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.

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.

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.

Caracterización detallada de algunos anticuerpos monoclonales que reconocen sitios antigénicos en el VP1 del virus de la fiebre aftosa

La mayoría de los anticuerpos monoclonales (AcMs) desarrollados contra las cepas O1 Campos, A24 Cruzeiro y C Indaial de la fiebre aftosa y que previamente mostraron reacción con el VP1 en "Western Blots", reconocieron péptidos sintéticos correspondientes a la secuencia 141-158 de esta proteína. Una número significativo de los AcMs que reconocieron péptidos sintéticos, lo hicieron de forma heteróloga u homóloga. Así muchos de los AcMs A24 Cruzeiro reconocieron un péptido O1 Kaufbeuren y atribuyeron su reacción cruzadas a la región 141-158 del VP1. Un AcMc3 Indaial reconoció péptidos correspondientes a tres serotipos y la actividad cruzada fue atribuida a la secuencia 200-213 del VP1. Generalmente, la reacción cruzada de un AcM dado se observó con el virus y con el péptido, pero no con los dos antígenos.

Most monoclonal antibodies (MAbs) raised against the O1 Campos, A24 Cruzeiro and C3 Indaial strains of foot-and-mouth disease virus (FMD), and which were previously shown to react with VP, in Western Blots, recognized synthetic peptides corresponding to the 141-158 sequence of this protein. A significant number of the MAbs which recognized synthetic peptides did so in a heterologous as well as homologous manner. Thus, many of the A24 Cruzeiro MAbs recognized an O 1 Kaufbeuren peptide and the cross-reactivity was ascribed to thE 141-158 region of the VP1. One C3 Indaial MAb recognized peptides corresponding to three serotypes and the cross-reactivity was ascribed to the 200-213 sequence of the VP1. Usually, the cross-reactivity of a given MAb was observed with either the virus or the peptide but not with both antigens.

Producción y caracterización de anticuerpos monoclonales contra la fiebre aftosa

Se describen los anticuerpos monoclonales (AcMs) producidos contra los virus de la fiebre aftosa tipo O, A y C. Para la caracterización preliminar se utilizaron las pruebas de virus-neutralización y seroprotección, y para la reacción de los AcMs, la prueba EITB con la proteína desnaturalizada del virus. Algunos AcMs que no neutralizaron la infección del virus en cultivos de tejidos se mostraron capaces de neutralizarla en ratones lactantes. Los AcMs designados 7EE6 y 7jA1, originarios del virus C3 Indaial, mostraron reacción cruzada con los virus heterólogos O1 Campos y A24 Cruzeiro de la fiebre aftosa.

Monoclonal antibodies (MAbs) produced against foot-and-mouth disease virus (FMDV) types O, A, and C are described. The preliminary characterization id discussed in terms of virus neutralization and mouse protection test results and the reactivity of MAbs in enzyme-linked immnoelectrotransfer blot assays with the denatured protein of the virus. Some MAbs which did not neutralize virus infectivity in tissue culture were able to neutralize virus infectivity in suckling mice. The MAbs designated as 7EE6 and 7JA1 which originated from C, Indaial virus cross-reacted with heterologus FMDV O(1) Campos and A(24) Cruzeiro viruses.

Producción de anticuerpos monoclonales contra antigenos grupo especificos del virus de la lengua azul

Se desarrollaron anticuerpos monoclonales contra el antígeno grupo específico del virus de la lengua azul serotipo 4. Los hibridomas se obtuvieron a partir de células de bazo de ratones BALB/c inmunizados, fusionadas con células de mieloma Sp2/O-Ag 14, usando polietilenglicol 1500 y clonados por dilución limitante. El sobrenadante de ocho anticuerpos monoclonales se probó, por inmunodifusión en gel de agar, contra antígenos grupo específicos procedentes de cuatro laboratorios diferentes. Todos mostraron una buena reacción con el antígeno grupo específico. Por lo tanto, estos anticuerpos monoclonales tienen gran potencial para ser usados en la identificación de anticuerpos grupos específicos mediante la prueba ELISA de competición.

Monoclonal antibodies (MAbs) against the group-specific antigen of Bluetong virus were developed using Bluetong virus serotype 4. The hybridomas were abtained from immunized BALB/c spleen cells fused with myeloma cells line Sp2/O-Ag14 using polyethylenglycol 1500, and cloned by limiting dilution. The supernatant from eight MAbs was tested against group-specific antigens used in agar gel immunodiffusion tests by different laboratories. The eight monoclonal antibodies presented good reactions to the group-specific antigen. Therefore, these MAbs have great potential for application in the identification of group-specific antibodies by competitive ELISA tests.