Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.

Characteristics of IEF, SDS and SAR-PAGE of Cuba EPO biosimilar

EPO is a glycoprotein produced in the kidney, which stimulates the division and differentiation of red cells in the bone marrow. Erythropoietin is available as a therapeutic agent produced by recombinant DNA technology in mammalian cell culture into which the human EPO gene has been transfected. Biosimilar Epoetins are mostly erythropoietins of the Epoetin alfa, beta or omega type, which are being produced at much lower cost due to expired patents. Recombinant human erythropoietin (rh-EPO) contains the identical amino acid sequence of natural EPO: 165 amino acids, with a molecular weight of 30,400 Da. Since glycosylation is not only dependent on the cell-line used for the expression of Epoetins but also on the entire biotechnological process the glycosylation patterns of biosimilars do not necessarily reflect the patterns of the originator compounds. Today biosimilar Epoetins are manufactured and distributed worldwide and under many different names. The use of recombinant EPOs for doping is prohibited because of its performance enhancing effect. The aim of the present study was to investigated whether biosimilar alpha r-HuEPO – ior®-EPOCIM, produced in Cuba and also available in other countries in all continents, could be differentiated from endogenous one by iso-electro-focusing plus double blotting, SDS-PAGE and SAR-PAGE for antidoping analysis.

A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.