Axonal autophagosomes use the ride-on service for retrograde transport toward the soma

Degradation of autophagic vacuoles (AVs) via lysosomes is an important homeostatic process in cells. Neurons are highly polarized cells with long axons, thus facing special challenges to transport AVs generated at distal processes toward the soma where mature acidic lysosomes are relatively enriched. We recently revealed a new motor-adaptor sharing mechanism driving autophagosome transport to the soma. Late endosome (LE)-loaded dynein-SNAPIN motor-adaptor complexes mediate the retrograde transport of autophagosomes upon their fusion with LEs in distal axons. This motor-adaptor sharing mechanism enables neurons to maintain effective autophagic clearance in the soma, thus reducing autophagic stress in axons. Therefore, our study reveals a new cellular mechanism underlying the removal of distal AVs engulfing aggregated misfolded proteins and dysfunctional organelles associated with several major neurodegenerative diseases.     

Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization

Variable (Fv) domain N-glycosylation sites are found in approximately 20% of human immunoglobulin Gs (IgGs) in addition to the conserved N-glycosylation sites in the C(H)2 domains. The carbohydrate structures of the Fv glycans and their impact on in vivo half-life are not well characterized. Oligosaccharide structures in a humanized anti-Abeta IgG1 monoclonal antibody (Mab) with an N-glycosylation site in the complementary determining region (CDR2) of the heavy chain variable region were elucidated by LC/MS analysis following sequential exoglycosidase treatments of the endoproteinase Lys-C digest. Results showed that the major N-linked oligosaccharide structures in the Fv region have three characteristics (core-fucosylated biantennary oligosaccharides with one or two N-glycolylneuraminic acid [NeuGc] residues, zero or one alpha-linked Gal residue, and zero or one beta-linked GalNAc residue), whereas N-linked oligosaccharides in the Fc region contained typical Fc glycans (core-fucosylated, biantennary oligosaccharides with zero to two Gal residues). To elucidate the contribution of Fv glycans to the half-life of the antibody, a method that allows capture of the Mab and determination of its glycan structures at various time points after administration to mice was developed. Anti-Abeta antibody in mouse serum was immunocaptured by immobilized goat anti-human immunoglobulin Fc(gamma) antibody resin, and the captured material was treated with papain to generate Fab and Fc for LC/MS analysis. Different glycans in the Fc region showed the same clearance rate as demonstrated previously. In contrast to many other non-antibody glycosylated therapeutics, there is no strong correlation between oligosaccharide structures in the Fv region and their clearance rates in vivo. Our data indicated that biantennary oligosaccharides lacking galactosylation had slightly faster clearance rates than other structures in the Fv domain.     

Interconversion and tissue distribution of pentoxifylline and lisofylline in mice

The aim of this study was to assess the interconversion pharmacokinetics and tissue distribution of pentoxifylline and the active (R)-enantiomer of its metabolite M1, lisofylline in male CD-1 mice. Both compounds were administered intravenously at a dose of 50 mg/kg on two separate occasions. Serum and tissues were collected at different time points following drug administration. In addition, the (S)-enantiomer of M1 was administered to a group of mice and serum samples were obtained. Analyte concentrations were measured by chiral HPLC. All serum concentration versus time data were fitted simultaneously to a pharmacokinetic model incorporating interconversion processes of parent drug and metabolites. The estimated conversion clearance of (-)-(R)-M1 to pentoxifylline (CL21) was six times greater than that for the reverse process (CL12). The interconversion of pentoxifylline and (+)-(S)-M1 was faster as reflected by the values of conversion clearances CL13 and CL31 which were approximately 16 and 7 times greater in comparison with the corresponding clearances for the interconversion of pentoxifylline and (-)-(R)-M1. When fitting pharmacokinetic data of both parent compounds to a one-compartment model, the values of elimination clearances assessed were close to those obtained on the basis of the interconversion model. After administration of pentoxifylline, tissue-to-serum AUC ratios ranged from 0.1 for liver and lungs to 0.32 for brain tissue. Serum levels of its metabolite, (-)-(R)-M1 were very low, whereas its tissue levels exceeded serum concentrations. The highest value of metabolite-to-parent AUC ratio (4.98) was observed in lungs. When (-)-(R)-M1 was given as a parent drug, tissue-to-serum AUC ratios in liver, kidney, and lungs were very close and ranged from 0.64 to 0.72. At the same time, levels of its metabolite, pentoxifylline were relatively low both in serum and all tissues studied. In consequence, metabolite-to-parent AUC ratios did not exceed the value of 0.27. In conclusion, reversible metabolism plays a modest role in the disposition of pentoxifylline and (-)-(R)-M1. It seems that pentoxifylline has less favourable pharmacokinetic properties than (-)-(R)-M1 due to lower concentrations attained in target organs. High levels of (-)-(R)-M1 observed after pentoxifylline administration in certain tissues such as liver or lungs suggest that pentoxifylline may constitute an effective prodrug for (-)-(R)-M1 in these organs.     

Seasonal change in the gut clearance rate of three stonefly species in a Japanese stream

We measured seasonal changes in the gut clearance rate of three stonefly species in a stream. The rate declined with water temperature. Based on the gut clearance rate obtained here, we estimated the half-reduction time of gut contents and consumption rate of stoneflies on algae. Irrespective of stonefly species, the half-reduction time in winter (2 days) was twice as long as that in summer (1 day). The areal consumption rates of stoneflies varied depending on species and season. These results imply that the seasonal consumption impact of aquatic insects can be different, despite their daily ration being similar among species.     

Galectin-3 functions as an opsonin and enhances the macrophage clearance of apoptotic neutrophils

Galectin-3, a β-galactoside binding, endogenous lectin,takes part in various inflammatory events and is produced insubstantial amounts at inflammatory foci. We investigated whetherextracellular galectin-3 could participate in the phagocyticclearance of apoptotic neutrophils by macrophages, a processof crucial importance for termination of acute inflammation.Using human leukocytes, we show that exogenously added galectin-3increased the uptake of apoptotic neutrophils by monocyte-derivedmacrophages (MDM). Both the proportion of MDM that engulfedapoptotic prey and the number of apoptotic neutrophils thateach MDM engulfed were enhanced in the presence of galectin-3.The effect was lactose-inhibitable and required galectin-3 affinityfor N-acetyllactosamine, a saccharide typically found on cellsurface glycoproteins, since a mutant lacking this activitywas without effect. The enhanced uptake relied on the presenceof galectin-3 during the cellular interaction and was paralleledby lectin binding to apoptotic cells as well as MDM in a lactose-dependentmanner. These findings suggest that galectin-3 functions asa bridging molecule between phagocyte and apoptotic prey, actingas an opsonin. The process of clearance, whereby apoptotic neutrophilsare removed by macrophages, is crucial for the resolution ofacute inflammation and our data imply that the increased levelsof galectin-3 often found at inflammatory sites could potentlyaffect this process.     

Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products

The mammalian cell-lines used to produce biopharmaceutical products are known to produce endogenous retrovirus-like particles and have the potential to foster adventitious viruses as well. To ensure product safety and regulatory compliance, recovery processes must be capable of removing or inactivating any viral impurities or contaminants which may be present. Anion exchange chromatography (AEX) is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal. To further characterize the robustness of viral clearance by AEX with respect to process variations, we have investigated the ability of an AEX process to remove three model viruses using various combinations of mAb products, feedstock conductivities and compositions, equilibration buffers, and pooling criteria. Our data indicate that AEX provides complete or near-complete removal of all three model viruses over a wide range of process conditions, including those typically used in manufacturing processes. Furthermore, this process provides effective viral clearance for different mAb products, using a variety of feedstocks, equilibration buffers, and different pooling criteria. Viral clearance is observed to decrease when feedstocks with sufficiently high conductivities are used, and the limit at which the decrease occurs is dependent on the salt composition of the feedstock. These data illustrate the robust nature of the AEX recovery process for removal of viruses, and they indicate that proper design of AEX processes can ensure viral safety of mAb products.     

Small molecule clearance in ultrafiltration/diafiltration in relation to protein interactions: Study of citrate binding to a Fab

Ultrafiltration/diafiltration (UFDF) is commonly utilized in the purification of recombinant proteins to concentrate and buffer exchange the product. It is often the final step in the purification process, placing the protein in its final formulation and clearing small molecules introduced in upstream purification steps. This article presents a case study of reduced small molecule clearance in ultrafiltration/diafiltration of an antigen‐binding fragment of a monoclonal antibody. Citrate, a commonly utilized small molecule in downstream processes, is shown to have reduced clearance due to specific interactions with the protein product. The study presents process solutions and utilizes a simple model to characterize clearance of small molecules which exhibit interactions with product protein. Biotechnol. Bioeng. 2009;102: 1718–1722. © 2008 Wiley Periodicals, Inc.