RESUMO
High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48â mM (27.2â g/L) in a small-scale batch process (200â µL) within 24â h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6â mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1â L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4â g/L UDP-galactose were produced within 23â h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26â /g.
Assuntos
Enzimas/metabolismo , Uridina Difosfato Galactose/biossíntese , Trifosfato de Adenosina/metabolismo , Reatores Biológicos , Sistema Livre de Células , Concentração de Íons de Hidrogênio , Oligossacarídeos/biossíntese , Estudo de Prova de Conceito , Uridina Difosfato Galactose/isolamento & purificaçãoRESUMO
High-dose glucocorticoids (GCs) can be a useful treatment for aggressive forms of chronic lymphocytic leukemia (CLL). However, their mechanism of action is not well understood, and resistance to GCs is inevitable. In a minimal, serum-free culture system, the synthetic GC dexamethasone (DEX) was found to decrease the metabolic activity of CLL cells, indicated by down-regulation of pyruvate kinase M2 (PKM2) expression and activity, decreased levels of pyruvate and its metabolites, and loss of mitochondrial membrane potential. This metabolic restriction was associated with decreased size and death of some of the tumor cells in the population. Concomitant plasma membrane damage increased killing of CLL cells by DEX. However, the nuclear receptor peroxisome proliferator activated receptor α (PPARα), which regulates fatty acid oxidation, was also increased by DEX, and adipocyte-derived lipids, lipoproteins, and propionic acid protected CLL cells from DEX. PPARα and fatty acid oxidation enzyme inhibitors increased DEX-mediated killing of CLL cells in vitro and clearance of CLL xenografts in vivo. These findings suggest that GCs prevent tumor cells from generating the energy needed to repair membrane damage, fatty acid oxidation is a mechanism of resistance to GC-mediated cytotoxicity, and PPARα inhibition is a strategy to improve the therapeutic efficacy of GCs.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Ácidos Graxos/metabolismo , Glucocorticoides/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , PPAR alfa/metabolismo , Adipócitos/citologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Meios de Cultivo Condicionados , Dexametasona/farmacologia , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Metabolismo dos Lipídeos , Potencial da Membrana Mitocondrial , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oxigênio/metabolismo , Fosforilação , Propionatos/química , Hormônios Tireóideos/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture-based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture-derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell-derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM-adsorbers were compared directly to column-based Cellufine sulfate and commercially available cation-exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM-capture step resulted in a higher reduction of dsDNA compared to the tested cation-exchange membrane adsorbers. The productivity of the SCM-based unit operation could be significantly improved by a 30-fold increase in volumetric flow rate during adsorption compared to the bead-based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell-derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM-adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine sulfate.