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1.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207579

RESUMO

Biomanufacturing processes may be optimized by storing cell culture media at room temperature, but this is currently limited by their instability and change in color upon long-term storage. This study demonstrates that one of the critical contributing factors toward media browning is tryptophan. LC-MS technology was utilized to identify tryptophan degradation products, which are likely formed primarily from oxidation reactions. Several of the identified compounds were shown to contribute significantly to color in solutions but also to exhibit toxicity against CHO cells. A cell-culture-compatible antioxidant, a-ketoglutaric acid, was found to be an efficient cell culture media additive for stabilizing components against degradation, inhibiting the browning of media formulations, and decreasing ammonia production, thus providing a viable method for developing room-temperature stable cell culture media.


Assuntos
Meios de Cultura/química , Triptofano/metabolismo , Animais , Células CHO , Cricetulus , Oxirredução , Triptofano/análise
2.
Free Radic Biol Med ; 160: 696-718, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-32911085

RESUMO

Tryptophan is one of the essential mammalian amino acids and is thus a required component in human nutrition, animal feeds, and cell culture media. However, this aromatic amino acid is highly susceptible to oxidation and is known to degrade into multiple products during manufacturing, storage, and processing. Many physical and chemical processes contribute to the degradation of this compound, primarily via oxidation or cleavage of the highly reactive indole ring. The central contributing factors are reactive oxygen species, such as singlet oxygen, hydrogen peroxide, and hydroxyl radicals; light and photosensitizers; metals; and heat. In a multi-component mixture, tryptophan also commonly reacts with carbonyl-containing compounds, leading to a wide variety of products. The purpose of this review is to summarize the current state of knowledge regarding the degradation and interaction products of tryptophan in complex liquid solutions and in proteins. For the purposes of context, a brief summary of the key pathways in tryptophan metabolism will be included, along with common methods and issues in tryptophan manufacturing. The review will focus on the conditions that lead to tryptophan degradation, the products generated in these processes, their known biological effects, and methods which may be applied to stabilize the amino acid.


Assuntos
Oxigênio Singlete , Triptofano , Animais , Humanos , Radical Hidroxila , Oxirredução , Espécies Reativas de Oxigênio , Triptofano/metabolismo
3.
Biotechnol Bioeng ; 116(6): 1537-1555, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30793282

RESUMO

Nowadays, chemically defined cell culture media (CCM) have replaced serum- and hydrolysate-based media that rely on complex ingredients, such as yeast extracts or peptones. Benefits include a significantly lower lot-to-lot variability, more efficient manufacturing by reduction to essential components, and the ability to exclude components that may negatively influence growth, viability, or productivity. Even though current chemically defined CCMs provide an excellent basis for various mammalian biotechnological processes, vitamin instabilities are known to be a key factor contributing to the variabilities still present in liquid CCM as well as to short storage times. In this review, the chemical degradation pathways and products for the most relevant vitamins for CCM will be discussed, with a focus on the effects of light, oxygen, heat, and other CCM compounds. Different approaches to stabilize vitamins in solution, such as replacement with analogs, encapsulation, or the addition of stabilizing compounds will also be reviewed. While these vitamins and vitamin stabilization approaches are presented here as particular for CCM, the application of these concepts can also be considered relevant for pharmaceutical, medical, and food supplement purposes. More precise knowledge regarding vitamin instabilities will contribute to stabilize future formulations and thus decrease residual lot-to-lot variability.


Assuntos
Meios de Cultura/química , Vitaminas/química , Animais , Biotecnologia/métodos , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Estabilidade de Medicamentos , Excipientes/química , Excipientes/metabolismo , Temperatura Alta , Humanos , Luz , Oxigênio/metabolismo , Vitaminas/metabolismo
4.
Elife ; 62017 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-28884684

RESUMO

Genetic analysis has revealed that the dual specificity protein kinase DYRK1A has multiple roles in the development of the central nervous system. Increased DYRK1A gene dosage, such as occurs in Down syndrome, is known to affect neural progenitor cell differentiation, while haploinsufficiency of DYRK1A is associated with severe microcephaly. Using a set of known and newly synthesized DYRK1A inhibitors, along with CRISPR-mediated gene activation and shRNA knockdown of DYRK1A, we show here that chemical inhibition or genetic knockdown of DYRK1A interferes with neural specification of human pluripotent stem cells, a process equating to the earliest stage of human brain development. Specifically, DYRK1A inhibition insulates the self-renewing subpopulation of human pluripotent stem cells from powerful signals that drive neural induction. Our results suggest a novel mechanism for the disruptive effects of the absence or haploinsufficiency of DYRK1A on early mammalian development, and reveal a requirement for DYRK1A in the acquisition of competence for differentiation in human pluripotent stem cells.


Assuntos
Diferenciação Celular , Neurônios/fisiologia , Células-Tronco Pluripotentes/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células Cultivadas , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores
5.
Chemistry ; 21(5): 1966-77, 2015 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-25487964

RESUMO

Glycoside hydrolase family 99 (GH99) was created to categorize sequence-related glycosidases possessing endo-α-mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N-glycan precursors (Glc1-3 Man9 GlcNAc2 ), releasing mono-, di- and triglucosylated-mannose (Glc1-3 -1,3-Man). GH99 family members have recently been implicated in the ability of Bacteroides spp., present within the gut microbiota, to metabolize fungal cell wall α-mannans, releasing α-1,3-mannobiose by hydrolysing αMan-1,3-αMan→1,2-αMan-1,2-αMan sequences within branches off the main α-1,6-mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel endo-α-1,2-mannanase activity of bacterial GH99 enzymes from Bacteroides thetaiotaomicron and xylanisolvens. These data reveal an approximate 5 kJ mol(-1) preference for mannose-configured substrates in the -2 subsite (relative to glucose), which inspired the development of a new inhibitor, α-mannopyranosyl-1,3-isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X-ray structures of ManIFG or a substrate in complex with wild-type or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to D-mannose- rather than D-glucose-configured substrates.


Assuntos
Bacteroides/metabolismo , Glicosídeo Hidrolases/metabolismo , Hidrolases/metabolismo , Manosidases/química , Manosidases/metabolismo , Modelos Moleculares
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