ABSTRACT
This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). The fusion proteins were constructed in the form of chimeric toxins, aiming to obtain bifunctional analgesic and antitumor activity. The fusion proteins consisted of luteinizing hormone-releasing hormone (LHRH), three different types of flexible linkers (L1, Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met; L2, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser; L3, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser), and BmKAGAP. The genes coding three fusion proteins were cloned and expressed in E. coli in soluble form. Following two successive column chromatographic separations, purified fusion proteins were obtained. These fusion proteins exhibited analgesic activity in mice and were cytotoxic to a hepatocellular carcinoma cell line Hep3B.
Subject(s)
Analgesics/administration & dosage , Analgesics/isolation & purification , Analgesics/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Liver Neoplasms, Experimental/drug therapy , Mice , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacology , Scorpion Venoms/administration & dosage , Scorpion Venoms/biosynthesis , Scorpion Venoms/chemistry , Scorpion Venoms/isolation & purification , Scorpion Venoms/pharmacology , ScorpionsABSTRACT
We evaluated the effect of a basic fibroblast growth factor (bFGF) and saporin conjugate (bFGF-SAP) on proliferation, migration and tubule formation in bovine choriocapillary endothelial cells (BCECs). Cell proliferation and MTS assays were done with 0.01, 0.1, 1, 10, and 100 nM bFGF-SAP, and an equimolar concentration of bFGF and saporin. TUNEL assay was performed to confirm apoptosis. Cells were treated with 1, 10, and 100 nM bFGF-SAP and migration assay and tubule formation assay were done. Results were evaluated with image analysis. All experiments were performed in triplicate and repeated three times. Viable cells (ID50 = 0.62) and cell proliferation by MTS assay (ID50 = 0.75 nM) were inhibited. Saporin caused cytotoxicity and inhibition of proliferation at high concentration. DNA fragmentation was identified by TUNEL assay. Migration and tubule formation were also inhibited. All mechanisms responsible for neovascularization were inhibited, and this could be applied in the management of subretinal choroidal neovascularization (SRN).
Subject(s)
Animals , Cattle , Apoptosis/drug effects , Cell Count , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Comparative Study , Cytotoxins/pharmacology , Endothelium, Vascular/drug effects , Fibroblast Growth Factor 2/pharmacology , In Situ Nick-End Labeling , Neovascularization, Physiologic/drug effects , Plant Proteins/pharmacology , Recombinant Fusion Proteins/pharmacologyABSTRACT
The cytokine pattern on viral antigen recognition is believed to exert a profound influence on the resolution of viral infections and viral clearance. This study was initiated to investigate whether a cytokine imbalance oriented toward Th2 type response plays a role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analysed by RT-PCR. Upon HBsAg stimulation, expression of IFN-gamma, IL-2, IL-4, and IL-10 was detected in 41%, 8%, 41%, and 50% of the patients, respectively. Among these cytokines, the expression of IFN-gamma was associated with high levels of serum AST/ALT. However, we could not prove that Th2 type cytokines had a protective effect on hepatocytes. Upon HBxAg stimulation, there was no recognizable association of cytokine patterns with AST/ALT levels. In conclusion, production of a Th1 cytokine, IFN-gamma, by HBsAg-reactive cells was associated with hepatocyte damage in chronic hepatitis B, while no counteracting effect of Th2 cytokines produced by those cells was observed.
Subject(s)
Humans , Cytokines/genetics , Cytokines/biosynthesis , Hepatitis B Surface Antigens/pharmacology , Hepatitis B Surface Antigens/immunology , Hepatitis B, Chronic/immunology , Interferon-gamma/genetics , Interferon-gamma/biosynthesis , Leukocytes, Mononuclear/immunology , Liver/cytology , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/immunology , Th1 Cells/immunology , Th1 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/drug effects , Trans-Activators/pharmacology , Trans-Activators/immunologyABSTRACT
En la insuficiencia renal crónica (IRC), la anemia es una de las complicaciones más frecuentes, ésta es de tipo hipoproliferativo y su principal causa es el déficit relativo de eritropoyetina. El tratamiento fue, hasta la obtención de eritropoyetina por métodos recombinantes (rHuEpo), las transfusiones y los estimulantes eritropoyéticos, como los andrógenos. Actualmente existen muchas evidencias clínicas de la utilidad de la rHuEpo en el tratamiento de la anemia de la IRC. La mayoría de estos ensayos clínicos fueron realizados en adultos y en ellos se mostró no sólo la mejoría de los datos hematimétricos, sino también de la calidad de vida. Este trabajo se realizó en 18 pacientes pediátricos que estaban sometidos a regímenes de hemodiálisis crónica. El objetivo del mismo fue demostrar la seguridad y eficacia de la rHuEpo en los niños durante un año de tratamiento. El 94,2 por ciento de los pacientes evaluados alcanzaron la tasa de hemoglobina que habíamos planeado (10 gr por ciento), el único paciente que no lo logró tenía fibrosis medular. Los efectos adversos fueron similares a los observados en la población de adultos siendo la exacerbación de la hipertensión arterial el más frecuente. En seis niños se evaluó la capacidad física post tratamiento, todos se demosntró una mejoría de los índices de eficiencia. Se concluye que la rHuEpo es efectiva y segura en niños con IRC y anemia aumentando el número de glóbulos rojos evitando las transfusiones y mejorando el rendimiento físico que se relaciona con mejoría en la calidad de vida