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Biotechnol Adv ; : 107552, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32416132


The manufacturing of recombinant protein is traditionally undertaken in mammalian cell culture. Today, speed, cost and safety are the primary considerations for process improvements in both upstream and downstream manufacturing. Leaders in the biopharmaceutical industry are striving for continuous improvements to increase throughput, lower costs and produce safer more efficacious drugs. This can be achieved through advances in cell line engineering, process development of cell culture, development of chemically defined media and increased emphasis on product characterization. In the first part, this review provides a historical perspective on approved biotherapeutics by regulatory bodies which pave the way for next-generation products (including gene therapy). In the second part, it focuses on the application of in vitro and in vivo cell line engineering approaches, modern process development improvements including continuous manufacturing, recent developments in media formulation, and improvements in critical quality attribute determinations for products produced predominantly in mammalian cells.

ISA Trans ; 97: 251-260, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31303338


Steam generator water level control incorporates the system that controls the speed of the main water feed pump and the one that controls the degree to which the main water feed valve is open. Although these two control systems are set independently, they have a reciprocal influence. A key problem is that, in the actuator for the regulation of the two systems, the regulated variables tend to oscillate with a relatively large amplitude even under stable conditions. In this paper, a pervasive regulating resonance phenomenon is inspected and analyzed by means of simulating the real working conditions. And using a mathematical model of the main water feed flow, it reveals that the two factors which directly cause the regulation resonance phenomenon are the frequency characteristics of the control system and the phase difference. On the basis of this, three potential optimization schemes have been proposed: the optimization of the regulation parameters; changing the phase difference; and setting a regulation dead-band which can improve the stability of the control system.

Bioorg Chem ; 66: 41-5, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27014866


The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.

Frutosefosfatos/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glucose-6-Fosfato/metabolismo , Thermococcus/enzimologia , Animais , Relação Dose-Resposta a Droga , Frutosefosfatos/química , Glucose-6-Fosfato/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Coelhos , Estereoisomerismo , Relação Estrutura-Atividade
J Biol Chem ; 290(47): 28343-52, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26221033


Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12(undiff)). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species.

Amiloide/metabolismo , Biopolímeros/metabolismo , Morte Celular , Amiloide/química , Animais , Biopolímeros/química , Células PC12 , Ratos
Proteins ; 81(1): 1-17, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22777854


Human α-lactalbumin made lethal to tumor cells (HAMLET) and its analogs are partially unfolded protein-oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge-specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively-charged lysine residues to negatively-charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild-type α-lactalbumin-oleic acid complex. With the addition of OA, the wild-type and guanidinated α-lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α-lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively-charged basic groups on α-lactalbumin and the negatively-charged carboxylate groups on OA molecules play an essential role in the binding of OA to α-lactalbumin and that these interactions appear to be as important as hydrophobic interactions.

Lactalbumina/química , Lactalbumina/metabolismo , Ácidos Oleicos/química , Ácidos Oleicos/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lactalbumina/farmacologia , Lisina/química , Modelos Moleculares , Ácidos Oleicos/farmacologia , Conformação Proteica , Desdobramento de Proteína , Espectrometria de Fluorescência , Eletricidade Estática , Termodinâmica
Biochem Soc Trans ; 40(4): 746-51, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22817727


HAMLET (human α-lactalbumin made lethal to tumour cells) and its related partially unfolded protein-fatty acid complexes are novel biomolecular nanoparticles that possess relatively selective cytotoxic activities towards tumour cells. One of the key characteristics is the requirement for the protein to be partially unfolded, hence endowing native proteins with additional functions in the alternatively folded states. Beginning with the history of its discovery and development, the cellular targets that appear to be strongly correlated with tumour cell death are introduced in the present article.

Lactalbumina/química , Lactalbumina/metabolismo , Ácido Oleico/química , Ácido Oleico/metabolismo , Ácidos Oleicos/química , Ácidos Oleicos/metabolismo , Animais , Apoptose , Bovinos , Humanos , Dobramento de Proteína