were lost in D. odorata simulations than in the field. The different impacts and periods of recovery for each species support the idea that ecological and genetic information are essential at species, ecological guild or reproductive group levels to help derive sustainable management scenarios for tropical forests.

Ação antiproliferativado flavonoide morina e do extrato da folha de oliveira (Oleaeuropaea L. ) contra a linhagem de célula H460 / The antiproliferative action of morin flavonoid and olive leaf extract (Olea europaea L. ) against the cell line H460

RESUMO O presente estudo investigou a indução de morte celular por apoptose pelo flavonóide morina e pelo extrato da folha de oliveira (Oleaeuropaea L.) em linhagens de células de câncer de pulmão do tipo não pequenas (H460). O tratamento com morina e o extrato de oliveira em células H460 resultou na redução do crescimento tumoral e indução de morte celular avaliados pelos ensaios de MTT e lactato desidrogenase (LDH) e a morte celular por apoptose confirmada por microscopia de fluorescência e análise por citometria de fluxo. Os dados indicaram que o flavonóide morina e o extrato de oliveira diminuíram a viabilidade celular para taxas percentuais de 7,22± 1,54% e 62,37± 2,85% nas concentrações de 800µM e µg/mL, respectivamente. As maiores taxas percentuais de morte celular por apoptose foram100% para morina na concentração de 800µM e 70,49 ± 5,91% para o extrato de oliveira na concentração de 800 µg/mL. Estes resultados foram associados com a alteração do potencial de membrana mitocondrial, cujos valores são de 54,91% para morina na concentração de 400µM e 42,2% para o extrato de oliveira na concentração de 800 µg/mL sugerindo envolvimento da via intrínseca da morte celular por apoptose. Portanto, morina e o extrato de oliveira afetaram a viabilidade celular da linhagem H460 induzindo morte celular por apoptose.

ABSTRACT This study investigates possible apoptosis induction mechanism by the flavonoid morin and the olive leaf extract (Olea europaea L.) in non-small lung cancer cells (H460). The treatment with morin and olive leaves extract resulted in growth inhibition and induction of apoptosis in H460 cells lines measured by the MTT assay methods and confirmed by fluorescence microscopy, flow cytometry analysis. The data indicated that themurin and the extract of olive decreased the cell viability percentage rates of 7.22 ± 1,54% and 62.37 ± 2,85% in the concentrations of 800 µM and µg/mL, respectively. The highest percentage rates of cell death by apoptosis were 100% for themorin in a concentration of 800 µM and 70.49 ± 5.91% for the olive extract in a concentration of 800 µg/mL. These findings were associated with altered mitochondrial membrane potential, whose value is 54.91% for the murin concentration of 400 µM and 42.2% for the olive extract in a concentration of 800 mg / mL, suggesting involvement of the intrinsic pathway of cell death by apoptosis. Therefore, the morin and the olive extract affect the cell viability of H460 cell lines inducing cell death by apoptosis.

Electromyographic analysis of rectus femoris activity during seated to standing position and walking in water and on dry land in healthy children and children with cerebral palsy.

PURPOSE: To analyze rectus femoris activity during seated to standing position and walking in water and on dry land comparing a group of children with the spastic diparesis type of cerebral palsy (CP) and a group of children without neurological disorders. METHODS: This study included a group of nine children with CP and a control group of 11 children. The study compared the electromyographic activity of the rectus femoris during seated to standing position and walking, in water and on land. RESULTS: A greater activation of the rectus femoris was observed in the group of children with CP compared with the control group when moving from a seated position to a standing position in water (p=0.0039) and while walking on land (p=0.0014) or in the pool (p=0.007). CONCLUSION: This study demonstrated the activation of the rectus femoris while walking or standing up from a seated position in water was greater in the group of children with CP. Further studies should be performed to better understand the extent of muscular activation during body immersion in individuals with neurological disorders.

Mendelian inheritance, linkage and genotypic disequilibrium in microsatellite loci isolated from Hymenaea courbaril (Leguminosae).

The Neotropical tree Hymenaea courbaril, locally known as Jatobá, is a valuable source of lumber and also produces comestible and medicinal fruit. We characterized Mendelian inheritance, linkage and genotypic disequilibrium at nine microsatellite loci isolated from H. courbaril, in order to determine if they would provide accurate estimates of population genetic parameters of this important Amazon species. The study was made on 250 open-pollinated offspring originated from 14 seed trees. Only one of nine loci presented significant deviation from the expected Mendelian segregation (1:1). Genotypic disequilibrium between pairwise loci was investigated based on samples from 55 adult and 56 juvenile trees. No genetic linkage between any paired loci was observed. After Bonferroni's corrections for multiple tests, we found no evidence of genotypic disequilibrium between pairs of loci. We conclude that this set of loci can be used for genetic diversity/ structure, mating system, gene flow, and parentage analyses in H. courbaril populations.

Impact of selective logging on inbreeding and gene dispersal in an Amazonian tree population of Carapa guianensis Aubl.

Selective logging may impact patterns of genetic diversity within populations of harvested forest tree species by increasing distances separating conspecific trees, and modifying physical and biotic features of the forest habitat. We measured levels of gene diversity, inbreeding, pollen dispersal and spatial genetic structure (SGS) of an Amazonian insect-pollinated Carapa guianensis population before and after commercial selective logging. Similar levels of gene diversity and allelic richness were found before and after logging in both the adult and the seed generations. Pre- and post-harvest outcrossing rates were high, and not significantly different from one another. We found no significant levels of biparental inbreeding either before or after logging. Low levels of pollen pool differentiation were found, and the pre- vs. post-harvest difference was not significant. Pollen dispersal distance estimates averaged between 75 m and 265 m before logging, and between 76 m and 268 m after logging, depending on the value of tree density and the dispersal model used. There were weak and similar levels of differentiation of allele frequencies in the adults and in the pollen pool, before and after logging occurred, as well as weak and similar pre- and post-harvest levels of SGS among adult trees. The large neighbourhood sizes estimated suggest high historical levels of gene flow. Overall our results indicate that there is no clear short-term genetic impact of selective logging on this population of C. guianensis.

Lipid peroxidation may predict restenosis after coronary balloon angioplasty.

The present study assessed whether lipid peroxidation in plasma might predict restenosis after coronary balloon angioplasty. A total of 87 patients, who had undergone successful coronary balloon angioplasty using standard techniques, were enrolled. Fasting blood samples before the intervention were measured for plasma levels of thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation). Angiography was carried out before and 15 min after angioplasty, and at follow-up (4 months after angioplasty), and evaluated using a quantitative approach. There were 23 patients with restenosis (group R) and 64 patients without restenosis (group N) after coronary balloon angioplasty. The plasma TBARS level (mean+/-SEM) of 4.3+/-0.1 micromol/L in group R was significantly higher than that of 3.2+/-0.1 micromol/L in group N (p<0.01). There were no significant differences in other parameters, including plasma lipid levels, between the 2 groups. The plasma level of TBARS positively correlated with lumen loss of the coronary artery at the time of follow-up angiography (r=0.57, p<0.01). Our results suggest that oxidative stress contributes to restenosis and indicate that an elevated plasma level of TBARS may be a reliable predictor of restenosis.

Two related thrombin-like enzymes present in Bothrops atrox venom

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80 percent homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii

Two related thrombin-like enzymes present in Bothrops atrox venom.

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical K(m) on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

Edema induction by the disintegrin-like/cysteine-rich domains from a Bothrops atrox hemorrhagin.

Viperine and crotaline snake venoms contain one or more hemorrhagic metalloproteases called hemorrhagins. The most potent hemorrhagins belong to the P-III class and have, in addition to the protease domain, disintegrin-like and cysteine-rich domains. Although proteolytic degradation of vascular endothelium basement membrane has been established to be the main factor responsible for hemorrhage, several studies reveal other factors that actually do facilitate this process. Recent evidence has shown that the nonprotease domains of the P-III class hemorrhagins are able to inhibit the platelet aggregation by blocking essential procoagulant integrins on platelets. In this study we report the identification of a hemorrhagin from Bothrops atrox venom. This enzyme, a P-III class metalloprotease, undergoes an apparent spontaneous degradation, releasing a proteic fragment containing the disintegrin-like/cysteine-rich domains. This fragment shows the capability to induce an edematogenic process, suggesting the existence of a still unknown nonenzymatic mechanism of vascular permeability increase.

Nitrosyl hemoglobin detected by near-infrared spectroscopy in rat liver allografts.

Near-infrared spectroscopy (NIRS) is a noninvasive biomeasurement system with rays in the near-infrared region that possess high permeability to biological tissues. NIRS was applied to liver allografts undergoing rejection in rats treated with deoxyspergualin (DSG) or tacrolimus (FK506). The nitrosyl hemoglobin (Hb) levels detected in the liver grafts increased 3 days and 5 days after grafting in both allogeneic and syngeneic transplantation. The levels on day 8 remained high in the allogeneic graft, but markedly decreased in the syngeneic graft. Although the serum levels of nitrite and nitrate were extremely low 8 days after grafting in allografted recipients treated with DSG or FK506, the nitrosyl-Hb level in DSG-treated graft was much higher than that in FK506-treated graft. There was no significant difference in survival time between DSG-treated and FK506-treated recipients. In conclusion, DSG and FK506 have a different effect on NO production in allografted liver with ongoing rejection, and circulating nitrite and /nitrate levels do not reflect the local levels of NO in the graft.

Development of snake antivenom antibodies in chickens and their purification from yolk.

Adult white leghorn hens hyperimmunised with Brazilian snake venoms of the genus Bothrops and/or Crotalus produced antibodies capable of recognising, combining with and neutralising the toxic and lethal components of the venoms. The antibodies were first detected by an enzyme-linked immunosorbent assay two weeks after starting the immunisation schedule, reached the highest titres by the third week and remained high for at least 24 weeks. These antibodies are transferred to the egg yolk from which they were isolated as enriched IgY preparations by a combination of methods using positive and negative precipitation with sodium sulphate and/or caprylic acid. The yolk-derived IgY preparations contained antibodies which blocked the phospholipase A2-dependent haemolytic activity of both venoms and the haemorrhagic activity of Bothrops venom, and neutralised the toxic lethal activities of the venoms with good efficacy. The median effective dose (ED50) of the IgY anti-Bothrops venom was 592.5 microliters/2LD50 and, 1.0 ml neutralised 0.0675 mg of venom. The ED50 of the IgY anti-Crotalus venom was 457.5 microliters/3LD50 and 1.0 ml neutralised 0.075 mg of venom.

The effects of atrial natriuretic peptide on brain edema, intracranial pressure and cerebral energy metabolism in rat congenital hydrocephalus.

Atrial natriuretic peptide (ANP) regulates fluid and electrolyte homeostasis in the central nervous system. In this study, we evaluated the effects of ANP on brain edema, intracranial pressure (ICP) and cerebral energy metabolism in congenital hydrocephalus in rats. Brain edema, indicated by the longitudinal relaxation time (T1), was evaluated by 1H-magnetic resonance imaging (MRI). The ICP was monitored with a miniature pressure-transducer with telemetric system. Cerebral energy metabolism, indicated by PCr/Pi ratio, was measured by 31P-magnetic resonance spectroscopy (MRS). The rats were given 10 microliters of ANP in the left cerebral ventricle. Three different concentrations of ANP were given; 0.2 (group I), 2.0 (group II) and 20.0 (group III) micrograms/10 microliters, respectively. 10 microliters of saline was injected into the ventricle of the control group rats. There were no significant changes of ICP, T1 value and PCr/Pi ratio among the control group, group I and group II. In group III, in contrast, ICP decreased significantly at 20 minutes after ANP administration and stayed at this ICP level for 60 minutes. The T1 value decreased and PCr/Pi ratio increased 30 minutes after ANP administration. This study revealed that intraventricularly administered ANP could decrease ICP, reduce brain edema and improve the cerebral energy metabolism in rats with congenital hydrocephalus.

In vivo near-infrared monitoring of nitric oxide production and tissue oxygen sufficiency in rat liver allografts during the acute rejection reaction.

We established a new technique of in vivo near-infrared (NIR) spectroscopy that can estimate both nitric oxide (NO) production and tissue oxygen sufficiency in living organs during the alloimmune response. The present study was aimed at evaluating the potential of this technique for monitoring the rejection response utilizing the rat model of orthotopic liver transplantation without arterialization. The relative changes of nitrosyl-hemoglobin, oxyhemoglobin and oxidized-cytochrome oxidase in the graft livers were quantified by use of this method. Nitrosyl-hemoglobin in the allogenic grafts was elevated at the onset of the rejection response and was suppressed when the rejection reaction was treated by the administration of 15-deoxyspergualin. Oxy-hemoglobin and oxidized-cytochrome oxidase were decreased in accordance with parenchymal disorder determined histologically. These results demonstrated that the new technique of in vivo NIR spectroscopy can assess simultaneously both the immune response and graft function after liver transplantation.