Development of polyclonal antibodies for the detection of recombinant human erythropoietin

Recombinant human erythropoietin (rHuEPO) is detected by using direct pharmacological assays and indirect haematological assays. However, both methods have several limitations including technical challenges and  cost-related issues. The aim of this study was to develop polyclonal antibodies against rHuEPO (anti-rHuEPO pAb) that can be used in immunoassays. In this study, we purified anti-rHuEPO pAb that could be used in immunoblotting assays to efficiently detect rHuEPO. Furthermore, these antirHuEPO pAb which could also detect rHuEPO that was expressed in a eukaryotic expression system (CHO cells). Thus, the anti-rHuEPO pAb developed in this study may be useful for rHuEPO detection.Keywords: Antibodies, rHuEPO, immunoassays, pAb.African Journal of Biotechnology Vol. 12(37), pp. 5595-559

Testis-mediated gene transfer in mice : comparison of transfection reagents regarding transgene transmission and testicular damage

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PCR and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT

Testis-mediated gene transfer in mice : comparison of transfection reagents regarding transgene transmission and testicular damage

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PCR and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT

Methotrexate diethyl ester-loaded lipid-core nanocapsules in aqueous solution increased antineoplastic effects in resistant breast cancer cell line

Breast cancer is the most frequent cancer affecting women. Methotrexate (MTX) is an antimetabolic drug that remains important in the treatment of breast cancer. Its efficacy is compromised by resistance in cancer cells that occurs through a variety of mechanisms. This study evaluated apoptotic cell death and cell cycle arrest induced by an MTX derivative (MTX diethyl ester [MTX(OEt)2]) and MTX(OEt)2-loaded lipid-core nanocapsules in two MTX-resistant breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231. The formulations prepared presented adequate granulometric profile. The treatment responses were evaluated through flow cytometry. Relying on the mechanism of resistance, we observed different responses between cell lines. For MCF-7 cells, MTX(OEt)2 solution and MTX(OEt)2-loaded lipid-core nanocapsules presented significantly higher apoptotic rates than untreated cells and cells incubated with unloaded lipid-core nanocapsules. For MDA-MB-231 cells, MTX(OEt)2-loaded lipid-core nanocapsules were significantly more efficient in inducing apoptosis than the solution of the free drug. S-phase cell cycle arrest was induced only by MTX(OEt)2 solution. The drug nanoencapsulation improved apoptosis induction for the cell line that presents MTX resistance by lack of transport receptors

Methotrexate diethyl ester-loaded lipid-core nanocapsules in aqueous solution increased antineoplastic effects in resistant breast cancer cell line

Breast cancer is the most frequent cancer affecting women. Methotrexate (MTX) is an antimetabolic drug that remains important in the treatment of breast cancer. Its efficacy is compromised by resistance in cancer cells that occurs through a variety of mechanisms. This study evaluated apoptotic cell death and cell cycle arrest induced by an MTX derivative (MTX diethyl ester [MTX(OEt)2]) and MTX(OEt)2-loaded lipid-core nanocapsules in two MTX-resistant breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231. The formulations prepared presented adequate granulometric profile. The treatment responses were evaluated through flow cytometry. Relying on the mechanism of resistance, we observed different responses between cell lines. For MCF-7 cells, MTX(OEt)2 solution and MTX(OEt)2-loaded lipid-core nanocapsules presented significantly higher apoptotic rates than untreated cells and cells incubated with unloaded lipid-core nanocapsules. For MDA-MB-231 cells, MTX(OEt)2-loaded lipid-core nanocapsules were significantly more efficient in inducing apoptosis than the solution of the free drug. S-phase cell cycle arrest was induced only by MTX(OEt)2 solution. The drug nanoencapsulation improved apoptosis induction for the cell line that presents MTX resistance by lack of transport receptors

Npy and sbgnrh gene expression in juvenile and adult male brazilian flounder paralichthys orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.O objetivo deste estudo foi avaliar a expressão gênica do neuropeptídeo Y (NPY) e da variante sea bream do hormônio liberador de gonadotrofinas (sbGnRH) em linguados machos juvenis e adultos. O hipotálamo foi isolado para a extração de RNA total. Após a síntese de cDNA, a PCR em tempo real foi usada para avaliar a expressão gênica. Foi observado um aumento de aproximadamente duas vezes nos níveis de NPY e de aproximadamente três vezes nos níveis de sbGnRH nos peixes adultos. Esses resultados demonstram que estes peptídeos podem estar envolvidos na regulação, via hipotálamo, da maturação sexual no linguado