Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Delayed post-conditioning reduces post-ischemic glutamate level and improves protein synthesis in brain

In the clinic delayed post-conditioning would represent an attractive strategy for the survival of vulnerable neurons after an ischemic event. In this paper we studied the impact of ischemia and delayed post-conditioning on blood and brain tissue concentrations of glutamate and protein synthesis. We designed two groups of animals for analysis of brain tissues and blood after global ischemia and post-conditioning, and one for analysis of blood glutamate after transient focal ischemia.     

An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes

Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G-beaded agarose, which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein–protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins and their interacting proteins, can be widely used for unraveling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states.     

Recent Dinoflagellate cyst distribution in the North Canary Basin,NW Africa

The North Canary Basin (NW Africa) falls within a major eastern boundary upwelling system. This part of the coastal upwelling system is seasonal and is characterised by the development of large filaments migrating seawards. Hence, 16 samples from this location were selected to identify an “upwelling signal” in the composition of the dinoflagellate cyst assemblages.

Samples closest to the most intense upwelling cells are dominated by L. machaerophorum and G. catenatum and Protoperidinium spp. These make up the “upwelling signal” characteristic for the system. Moreover, the “upwelling signal” can be advected offshore, with filaments that may extend as far as 300 km. Finally, the finding of cysts from G. catenatum, a toxic dinoflagellate, raises the need for a better understanding of the relationship between its presence and distribution in the region, and the coastal upwelling system.     

Effect of enzyme dehydration on alcalase‐catalyzed dipeptide synthesis in near‐anhydrous organic media

The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N‐protected phenylalanine in near‐anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0–80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration‐induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration‐induced inactivation that takes place during the first activity measurement, (2) a phase of first‐order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:870–875, 2013     

Iron Is a Sensitive Biomarker for Inflammation in Multiple Sclerosis Lesions

MRI phase imaging in multiple sclerosis (MS) patients and in autopsy tissue have demonstrated the presence of iron depositions in white matter lesions.The accumulation of iron in some but not all lesions suggests a specific, potentially disease-relevant process, however; its pathophysiological significance remains unknown.Here, we explore the role of lesional iron in multiple sclerosis using multiple approaches: immunohistochemical examination of autoptic MS tissue, an in vitro model of iron-uptake in human cultured macrophages and ultra-highfield phase imaging of highly active and of secondary progressive MS patients.Using Perls'' stain and immunohistochemistry, iron was detected in MS tissue sections predominantly in non-phagocytosing macrophages/microglia at the edge of established, demyelinated lesions. Moreover, iron-containing macrophages but not myelin-laden macrophages expressed markers of proinflammatory (M1) polarization.Similarly, in human macrophage cultures, iron was preferentially taken up by non-phagocytosing, M1-polarized macrophages and induced M1 (super) polarization. Iron uptake was minimal in myelin-laden macrophages and active myelin phagocytosis led to depletion of intracellular iron.Finally, we demonstrated in MS patients using GRE phase imaging with ultra-highfield MRI that phase hypointense lesions were significantly more prevalent in patients with active relapsing than with secondary progressive MS.Taken together, our data provide a basis to interpret iron-sensitive GRE phase imaging in MS patients: iron is present in non-phagocytosing, M1-polarized microglia/macrophages at the rim of chronic active white matter demyelinating lesions. Phase imaging may therefore visualize specific, chronic proinflammatory activity in established MS lesions and thus provide important clinical information on disease status and treatment efficacy in MS patients.