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1.
Amino Acids ; 56(1): 51, 2024 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-39198298

RESUMEN

Branched-chain amino acids (BCAAs)-leucine (Leu), isoleucine (Ile), and valine (Val)-are essential nutrients with significant roles in protein synthesis, metabolic regulation, and energy production. This review paper offers a detailed examination of the physico-chemical properties of BCAAs, their industrial synthesis, and their critical functions in various biological processes. The unique isomerism of BCAAs is presented, focusing on analytical challenges in their separation and quantification as well as their solubility characteristics, which are crucial for formulation and purification applications. The industrial synthesis of BCAAs, particularly using bacterial strains like Corynebacterium glutamicum, is explored, alongside methods such as genetic engineering aimed at enhancing production, detailing the enzymatic processes and specific precursors. The dietary uptake, distribution, and catabolism of BCAAs are reviewed as fundamental components of their physiological functions. Ultimately, their multifaceted impact on signaling pathways, immune function, and disease progression is discussed, providing insights into their profound influence on muscle protein synthesis and metabolic health. This comprehensive analysis serves as a resource for understanding both the basic and complex roles of BCAAs in biological systems and their industrial application.


Asunto(s)
Aminoácidos de Cadena Ramificada , Transducción de Señal , Aminoácidos de Cadena Ramificada/metabolismo , Aminoácidos de Cadena Ramificada/biosíntesis , Corynebacterium glutamicum/metabolismo , Metabolismo Energético , Humanos , Animales , Leucina/metabolismo , Leucina/química
2.
Biotechnol Bioeng ; 120(11): 3163-3176, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37489835

RESUMEN

Fc-fusion proteins are highly complex molecules, difficult to manufacture at scale. In this work, undesired proteoforms were detected during the manufacture of a therapeutic fusion protein produced in CHO cells. These species were characterized using gel electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry leading to the identification of low molecular weight proteoforms presenting low N- and O-glycan site occupancy, as well as a low sialylation content. Upstream process parameters were investigated, and fusion protein quality was shown to be linked to the sodium chloride content of the medium. A mitigation strategy was developed to avoid formation of unwanted glyco-variants, resulting in an increased yield of highly glycosylated Fc-fusion protein. The effect of sodium chloride was shown to be independent of the osmolality increase and was hypothesized to be linked to a modulation of Golgi acidity, which is required for the correct localization and function of glycosyltransferases. Altogether, this study highlights the importance of the salt balance in cell culture media used to produce highly sialylated and occupied glycoproteins, helping to maximize the yield and increase robustness of processes aiming at producing biopharmaceutical complex therapeutic molecules.


Asunto(s)
Glicoproteínas , Cloruro de Sodio , Cricetinae , Animales , Glicosilación , Cricetulus , Glicoproteínas/metabolismo , Polisacáridos/química , Células CHO
3.
Biotechnol Bioeng ; 118(5): 1818-1831, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33501689

RESUMEN

The reduction of antibody core-fucosylation is known to enhance antibody-dependent cellular cytotoxicity (ADCC). In this study, 5-Thio-l-Fucose (ThioFuc) was investigated as a media and feed supplement for modulating the fucosylation profile of therapeutic proteins and, thereby, improving the resulting effector functions. Glycan analysis of five different therapeutic proteins produced by a diverse set of Chinese hamster ovary cell lines demonstrated a clone dependent impact of ThioFuc treatment. Using rituximab as a model, an efficient dose- and time-dependent reduction of core-fucosylation up to a minimum of 5% were obtained by ThioFuc. Besides a concomitant increase in the afucosylation level up to 48%, data also revealed up to 47% incorporation of ThioFuc in place of core-fucosylation. In accordance with the glycan data, antibodies produced in the presence of ThioFuc revealed an enhanced FcγRIIIa binding up to 7.7-fold. Furthermore, modified antibodies subjected to a cell-based ADCC reporter bioassay proved to exert both a 1.5-fold enhanced ADCC efficacy and 2.6-fold enhancement in potency in comparison to their native counterparts-both of which contribute to an improvement in the ADCC activity. In conclusion, ThioFuc is a potent fucose derivative with potential applications in drug development processes.


Asunto(s)
Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Fucosa/análogos & derivados , Receptores de IgG , Proteínas Recombinantes , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , Fucosa/química , Fucosa/metabolismo , Fucosa/farmacología , Glicosilación/efectos de los fármacos , Humanos , Unión Proteica , Receptores de IgG/química , Receptores de IgG/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
4.
Biotechnol Bioeng ; 118(9): 3395-3408, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-33738790

RESUMEN

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Técnicas de Cultivo de Célula , Medios de Cultivo , Isoleucina , Leucina , Animales , Células CHO , Cricetulus , Medios de Cultivo/química , Medios de Cultivo/farmacología , Isoleucina/análogos & derivados , Isoleucina/química , Isoleucina/farmacología , Leucina/análogos & derivados , Leucina/química , Leucina/farmacología , Proteínas Recombinantes/biosíntesis
5.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-34207579

RESUMEN

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.


Asunto(s)
Medios de Cultivo/química , Triptófano/metabolismo , Animales , Células CHO , Cricetulus , Oxidación-Reducción , Triptófano/análisis
6.
J Neurochem ; 155(4): 348-369, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32320074

RESUMEN

Amyloid-ß (Aß) dysmetabolism is tightly associated with pathological processes in Alzheimer's disease (AD). Currently, it is thought that, in addition to Aß fibrils that give rise to plaque formation, Aß aggregates into non-fibrillar soluble oligomers (AßOs). Soluble AßOs have been extensively studied for their synaptotoxic and neurotoxic properties. In this review, we discuss physicochemical properties of AßOs and their impact on different brain cell types in AD. Additionally, we summarize three decades of studies with AßOs, providing a compelling bulk of evidence regarding cell-specific mechanisms of toxicity. Cellular models may lead us to a deeper understanding of the detrimental effects of AßOs in neurons and glial cells, putatively shedding light on the development of innovative therapies for AD.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Células Cultivadas , Humanos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Neuronas/patología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología
7.
Clin Transplant ; 34(8): e13997, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32484985

RESUMEN

Immunosuppressive therapy is used in solid organ transplant treatment, and mycophenolic acid (MPA) is one of the immunosuppressive drugs most used worldwide. It is a potent, selective, non-competitive, and reversible inosine monophosphate dehydrogenase (IMPDH) inhibitor that acts to inhibit guanine synthesis. To improve solubility, MPA is used as the prodrug mycophenolate mofetil (MMF) or as an enteric-coated mycophenolate sodium salt (EC-MPS). It is metabolized into mycophenolic acid phenyl glucuronide (MPAG), the inactive and major metabolite, and into acyl glucuronide (AcMPAG), pharmacologically active. In kidney transplantation, combined immunosuppressive therapy with cyclosporine (CsA) and tacrolimus (Tac) is widely used, showing beneficial effects. This paper aimed to review papers published in the last two decades and discuss factors that can interfere with the pharmacokinetics of MPA. Data collected confirm that MPA plasma levels should be monitored to evaluate immunosuppressive therapy since pharmacokinetics can be influenced by factors such as interpatient variability, coadministration of other immunosuppressive agents, post-transplant period, renal function, and dose. However, to perform drug monitoring, costs and facility may be limitations. Monitoring MPAG together with MPA would be a great improvement in therapy as it represents a big part of MPA levels and can be related to the increase of adverse effects.


Asunto(s)
Trasplante de Riñón , Ácido Micofenólico , Ciclosporina , Humanos , Inmunosupresores , Tacrolimus
8.
Mycoses ; 63(2): 197-211, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31657052

RESUMEN

BACKGROUND: Fungal infections are highly prevalent and are responsible for high rates of morbidity and mortality. In this context, the search for new treatment alternatives is very relevant. OBJECTIVES: Analyse chemical compounds for antifungal potential against dermatomycosis fungi. METHODS: The antifungal activity of 121 compounds, intermediates or derivatives of 1,3-bis(aryloxy)propane substituted at C-2 (111 compounds) and isothiouronium derivatives (10 compounds) was investigated through susceptibility tests, mechanism of action, toxicity and hydrogel incorporation. RESULTS: The compound 1,3-bis(3,4-dichlorophenoxy)propan-2-aminium chloride (2j) was the most active fungicide against dermatophytes and Candida spp., at very low concentrations (0.39-3.12 µg/mL), including action on resistant and multidrug-resistant clinical strains. Compound 2j has presented a promising toxicity profile, showing selectivity index >10, relative to human lymphocytes. The compound was classified as non-irritant by the HET-CAM test and did not cause histopathological alterations in pig ear skin, thus presenting an excellent perspective for topical application. 2j targets the fungal cell wall, which was confirmed by scanning electron microscopy, which also indicated the additional ability of 2j to inhibit the Candida albicans pseudohyphae formation and biofilm of Microsporum canis. Compound 2j was incorporated in a hydrogel with bioadhesive potential. The results of the human skin permeation showed that 2j remained significantly in the epidermis, ideally for the dermatomycosis treatment. CONCLUSIONS: Therefore, the compound 2j demonstrated the potential for antifungal drug development, with a action mechanism elucidated and already applied in a semisolid formulation as a new therapeutic option for fungal skin infections.


Asunto(s)
Antifúngicos/farmacología , Arthrodermataceae/efectos de los fármacos , Candida/efectos de los fármacos , Linfocitos/efectos de los fármacos , Propano/análogos & derivados , Animales , Antifúngicos/química , Supervivencia Celular , Células Cultivadas , Pollos , Membrana Corioalantoides/efectos de los fármacos , Oído Externo/efectos de los fármacos , Epidermis/efectos de los fármacos , Ergosterol/metabolismo , Femenino , Citometría de Flujo , Humanos , Hidrogeles , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Masculino , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Propano/química , Propano/farmacología , Reología , Relación Estructura-Actividad , Porcinos
9.
BMC Biotechnol ; 19(1): 33, 2019 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-31174517

RESUMEN

BACKGROUND: Foot-and-mouth disease is a highly contagious and economically devastating disease with endemic occurrence in many parts of the world. Vaccination is the method of choice to eradicate the disease and to limit the viral spread. The vaccine production process is based on mammalian cell culture, in which the viral yield varies in dependence of the composition of the culture media. For foot-and-mouth disease virus (FMDV), very little is known about the culture media components that are necessary to grow the virus to high titers in cell culture. RESULTS: This study examined the influence of increasing concentrations of glucose, glutamine, ammonium chloride and different cell densities on the yield of FMDV. While an excess of glucose or glutamine does not affect the viral yield, increasing cell density reduces the viral titer by a log10 step at a cell density of 3 × 106 cells/mL. This can be mitigated by performing a 100% media exchange before infection of the cells. CONCLUSIONS: The reasons for the diminished viral growth, if no complete media exchange has been performed prior to infection, remain unclear and further studies are necessary to investigate the causes more deeply. For now, the results argue for a vaccine production process with 100% media exchange to reliably obtain high viral titers.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Vacunas Virales/inmunología , Replicación Viral/inmunología , Compuestos de Amonio/farmacología , Animales , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Cricetinae , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Fiebre Aftosa/inmunología , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/metabolismo , Virus de la Fiebre Aftosa/fisiología , Glucosa/farmacología , Glutamina/farmacología , Vacunación/métodos , Vacunas Virales/administración & dosificación , Vacunas Virales/metabolismo , Replicación Viral/efectos de los fármacos
10.
Biotechnol Bioeng ; 116(4): 816-830, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30552760

RESUMEN

Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half-life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed-batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high-mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2-F-peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.


Asunto(s)
Medios de Cultivo/metabolismo , Inmunoglobulina G/metabolismo , Animales , Reactores Biológicos , Biotecnología/métodos , Células CHO , Técnicas de Cultivo de Célula/métodos , Cricetinae , Cricetulus , Medios de Cultivo/química , Glicosilación , Humanos , Inmunoglobulina G/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
11.
Biotechnol Bioeng ; 116(6): 1537-1555, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30793282

RESUMEN

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.


Asunto(s)
Medios de Cultivo/química , Vitaminas/química , Animales , Biotecnología/métodos , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/metabolismo , Estabilidad de Medicamentos , Excipientes/química , Excipientes/metabolismo , Calor , Humanos , Luz , Oxígeno/metabolismo , Vitaminas/metabolismo
12.
Eur J Clin Pharmacol ; 75(4): 553-559, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30610275

RESUMEN

PURPOSE: Mycophenolic acid is one of the most used immunosuppressive drugs in solid organ transplant treatments in the world. Developing a highly sensitive analytical method to analyse the drug and its metabolites in oral fluid and plasma is important to evaluate the possibility of using oral fluid as a biological matrix in therapeutic drug monitoring, instead of plasma. METHOD: The liquid chromatography coupled to mass spectrometry (LC-MS) method was developed and validated for determining mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) in oral fluid and plasma, with both matrices presenting a detection limit of 1 ng/mL for MPA and 5 ng/mL for MPAG. Both analytes were analysed after a simple protein precipitation procedure. Transplanted-kidney samples of oral fluid and blood were collected from 13 patients that were hospitalised and kept at - 80 °C until analyses. RESULTS: The proposed method was linear in the concentration range of 5-500 ng/mL for MPA and 10-500 ng/mL for MPAG, with correlation coefficients (r) between 0.9925 and 0.9973. It was then applied to samples collected from kidney-transplanted patients and used for calculation of pharmacokinetics parameters. CONCLUSION: After comparing plasma and oral fluid concentrations as well as performing a non-compartmental pharmacokinetic analysis of the average curves, it is possible to suggest that oral fluid concentration may be used as an alternative for MPA and MPAG monitoring in kidney transplant patients.


Asunto(s)
Glucurónidos/metabolismo , Trasplante de Riñón , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacocinética , Saliva/metabolismo , Cromatografía Liquida/métodos , Glucurónidos/análisis , Glucurónidos/sangre , Glucurónidos/farmacocinética , Humanos , Ácido Micofenólico/análisis , Ácido Micofenólico/sangre , Espectrometría de Masas en Tándem/métodos
13.
Virol J ; 15(1): 46, 2018 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-29548334

RESUMEN

BACKGROUND: Suspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease. METHODS: Changes to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation. RESULTS: Capsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A24-2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media. CONCLUSION: The selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization profile were unaffected, but the 146S particle yield differed for one of the tested serotypes.


Asunto(s)
Virus de la Fiebre Aftosa/fisiología , Fiebre Aftosa/inmunología , Fiebre Aftosa/virología , Vacunas Virales/inmunología , Animales , Células CHO , Cápside/química , Cápside/inmunología , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Línea Celular , Células Cultivadas , Cricetulus , Fiebre Aftosa/prevención & control , Virus de la Fiebre Aftosa/clasificación , Modelos Moleculares , Mutación , Pruebas de Neutralización , Conformación Proteica , Virión , Replicación Viral
14.
Horm Behav ; 66(2): 383-92, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24937439

RESUMEN

Nandrolone decanoate (ND), an anabolic androgenic steroid (AAS), induces an aggressive phenotype by mechanisms involving glutamate-induced N-methyl-d-aspartate receptor (NMDAr) hyperexcitability. The astrocytic glutamate transporters remove excessive glutamate surrounding the synapse. However, the impact of supraphysiological doses of ND on glutamate transporters activity remains elusive. We investigated whether ND-induced aggressive behavior is interconnected with GLT-1 activity, glutamate levels and abnormal NMDAr responses. Two-month-old untreated male mice (CF1, n=20) were tested for baseline aggressive behavior in the resident-intruder test. Another group of mice (n=188) was injected with ND (15mg/kg) or vehicle for 4, 11 and 19days (short-, mid- and long-term endpoints, respectively) and was evaluated in the resident-intruder test. Each endpoint was assessed for GLT-1 expression and glutamate uptake activity in the frontoparietal cortex and hippocampal tissues. Only the long-term ND endpoint significantly decreased the latency to first attack and increased the number of attacks, which was associated with decreased GLT-1 expression and glutamate uptake activity in both brain areas. These alterations may affect extracellular glutamate levels and receptor excitability. Resident males were assessed for hippocampal glutamate levels via microdialysis both prior to, and following, the introduction of intruders. Long-term ND mice displayed significant increases in the microdialysate glutamate levels only after exposure to intruders. A single intraperitoneal dose of the NMDAr antagonists, memantine or MK-801, shortly before the intruder test decreased aggressive behavior. In summary, long-term ND-induced aggressive behavior is associated with decreased extracellular glutamate clearance and NMDAr hyperexcitability, emphasizing the role of this receptor in mediating aggression mechanisms.


Asunto(s)
Agresión/efectos de los fármacos , Anabolizantes/farmacología , Espacio Extracelular/metabolismo , Ácido Glutámico/metabolismo , Homeostasis/efectos de los fármacos , Nandrolona/farmacología , Animales , Química Encefálica/efectos de los fármacos , Transportador 1 de Aminoácidos Excitadores/metabolismo , Espacio Extracelular/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Actividad Motora/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos
15.
Biomed Chromatogr ; 28(12): 1728-37, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24788676

RESUMEN

This study describes and characterizes methods for high-performance liquid chromatography diode array detection (HPLC-DAD) analysis of formulations containing molecules with antifungal activity of three different classes: terbinafine and butenafine (allylamines), miconazole and fluconazole (azoles), and geraniol, neral and geranial (monoterpenes). All methods used the same chromatographic column (RP18 ), enabling the analysis to be performed in a single batch. The specificity was extensively discussed through the establishment of purity peak methods. The analytical parameters (linearity, precision and accuracy) were calculated and discussed in detail using specific statistical approaches. All substances showed satisfactory results for chromatographic and analytical parameters. Limits of 1.3% to mean repeatability and 2.0% for intermediate precision are suggested as acceptance criteria in validation of methods by HPLC-DAD, in situations where there is no extensive pretreatment of the samples. The methods proved to be robust and significant factors were discussed regarding their influence on chromatographic parameters (retention time, resolution, tailing factor and column efficiency). Finally, the application of the developed methods was demonstrated by the results of a permeation study of the antifungal agents through bovine hoof membranes.


Asunto(s)
Antifúngicos/análisis , Cromatografía Líquida de Alta Presión/métodos , Animales , Antifúngicos/química , Antifúngicos/farmacocinética , Bovinos , Pezuñas y Garras/metabolismo , Concentración de Iones de Hidrógeno , Modelos Lineales , Permeabilidad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
16.
J Immunol ; 186(7): 3966-76, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-21368225

RESUMEN

We characterized a new pathway to induce tolerogenic dendritic cells (DCs) following treatment of human monocyte-derived DCs with proteases from the fungus Aspergillus oryzae (ASP). ASP-treated DCs (ASP-DCs) exhibit a CD80(-)CD83(-)CD86(-)Ig-like transcript (ILT)2(-)ILT3(-)ILT4(+) phenotype, do not secrete cytokines or chemokines, and express tolerogenic markers such as glucocorticoid-induced leucine zipper, NO synthetase-2, retinaldehyde dehydrogenase-1 or retinaldehyde dehydrogenase-2. When cocultured with naive CD4(+) T cells, ASP-DCs induce an anergic state that can be reversed by IL-2. Generated T cells mediate a suppressive activity in third-party experiments that is not mediated by soluble factors. A comparison between dexamethasone-treated DCs used as a reference for regulatory T cell-inducing DCs and ASP-DCs reveals two distinct phenotypes. In contrast to dexamethasone, ASP treatment induces glucocorticoid-induced leucine zipper independently of glucocorticoid receptor engagement and leads to NF-κB p65 degradation. Abrogation of protease activities in ASP using specific inhibitors reveals that aspartic acid-containing proteases are key inducers of regulatory genes, whereas serine, cysteine, and metalloproteases contribute to NF-κB p65 degradation. Collectively, those features correspond to a previously unreported anergizing phenotype for human DCs. Such regulatory mechanisms may allow fungi to downregulate host immune responses and provide clues for new approaches to treat proinflammatory disorders.


Asunto(s)
Aspergillus oryzae/enzimología , Aspergillus oryzae/inmunología , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Tolerancia Inmunológica , Inmunofenotipificación , Péptido Hidrolasas/fisiología , Aspergillus oryzae/genética , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/microbiología , Dexametasona/farmacología , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/fisiología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/genética , Péptido Hidrolasas/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Linfocitos T/inmunología , Transfección
17.
J Allergy Clin Immunol ; 129(4): 1020-30, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22464673

RESUMEN

BACKGROUND: Given their pivotal role in the polarization of T-cell responses, molecular changes at the level of dendritic cells (DCs) could represent an early signature indicative of the subsequent orientation of adaptive immune responses during immunotherapy. OBJECTIVE: We sought to investigate whether markers of effector and regulatory DCs are affected during allergen immunotherapy in relationship with clinical benefit. METHODS: Differential gel electrophoresis and label-free mass spectrometry approaches were used to compare whole proteomes from human monocyte-derived DCs differentiated toward either regulatory or effector functions. The expression of those markers was assessed by using quantitative PCR in PBMCs from 79 patients with grass pollen allergy enrolled in a double-blind, placebo-controlled clinical study evaluating the efficacy of sublingual tablets in an allergen exposure chamber over a 4-month period. RESULTS: We identified several markers associated with DC1 and/or DC17 effector DCs, including CD71, FSCN1, IRF4, NMES1, MX1, TRAF1. A substantial phenotypic heterogeneity was observed among various types of tolerogenic DCs, with ANXA1, Complement component 1 (C1Q), CATC, GILZ, F13A, FKBP5, Stabilin-1 (STAB1), and TPP1 molecules established as shared or restricted regulatory DC markers. The expression of 2 of those DCs markers, C1Q and STAB1, was increased in PBMCs from clinical responders in contrast to that seen in nonresponders or placebo-treated patients. CONCLUSION: C1Q and STAB1 represent candidate biomarkers of early efficacy of allergen immunotherapy as the hallmark of a regulatory innate immune response predictive of clinical tolerance.


Asunto(s)
Biomarcadores/metabolismo , Células Dendríticas/inmunología , Desensibilización Inmunológica/métodos , Administración Sublingual , Células Dendríticas/clasificación , Células Dendríticas/metabolismo , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Hipersensibilidad/terapia , Tolerancia Inmunológica/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Proteoma/metabolismo , Resultado del Tratamiento , Tripeptidil Peptidasa 1
18.
Biotechnol Adv ; 65: 108141, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37001570

RESUMEN

Chinese hamster ovary (CHO) cells are the preferred mammalian host for the large-scale production of recombinant proteins in the biopharmaceutical industry. Research endeavors have been directed to the optimization of CHO-based bioprocesses to increase protein quantity and quality, often in an empirical manner. To provide a rationale for those achievements, a myriad of CHO proteomic studies has arisen in recent decades. This review gives an overview of significant advances in LC-MS-based proteomics and sheds light on CHO proteomic studies, with a particular focus on CHO cells with superior bioprocessing phenotypes (growth, viability, titer, productivity and cQA), that have exploited novel proteomic or sub-omic techniques. These proteomic findings expand the current knowledge and understanding about the underlying protein clusters, protein regulatory networks and biological pathways governing such phenotypic changes. The proteomic studies, highlighted herein, will help in the targeted modulation of these cell factories to the desired needs.


Asunto(s)
Proteómica , Cricetinae , Animales , Cricetulus , Células CHO , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fenotipo
19.
Cells ; 12(2)2023 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-36672269

RESUMEN

Thiamin is susceptible to heat and oxidation, which is a concern for the development of concentrated and room temperature stable feeds used to produce recombinant proteins. Hence, it is critical to understand the reactivity and necessity of the vitamin in liquid feeds to be able to either develop mitigation strategies to stabilize the vitamin or to remove thiamin from formulations if it is unnecessary. LC-MS/MS was used to investigate thiamin stability in different liquid feed formulations and to identify thiamin degradation products. Results indicate oxidation of thiamin and interaction with amino acids, keto acids, and sulfur containing components. Thiamin necessity in feed was assessed during a fed batch experiment, focusing on cell performance and critical quality attributes of the produced recombinant proteins. The impact of thiamin depletion in the feed on the intra- and extracellular metabolome was investigated using untargeted LC-MS/MS. Results indicate that thiamin can be removed from the feed without affecting the performance or the intra- and extracellular metabolome of the tested cell lines. Overall, profound insights on thiamin reactivity and necessity are presented in this study, suggesting the removal of the dispensable and instable vitamin as a simple means for the development of next generation feeds used to produce therapeutic biological entities.


Asunto(s)
Espectrometría de Masas en Tándem , Tiamina , Tiamina/metabolismo , Cromatografía Liquida , Técnicas de Cultivo de Célula , Vitaminas , Proteínas Recombinantes
20.
Front Bioeng Biotechnol ; 11: 1230422, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37680342

RESUMEN

S-Sulfocysteine (SSC), a bioavailable L-cysteine derivative (Cys), is known to be taken up and metabolized in Chinese hamster ovary (CHO) cells used to produce novel therapeutic biological entities. To gain a deeper mechanistic insight into the SSC biological activity and metabolization, a multi-omics study was performed on industrially relevant CHO-K1 GS cells throughout a fed-batch process, including metabolomic and proteomic profiling combined with multivariate data and pathway analyses. Multi-layered data and enzymatical assays revealed an intracellular SSC/glutathione mixed disulfide formation and glutaredoxin-mediated reduction, releasing Cys and sulfur species. Increased Cys availability was directed towards glutathione and taurine synthesis, while other Cys catabolic pathways were likewise affected, indicating that cells strive to maintain Cys homeostasis and cellular functions.

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