A microcarrier-based cell culture process for the production of a bovine respiratory syncytial virus vaccine

Veterinary viral vaccines generally comprise either attenuated or chemically inactivated viruses which have been propagated on mammalian cell substrates or specific pathogen free (SPF) eggs. New generation vaccines include chemically inactivated virally-infected whole cell vaccines. The NM57 cell line is a bovine nasal turbinate persistently infected (non-lytic infection) with a strain of the respiratory syncytial virus (RSV). The potential of microcarrier technology for the cultivation in bioreactors of this anchorage dependent cell line for RSV vaccine production has been investigated. Both Cytodex 3 and Cultispher S microcarriers proved most suitable from a selection of microcarriers as growth substrates for this NM57 cell line. Maximum cell densities of 4.12×105 cells ml-1and 5.52×105 cells ml-1 respectively were obtained using Cytodex 3 (3 g l-1) and and Cultispher S (1 g l-1) in 5 l bioreactor cultures. The fact that cell growth was less sensitive to agitation rate when cultured on Cultispher S microcarriers, and that cells were efficiently harvested from this microcarrier by an enzymatic method, suggested Cultispher S is suitable for further evaluation at larger bioreactor scales (>5 l) than that described here. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lithium activates the c-Jun NH2-terminal kinases in vitro and in the CNS in vivo

The therapeutic efficacy of lithium in the treatment of mood disorders is delayed and only observed after chronic administration, a temporal profile that suggests alterations at the genomic level. Lithium has been demonstrated to modulate AP-1 DNA binding activity as well as the expression of genes regulated by AP-1, but the mechanisms underlying these effects have not been fully elucidated. In the present study, we found that the lithium-induced increases in AP-1 DNA binding activity were accompanied by increases in p-cJun and cJun levels in SH-SY5Y cells. Lithium also increased cJun-mediated reporter gene expression in a dose-dependent manner, with significant effects observed at therapeutically relevant concentrations. Lithium's effects on cJun-mediated reporter gene expression in SH-SY5Y cells were more pronounced in the absence of myo-inositol and were blocked by protein kinase C (PKC) inhibitors and by cotransfection with a PKCalpha dominant-negative mutant. Chronic in vivo lithium administration increased AP-1 DNA binding activity in frontal cortex and hippocampus and also increased the levels of the phosphorylated, active forms of c-Jun NH2-terminal kinases (JNKs) in both brain regions. These results demonstrate that lithium activates the JNK signaling pathway in rat brain during chronic in vivo administration and in human cells of neuronal origin in vitro; in view of the role of JNKs in regulating various aspects of neuronal function and their well-documented role in regulating gene expression, these effects may play a major role in lithium's long-term therapeutic effects.     

Redistribution of a 120 kDa Phosphoprotein in the Parietal Cell Associated with Stimulation

When rabbit isolated gastric glands were stimulated via the cyclic AMP pathway, a phosphorylated protein band of about 120 kDa (pp120) was markedly increased in the apical membrane-rich fraction, concomitant with an increase in the amount of H,K-ATPase and the phosphorylation of the cytoskeletal protein ezrin in the same fraction. The cytosolic fraction, but not other membrane fractions, also contained a protein with common features to the membrane-bound pp120, i.e., comigration in two-dimensional gels with a pI of ∼4.5, anomalous mobility in SDS-PAGE, reactivity to antibodies, and phosphorylation, indicating that these two proteins were identical. The possibility that pp120 is vinculin was completely excluded. Using antibody against pp120, this protein was found to be almost exclusively in the gastric parietal cell. Biochemical and immunohistochemical analyses suggest that pp120 exists mainly in the cytosol, and that a small part of the protein binds to the apical membrane when the parietal cell is stimulated via the cyclic AMP pathway. In the presence of histone, purified pp120 produced phosphorylation on pp120 as well as histone. The inhibitor profile of this kinase activity is not consistent with any known kinase. We conclude that pp120 is closely associated with a new type of kinase, and translocates from cytosol to the apical membrane when the parietal cell is stimulated. Received: 9 September 1998/Revised: 29 December 1998     

Activation of cPLA2, PKC, and ERKs in the Rat Cerebral Cortex During Ischemia/Reperfusion

Release of the excitotoxic amino acid, glutamate, into the extracellular space during ischemia/reperfusion contributes to neuronal injury and death. To gain insights into the signal transduction pathways involved in glutamate release we examined the time course of changes in enzyme levels and activities of cPLA₂, PKC and ERKs in the rat cerebral cortex after four vessel (4VO) ischemia followed by reperfusion. Measurement both by enzymatic assay and Western blot analysis showed significant increases in the activity and protein levels of cPLA₂ during 10–20 min of ischemia. Activity remained elevated at 10 min and 20 min of reperfusion, whereas cPLA levels had returned to base line levels after 20 min of reperfusion. PKC activity increased significantly in the particulate, but not in the cytosolic, fractions both during ischemia and reperfusion. Increases in PKC levels were recorded in the particulate fraction during ischemia and reperfusion, and in the cytosolic fraction during ischemia. Western blot analysis with a phosphospecific antibody for characterization of MAPK (ERKs) activation revealed significantly increased phosphorylation of ERK₁, and ERK₂ in the particulate fraction, of ERK₂ in the cytosolic fraction, during ischemia and of both enzymes in the particulate and cytosolic fractions after 10 min of reperfusion. The relevance of the results to glutamate release is discussed.     

Polo-like kinase 3 is Golgi localized and involved in regulating Golgi fragmentation during the cell cycle

The Golgi apparatus undergoes extensive fragmentation during mitosis in animal cells. Protein kinases play a critical role during fragmentation of the Golgi apparatus. We reported here that Polo-like kinase 3 (Plk3) may be an important mediator during Golgi breakdown. Specifically, Plk3 was concentrated at the Golgi apparatus in HeLa and A549 cells during interphase. At the onset of mitosis, Plk3 signals disintegrated and redistributed in a manner similar to those of Golgi stacks. Nocodazole activated Plk3 kinase activity, correlating with redistribution of Plk3 signals and Golgi fragmentation. In addition, treatment with brefeldin A (BFA), a Golgi-specific poison, also resulted in disappearance of concentrated Plk3 signals. Plk3 interacted with giantin, a Golgi-specific protein. Expression of Plk3, but not the kinase-defective Plk3 (Plk3(K52R)), resulted in significant Golgi breakdown. Given its role in cell cycle progression, Plk3 may be a protein kinase involved in regulation of Golgi fragmentation during the cell cycle.     

Pervanadate-induced reverse translocation and tyrosine phosphorylation of phorbol ester-stimulated protein kinase C betaII are mediated by Src-family tyrosine kinases in porcine neutrophils

Protein kinase C (PKC), upon activation, translocates from the cytosol to the plasma membrane. Phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, is known to induce irreversible translocation of PKC to the plasma membrane, in contrast to the reversible translocation resulting from physiological stimuli and subsequent rapid return to the cytosol (reverse translocation). However, we have previously shown that tyrosine phosphatase (PTPase) inhibitors induce reverse translocation of PMA-stimulated PKCbetaII in porcine polymorphonuclear leukocytes (PMNs). In the present study, we showed that pervanadate, a potent PTPase inhibitor, also induces tyrosine phosphorylation of PMA-stimulated PKCbetaII in porcine PMNs. Furthermore, PP2, a specific inhibitor of Src-family tyrosine kinases (PTKs), was found to inhibit both pervanadate-induced reverse translocation and tyrosine phosphorylation of PMA-stimulated PKCbetaII, suggesting that these two pervanadate-induced responses are mediated by Src-family PTKs. Our findings provide novel insight into the relationship between the subcellular localization and tyrosine phosphorylation of PKC.     

Epigallocatechin-3-gallate, constituent of green tea, suppresses the LPS-induced phenotypic and functional maturation of murine dendritic cells through inhibition of mitogen-activated protein kinases and NF-kappaB

The effects of epigallocatechin-3-gallate (EGCG) on dendritic cells (DC) maturation were investigated. EGCG, in a dose-dependent manner, profoundly inhibited CD80, CD86, and MHC class I and II expression on bone marrow-derived murine myeloid DC. EGCG restored the decreased dextran-FITC uptake and inhibited enhanced IL-12 production by LPS-treated DC. EGCG-treated DC were poor stimulators of nai;ve allogeneic T-cell proliferation and reduced levels of IL-2 production in responding T cells. EGCG-pretreated DC inhibited LPS-induced MAPKs, such as ERK1/2, p38, JNK, and NF-kappaB p65 translocation. Therefore, the molecular mechanisms by which EGCG antagonized LPS-induced DC maturation appeared to involve the inhibition of MAPK and NF-kappaB activation. These novel findings provide new insight into the immunopharmacological role of EGCG and suggest a novel approach to the manipulation of DC for therapeutic application of autoimmune and allergic diseases.     

Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity

To generate industrially applicable new host cell lines for antibody production with optimizing antibody-dependent cellular cytotoxicity (ADCC) we disrupted both FUT8 alleles in a Chinese hamster ovary (CHO)/DG44 cell line by sequential homologous recombination. FUT8 encodes an alpha-1,6-fucosyltransferase that catalyzes the transfer of fucose from GDP-fucose to N-acetylglucosamine (GlcNAc) in an alpha-1,6 linkage. FUT8(-/-) cell lines have morphology and growth kinetics similar to those of the parent, and produce completely defucosylated recombinant antibodies. FUT8(-/-)-produced chimeric anti-CD20 IgG1 shows the same level of antigen-binding activity and complement-dependent cytotoxicity (CDC) as the FUT8(+/+)-produced, comparable antibody, Rituxan. In contrast, FUT8(-/-)-produced anti-CD20 IgG1 strongly binds to human Fcgamma-receptor IIIa (FcgammaRIIIa) and dramatically enhances ADCC to approximately 100-fold that of Rituxan. Our results demonstrate that FUT8(-/-) cells are ideal host cell lines to stably produce completely defucosylated high-ADCC antibodies with fixed quality and efficacy for therapeutic use.     

Comparisons of Two Large Phaeoviral Genomes and Evolutionary Implications

The evolution of viral genomes has recently attracted considerable attention. We compare the sequences of two large viral genomes, EsV-1 and FirrV-1, belonging to the family of phaeoviruses which infect different species of marine brown algae. Although their genomes differ substantially in size, these viruses share similar morphologies and similar latent infection cycles. In fact, sequence comparisons show that the viruses have more than 60% of their genes in common. However, the order of genes is completely different in the two genomes, suggesting that extensive recombinational events in addition to several large deletions had occurred during the separate evolutionary routes from a common ancestor. We investigated genes encoding components of signal transduction pathways and genes encoding replicative functions in more detail. We found that the two genomes possess different, although overlapping, sets of genes in both classes, suggesting that different genes from each class were lost, perhaps randomly, after the separate evolution from an ancestral genome. Random loss would also account for the fact that more than one-third of the genes in one viral genome has no counterparts in the other genome. We speculate that the ancestral genome belonged to a cellular organism that had once invaded a primordial brown algal host.