RESUMEN
Fibryga® is a new lyophilized fibrinogen concentrate for intravenous use for the treatment of congenital fibrinogen deficiency. fibryga® is produced from pooled human plasma and the final product is characterized by high purity, integrity, and pathogen safety. Functional activity of fibrinogen was demonstrated by cross-linking studies and thromboelastometry; integrity of the fibrinogen molecule was demonstrated by size exclusion chromatography and the detection of only trace amounts of activation markers in the final product. Pathogen safety of fibryga® was proved by downscaling studies for the two dedicated pathogen inactivation/removal steps, i.e. solvent detergent treatment and nanofiltration. Fibryga® is stable for at least three years when stored at room temperature. In conclusion, the performed studies demonstrated that fibryga® meets the requirements for a state-of-the-art fibrinogen concentrate, such as a satisfactory activity profile combined with a favorable pathogen safety profile and stability.
Asunto(s)
Desinfección/métodos , Fibrinógeno/química , Fibrinógeno/aislamiento & purificación , Estabilidad de Medicamentos , Humanos , Tromboelastografía/métodosRESUMEN
Collapsin response mediator proteins (CRMPs) are involved in cell differentiation and axonal growth. In this study, we investigated neurite outgrowth and the expression of CRMP-2 in PC12 cells. Nondifferentiated PC12 cells hardly express CRMP-2, but the expression of CRMP-2 increases with neurite outgrowth. We established a stable CRMP-2-overexpressing PC12 cell line and found that PC12 cells, which constitutively overexpress CRMP-2, were unable to extend neurites after stimulation with the nerve growth factor or the neural cell adhesion molecule, a procedure that promotes neurite outgrowth in untransfected cells. In contrast, a PC12 cell line deficient in CRMP-2 extends neurites after the stimulation of nerve growth factor or neural cell adhesion molecule.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/fisiología , Animales , Western Blotting , Recuento de Células , Aumento de la Célula , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Humanos , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Factor de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células PC12 , Ratas , TransfecciónRESUMEN
The most consistent neurochemical abnormality in Parkinson's disease is degeneration of dopaminergic neurons in the substantia nigra, leading to a reduction of striatal dopamine levels. The rate-limiting step in the biosynthesis of dopamine, noradrenalin, and adrenalin is catalyzed by tyrosine 3-monooxygenase (=tyrosine hydroxylase), which catalyzes the formation of L-DOPA. In earlier studies, we demonstrated that the novel synthetic sialic acid precursor N-propanoylmannosamine is a potent stimulator of axonal growth and promotes reestablishment of the perforant pathway from layer II of cortical neurons to the outer molecular layer of the dentate gyrus. Here we show that application of N-propanoylmannosamine leads to increased biosynthesis and secretion of dopamine. This increased biosynthesis of dopamine is due to decreased expression of O-linked N-acetylglucosamine on tyrosine 3-monooxygenase. Intracellular attachment of O-linked N-acetylglucosamine to serine and threonine residues hinders phosphorylation, thereby regulating the activity of the proteins concerned. We therefore propose a model in which the application of ManNProp leads to increased phosphorylation and activation of tyrosine 3-monooxygenase, which in turn leads to an increased synthesis of dopamine.
Asunto(s)
Acetilglucosamina/metabolismo , Dopamina/metabolismo , Glicoproteínas/metabolismo , Hexosaminas/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Animales , Dopamina/biosíntesis , Regulación hacia Abajo , Activación Enzimática , Células PC12 , Fosforilación/efectos de los fármacos , RatasRESUMEN
Neurite outgrowth (e.g. axonal or dendrite outgrowth) of neurons is necessary for the development and functioning of the central nervous system. It is well accepted that the differentiation of neurons and neurite outgrowth involve alterations in gene expression. Furthermore, mitochondria play a role in different aspects of neurite outgrowth. Here we show that the expression of Ndufb11, a gene encoding the mitochondrial protein NP15.6 is decreased in the course of neuronal differentiation. NP15.6 is homologous to the bovine protein ESSS, a component of the mitochondrial complex 1. The homologous human NDUFB11 gene is localized to Xp11.3-Xp11.23, a region associated with neurogenetic disorders. The down-regulation of NP15.6 correlates with neurite outgrowth of PC12 cells induced by nerve growth factor. Furthermore, we analyzed the expression of Ndufb11 in the embryonic and adult mouse.
Asunto(s)
Complejo I de Transporte de Electrón/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Neuritas/metabolismo , Animales , Células CHO , Diferenciación Celular , Cricetinae , Cricetulus , Embrión de Mamíferos/metabolismo , Hibridación in Situ , Proteínas Luminiscentes/genética , Ratones , Neuronas/citología , Células PC12 , Ratas , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Polysialic acid (polySia) is a unique and highly regulated posttranslational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration and plastic processes including learning and memory. Polysialylated NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. However, the impact of each enzyme in NCAM polysialylation is not understood. Here, we describe the selective cell-based in vitro inhibition of ST8SiaII using synthetic sialic acid precursors. We provide evidence for different substrate affinities of ST8SiaII and ST8SiaIV. These data open the possibility to study the individual role of the two enzymes during various aspects of brain development and function and in tumorigenesis.
Asunto(s)
Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/farmacología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Sialiltransferasas/antagonistas & inhibidores , Sialiltransferasas/metabolismo , Animales , Western Blotting , Células CHO , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Cricetinae , Células HL-60 , Hexosaminas/síntesis química , Hexosaminas/farmacología , Humanos , Estructura Molecular , Ácidos Neuramínicos/síntesis química , Ácidos Neuramínicos/farmacología , Neuronas/metabolismo , Células PC12 , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Ratas , Especificidad por SustratoRESUMEN
Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The precursor of all physiological sialic acids is N-acetyl-d-mannosamine. Using N-propanoyl mannosamine, a novel precursor of sialic acid, we showed earlier that sialic acids with a prolonged N-acyl side chain (e.g., N-propanoyl neuraminic acid) are incorporated into cell surface glycoconjugates. In this study, we report the structural and functional consequences of the incorporation of the nonphysiological sialic acid, N-propanoyl neuraminic acid, into glycoconjugates of HL60-I cells. These cells do not express UDP-GlcAc-2-epimerase, the key enzyme of the biosynthesis of N-acetyl-d-mannosamine. Therefore, they do not express sialyl-Lewis(x) structures and consequently do not bind to selectins. Application of N-acetyl-d-mannosamine leads to the expression of sialyl-Lewis(x) structures and to binding to selectins. Surprisingly, incorporation of N-propanoyl neuraminic acid into glycoconjugates of these cells leads to a dramatic increase of sialyl-Lewis(x) structures and to increased adhesion to selectins.
Asunto(s)
Selectina L/metabolismo , Ácido N-Acetilneuramínico/farmacocinética , Ingeniería de Proteínas/métodos , Ácidos Siálicos/metabolismo , Adhesión Celular , Epítopos , Expresión Génica , Células HL-60 , Humanos , Antígenos del Grupo Sanguíneo de Lewis , Ligandos , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas Recombinantes de Fusión/metabolismo , Selectinas/metabolismo , Ácidos Siálicos/química , Relación Estructura-ActividadRESUMEN
Sialylation is essential for development and regeneration in mammals. Using N-propanoylmannosamine, a novel precursor of sialic acid, we were able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into cell surface glycoconjugates. Here we report that this biochemical engineering of sialic acid leads to a stimulation of neuronal cells. Both PC12 cells and cerebellar neurons showed a significant increase in neurite outgrowth after treatment with this novel sialic acid precursor. Furthermore, also the reestablishment of the perforant pathway was stimulated in brain slices. In addition, we surprisingly identified several cytosolic proteins with regulatory functions, which are differentially expressed after treatment with N-propanoylmannosamine. Because sialic acid is the only monosaccharide that is activated in the nucleus, we hypothesize that transcription could be modulated by the unnatural CMP-N-propanoylneuraminic acid and that sialic acid activation might be a general tool to regulate cellular functions, such as neurite outgrowth.