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1.
Neurochem Res ; 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32130629

RESUMO

Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψm) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψm in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1-50 nM), caused depolarisation of Δψm. Small decreases in complex I activity caused an immediate and partial Δψm depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψm. A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψp) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψp were registered. These data indicate that in synaptosomes, both Δψm and Δψp are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson's disease in particular, where such reductions in complex I activity are present.

2.
J Neural Transm (Vienna) ; 127(2): 213-230, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894418

RESUMO

6-Hydroxydopamine (6-OHDA), which is a neurotoxin that selectively destroys catecholaminergic nerves in sympathetically innervated tissues, has been used to provide a model of Parkinson's disease in experimental animals. It is rapidly autoxidised to yield potentially toxic products and reactive oxygen species. Its ability to release Fe(II) from protein storage sites also results in the formation of hROS. This account will consider how this family of toxic products may contribute to the observed effects of 6-OHDA.

3.
Front Mol Neurosci ; 12: 219, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31619961

RESUMO

Mitochondrial dysfunction is a recognized hallmark of neurodegenerative diseases and abnormal mitochondrial fusion-fission dynamics have been implicated in the pathogenesis of neurodegenerative disorders. This study characterizes the effects of metabolic flux inhibitors and activators on mitochondrial fusion dynamics in the neuronal cell culture model of differentiated PC12 cells. Using a real time confocal microscopy assay, it was found that the carnitine palmitoyltransferase I (CPTI) inhibitor, etomoxir, reduced mitochondrial fusion dynamics in a time-dependent manner. Etomoxir also decreased JO2, ΔΨm and reactive oxygen species (ROS) production rates. The mitochondrial pyruvate carrier (MPC) inhibitor, UK5099, reduced fusion dynamics and in combination with etomoxir these inhibitory effects were amplified. Use of the pyruvate dehydrogenase (PDH) kinase inhibitor dichloroacetate, which is known to increase metabolic flux through PDH, reversed the etomoxir-induced effects on fusion dynamics, JO2, ΔΨm but not ROS production rates. Dichloroacetate also partially reversed inhibition of mitochondrial fusion dynamics caused by the parkinsonian-inducing neurotoxin, MPP+. These results suggest that dichloroacetate-induced activation of metabolic flux in the mitochondrion may be a mechanism to restore normal mitochondrial fusion-fission dynamics in metabolically challenged cells.

4.
Glycobiology ; 29(10): 726-734, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31245822

RESUMO

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers. Proteins obtained from whole saliva were subjected to PNGase F enzymatic digestion. The resulting N-glycans were analyzed with weak anion exchange chromatography, exoglycosidase digestions coupled to ultra-high performance liquid chromatography and/or mass spectrometry. To determine N-glycan changes, 23 individuals with or without cancerous oral lesions were analyzed using Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC), and peak-based area relative quantitation was performed. An abundant and complex salivary N-glycomic profile was identified. The main structures present in saliva were neutral oligosaccharides consisting of high mannose, hybrid and complex structures, followed by smaller fractions of mono and di-sialylated structures. To determine if differential N-glycosylation patterns distinguish between OC and control groups, Mann-Whitney testing and principle component analysis (PCA) were used. Eleven peaks were shown to be statistically significant (P ≤ 0.05), while PCA analysis showed segregation of the two groups based on their glycan profile. N-glycosylation changes are active in the oral carcinogenic process and may serve as biomarkers for early detection to reduce morbidity and mortality. Identifying which N-glycans contribute most in the carcinogenic process may lead to their use in the detection, prognosis and treatment of OC.

5.
PLoS Comput Biol ; 14(8): e1006348, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30074989

RESUMO

Glycosyltransferases are a class of enzymes that catalyse the posttranslational modification of proteins to produce a large number of glycoconjugate acceptors from a limited number of nucleotide-sugar donors. The products of one glycosyltransferase can be the substrates of several other enzymes, causing a combinatorial explosion in the number of possible glycan products. The kinetic behaviour of systems where multiple acceptor substrates compete for a single enzyme is presented, and the case in which high concentrations of an acceptor substrate are inhibitory as a result of abortive complex formation, is shown to result in non-Michaelian kinetics that can lead to bistability in an open system. A kinetic mechanism is proposed that is consistent with the available experimental evidence and provides a possible explanation for conflicting observations on the ß-1,4-galactosyltransferases. Abrupt switching between steady states in networks of glycosyltransferase-catalysed reactions may account for the observed changes in glycosyl-epitopes in cancer cells.


Assuntos
Glicosiltransferases/metabolismo , Glicosiltransferases/farmacocinética , Fenômenos Biofísicos/fisiologia , Catálise , Ativação Enzimática , Retroalimentação Fisiológica/fisiologia , Galactosiltransferases/metabolismo , Glicosilação , Glicosiltransferases/fisiologia , Humanos , Cinética , Especificidade por Substrato/fisiologia
6.
Mol Cell Proteomics ; 16(10): 1770-1788, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28576848

RESUMO

Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIaArg131/His131 (CD32a), RIIb (CD32b) and RIIIaPhe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIaPhe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIaPhe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a stabilizing role of FcγR glycans in the antibody binding interaction.


Assuntos
Polissacarídeos/imunologia , Receptores de IgG/imunologia , Rituximab/imunologia , Animais , Células CHO/metabolismo , Linhagem Celular , Cricetulus/imunologia , Glicosilação , Células HEK293 , Humanos , Imunidade Celular , Cinética , Camundongos , Polissacarídeos/metabolismo , Ligação Proteica , Receptores de IgG/metabolismo , Rituximab/metabolismo
7.
J Inflamm Res ; 9: 209-219, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27895507

RESUMO

Therapeutic antibodies hold great promise for the treatment of cancer and autoimmune diseases, and developments in antibody-drug conjugates and bispecific antibodies continue to enhance treatment options for patients. Immunoglobulin (Ig) G antibodies are proteins with complex modifications, which have a significant impact on their function. The most important of these modifications is glycosylation, the addition of conserved glycans to the antibody Fc region, which is critical for its interaction with the immune system and induction of effector activities such as antibody-dependent cell cytotoxicity, complement activation and phagocytosis. Communication of IgG antibodies with the immune system is controlled and mediated by Fc gamma receptors (FcγRs), membrane-bound proteins, which relay the information sensed and gathered by antibodies to the immune system. These receptors are also glycoproteins and provide a link between the innate and adaptive immune systems. Recent information suggests that this receptor glycan modification is also important for the interaction with antibodies and downstream immune response. In this study, the current knowledge on FcγR glycosylation is discussed, and some insight into its role and influence on the interaction properties with IgG, particularly in the context of biotherapeutics, is provided. For the purpose of this study, other Fc receptors such as FcαR, FcεR or FcRn are not discussed extensively, as IgG-based antibodies are currently the only therapeutic antibody-based products on the market. In addition, FcγRs as therapeutics and therapeutic targets are discussed, and insight into and comment on the therapeutic aspects of receptor glycosylation are provided.

8.
Curr Opin Struct Biol ; 40: 97-103, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27620650

RESUMO

Glycosylation is a common post-translational protein modification, in which glycans are built onto proteins through the sequential addition of monosaccharide units, in reactions catalysed by glycosyltransferases. Glycosylation influences the physicochemical and biological properties of proteins, with subsequent effects on subcellular and extracellular protein trafficking, cell-cell recognition, and ligand-receptor interactions. Glycan structures can be complex, as is the regulation of their biosynthesis, and it is only recently that the systems biology of metabolic flux control and glycosyltransferase networks has become a study in its own right. We review various models of glycosylation that have been proposed to date, based on current knowledge of Golgi structure and function, and consider how metabolic flux through glycosyltransferase networks regulates glycosylation events in the cell.


Assuntos
Glicosilação , Análise do Fluxo Metabólico/métodos , Animais , Enzimas/metabolismo , Humanos , Modelos Biológicos
9.
PLoS Comput Biol ; 12(4): e1004844, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27054587

RESUMO

O-linked glycosylation is an important post-translational modification of mucin-type protein, changes to which are important biomarkers of cancer. For this study of the enzymes of O-glycosylation, we developed a shorthand notation for representing GalNAc-linked oligosaccharides, a method for their graphical interpretation, and a pattern-matching algorithm that generates networks of enzyme-catalysed reactions. Software for generating glycans from the enzyme activities is presented, and is also available online. The degree distributions of the resulting enzyme-reaction networks were found to be Poisson in nature. Simple graph-theoretic measures were used to characterise the resulting reaction networks. From a study of in-silico single-enzyme knockouts of each of 25 enzymes known to be involved in mucin O-glycan biosynthesis, six of them, ß-1,4-galactosyltransferase (ß4Gal-T4), four glycosyltransferases and one sulfotransferase, play the dominant role in determining O-glycan heterogeneity. In the absence of ß4Gal-T4, all Lewis X, sialyl-Lewis X, Lewis Y and Sda/Cad glycoforms were eliminated, in contrast to knockouts of the N-acetylglucosaminyltransferases, which did not affect the relative abundances of O-glycans expressing these epitopes. A set of 244 experimentally determined mucin-type O-glycans obtained from the literature was used to validate the method, which was able to predict up to 98% of the most common structures obtained from human and engineered CHO cell glycoforms.


Assuntos
Bases de Conhecimento , Mucinas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Células CHO , Biologia Computacional , Simulação por Computador , Cricetulus , Técnicas de Inativação de Genes , Engenharia Genética , Glicosilação , Glicosiltransferases/deficiência , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Humanos , Redes e Vias Metabólicas/genética , Modelos Biológicos , Mucinas/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Terminologia como Assunto
10.
J Biol Chem ; 290(47): 28343-52, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26221033

RESUMO

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.


Assuntos
Amiloide/metabolismo , Biopolímeros/metabolismo , Morte Celular , Amiloide/química , Animais , Biopolímeros/química , Células PC12 , Ratos
11.
J Proteome Res ; 13(12): 5471-85, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25345863

RESUMO

FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa(Phe158/Val158), and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIa(Phe158/Val158) receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIa(Phe158) was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIa(Val158) remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.


Assuntos
Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Anticorpos Monoclonais Murinos/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Glicosilação , Células HEK293 , Humanos , Cinética , Mutação , Polissacarídeos/metabolismo , Ligação Proteica , Receptores de IgG/genética , Proteínas Recombinantes/metabolismo , Rituximab , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem
12.
J Cell Sci ; 127(Pt 23): 5014-26, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25271059

RESUMO

Protein N-glycosylation is a common post-translational modification that produces a complex array of branched glycan structures. The levels of branching, or antennarity, give rise to differential biological activities for single glycoproteins. However, the precise mechanism controlling the glycan branching and glycosylation network is unknown. Here, we constructed quantitative mathematical models of N-linked glycosylation that predicted new control points for glycan branching. Galactosyltransferase, which acts on N-acetylglucosamine residues, was unexpectedly found to control metabolic flux through the glycosylation pathway and the level of final antennarity of nascent protein produced in the Golgi network. To further investigate the biological consequences of glycan branching in nascent proteins, we glycoengineered a series of mammalian cells overexpressing human chorionic gonadotropin (hCG). We identified a mechanism in which galactosyltransferase 4 isoform regulated N-glycan branching on the nascent protein, subsequently controlling biological activity in an in vivo model of hCG activity. We found that galactosyltransferase 4 is a major control point for glycan branching decisions taken in the Golgi of the cell, which might ultimately control the biological activity of nascent glycoprotein.


Assuntos
Gonadotropina Coriônica/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismo , Animais , Células CHO , Gonadotropina Coriônica/química , Gonadotropina Coriônica/genética , Gonadotropina Coriônica/farmacologia , Simulação por Computador , Cricetulus , Glicosilação , Células HEK293 , Humanos , Isoenzimas , Cinética , Masculino , Modelos Biológicos , Modelos Moleculares , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/genética , Conformação Proteica , Ratos , Glândulas Seminais/efeitos dos fármacos , Glândulas Seminais/crescimento & desenvolvimento , Relação Estrutura-Atividade , Transfecção
13.
Curr Top Microbiol Immunol ; 382: 165-99, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25116100

RESUMO

Immunoglobulins and Fc receptors are critical glycoprotein components of the immune system. Fc receptors bind the Fc (effector) region of antibody molecules and communicate information within the innate and adaptive immune systems. Glycosylation of antibodies, particularly in the Fc region of IgG, has been extensively studied in health and disease. The N-glycans in the identical heavy chains have been shown to be critical for maintaining structural integrity, communication with the Fc receptor and the downstream immunological response. Less is known about glycosylation of the Fc receptor in either healthy or disease states, however, recent studies have implicated an active role for receptor associated oligosaccharides in the antibody-receptor interaction. Research into Fc receptor glycosylation is increasing rapidly, where Fc receptors are routinely used to analyze the binding of therapeutic monoclonal antibodies and where glycosylation of receptors expressed by cells of the immune system could potentially be used to mediate and control the differential binding of immunoglobulins. Here we discuss the glycosylation of immunoglobulin antibodies (IgA, IgE, IgG) and the Fc receptors (FcαR, FcεR, FcγR, FcRn) that bind them, the function of carbohydrates in the immune response and recent advances in our understanding of these critical glycoproteins.


Assuntos
Receptores Fc/metabolismo , Animais , Glicosilação , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia
15.
Biochem Soc Trans ; 40(4): 746-51, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22817727

RESUMO

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.


Assuntos
Lactalbumina/química , Lactalbumina/metabolismo , Ácido Oleico/química , Ácido Oleico/metabolismo , Ácidos Oleicos/química , Ácidos Oleicos/metabolismo , Animais , Apoptose , Bovinos , Humanos , Dobramento de Proteína
16.
Methods Mol Biol ; 899: 325-50, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22735963

RESUMO

Glycans are crucial to the functioning of multicellular organisms. They may also play a role as mediators between host and parasite or symbiont. As many proteins (>50%) are posttranslationally modified by glycosylation, this mechanism is considered to be the most widespread posttranslational modification in eukaryotes. These surface modifications alter and regulate structure and biological activities/functions of proteins/biomolecules as they are largely involved in the recognition process of the appropriate structure in order to bind to the target cells. Consequently, the recognition of glycans on cellular surfaces plays a crucial role in the promotion or inhibition of various diseases and, therefore, glycosylation itself is considered to be a critical protein quality control attribute for commercial therapeutics, which is one of the fastest growing segments in the pharmaceutical industry. With the development of glycobiology as a separate discipline, a number of databases and tools became available in a similar way to other well-established "omics." Alleviating the recognized shortcomings of the available tools for data storage and retrieval is one of the highest priorities of the international glycoinformatics community. In the last decade, major efforts have been made, by leading scientific groups, towards the integration of a number of major databases and tools into a single portal, which would act as a centralized data repository for glycomics, equipped with a number of comprehensive analytical tools for data systematization, analysis, and comparison. This chapter provides an overview of the most important carbohydrate-related databases and glycoinformatic tools.


Assuntos
Bases de Dados Factuais , Glicômica , Glicoproteínas , Polissacarídeos/química , Biologia Computacional , Bases de Dados como Assunto , Glicoproteínas/química , Glicoproteínas/uso terapêutico , Glicosilação , Internet , Estrutura Molecular , Polissacarídeos/análise , Software
17.
BMC Biotechnol ; 11: 95, 2011 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-22008152

RESUMO

BACKGROUND: The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. RESULTS: Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. CONCLUSIONS: The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.


Assuntos
Técnicas de Cultura de Células/métodos , Gonadotropina Coriônica/metabolismo , Glucose/metabolismo , Glutamina/deficiência , Glicólise , Proteínas Recombinantes/metabolismo , Uridina Difosfato N-Acetilgalactosamina/análise , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Glutamina/análise , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Nucleotídeos/metabolismo , Polissacarídeos/metabolismo , Uridina Difosfato N-Acetilgalactosamina/biossíntese
18.
Int Rev Neurobiol ; 100: 43-64, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21971002

RESUMO

Monoamine oxidase (MAO) inhibitors have proven to be valuable tools in pharmacology and therapeutics. This account concerns the behavior of the different types of reversible inhibitor and how an understanding of the kinetic mechanisms of MAO may help in their design.


Assuntos
Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/química , Monoaminoxidase/metabolismo , Animais , Humanos , Cinética , Monoaminoxidase/farmacocinética , Inibidores da Monoaminoxidase/síntese química , Inibidores da Monoaminoxidase/metabolismo , Relação Estrutura-Atividade
19.
Mol Neurodegener ; 6(1): 53, 2011 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-21791084

RESUMO

BACKGROUND: The activities of mitochondrial complex III (ubiquinol-cytochrome c reductase, EC 1.10.2.2) and complex IV (cytochrome c oxidase EC 1.9.3.1) are reduced by 30-70% in Huntington's disease and Alzheimer's disease, respectively, and are associated with excitotoxic cell death in these disorders. In this study, we investigated the control that complexes III and complex IV exert on glutamate release from the isolated nerve terminal. RESULTS: Inhibition of complex III activity by 60-90% was necessary for a major increase in the rate of Ca2+-independent glutamate release to occur from isolated nerve terminals (synaptosomes) depolarized with 4-aminopyridine or KCl. Similarly, an 85-90% inhibition of complex IV activity was required before a major increase in the rate of Ca2+-independent glutamate release from depolarized synaptosomes was observed. Inhibition of complex III and IV activities by ~ 60% and above was required before rates of glutamate efflux from polarized synaptosomes were increased. CONCLUSIONS: These results suggest that nerve terminal mitochondria possess high reserves of complex III and IV activity and that high inhibition thresholds must be reached before excess glutamate is released from the nerve terminal. The implications of the results in the context of the relationship between electron transport chain enzyme deficiencies and excitotoxicity in neurodegenerative disorders are discussed.

20.
Free Radic Biol Med ; 50(7): 899-902, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21145387

RESUMO

Glutathione is an important antioxidant in the brain that appears to be decreased, in conjunction with mitochondrial complex I activity, in Parkinson disease patients. In postmortem analysis, measurement of glutathione levels and complex I activity can be delayed up to 20h. We investigated whether depletion of glutathione in the preweanling rat induces a reduction in complex I activity in brain mitochondria and the effects that postmortem delay has on glutathione levels and electron transport chain activity. After injection with the glutamate-cysteine ligase inhibitor, buthionine sulfoximine (L-BSO), glutathione levels were decreased by 53% compared to the control values in whole-brain homogenates. During postmortem delay of 24h, in which animals were kept at 4°C, the levels of glutathione decreased in the control group by 58% and in the L-BSO-treated group by 79%. However, during this period, there were no changes in mitochondrial electron transport chain complex I, II-III, or IV activity in either group. These results suggest that a preexisting deficiency of glutathione or a loss of glutathione during postmortem delay does not influence mitochondrial respiratory chain activity in the brain.


Assuntos
Transporte de Elétrons , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutationa , Animais , Animais Lactentes/metabolismo , Encéfalo/metabolismo , Butionina Sulfoximina/administração & dosagem , Células Cultivadas , Complexo I de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/administração & dosagem , Glutamato-Cisteína Ligase/metabolismo , Glutationa/deficiência , Humanos , Masculino , Mitocôndrias/metabolismo , Oxirredução , Doença de Parkinson/metabolismo , Mudanças Depois da Morte , Ratos , Ratos Wistar
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