Media supplementation for targeted manipulation of monoclonal antibody galactosylation and fucosylation

Monoclonal antibodies are critically important biologics as the largest class of molecules used to treat cancers, rheumatoid arthritis, and other chronic diseases. Antibody glycosylation is a critical quality attribute that has ramifications for patient safety and physiological efficacy—one that can be modified by such factors as media formulation and process conditions during production. Using a design-of-experiments approach, we examined the effect of 2-F-peracetyl fucose (2FP), uridine, and galactose on cell growth and metabolism, titer, and gene expression of key glycosylation-related proteins, and report how the glycoform distribution changed from Days 4 to 7 in a batch process used for IgG1 production from Chinese hamster ovary cells. We observed major glycosylation changes upon supplement addition, where the addition of 2FP decreased antibody fucosylation by up to 48%, galactose addition increased galactosylation by up to 21%, and uridine addition decreased fucosylation and increased galactosylation by 6% and 2%, respectively. Despite having major effects on glycosylation, neither galactose nor 2FP significantly affected cell culture growth, metabolism, or titer. Uridine improved peak cell densities by 23% but also reduced titer by ∼30%. The supplements caused significant changes in gene expression by Day 4 of the cultures where 2FP addition significantly reduced fucosyltransferase 8 and nucleotide sugar transporter gene expression (by ∼2-fold), and uridine addition significantly increased expression of UDP-GlcNAcT (SLC35A3) and B4GALT1–6 genes (by 1.5–3-fold). These gene expression data alongside glycosylation, metabolic, and growth data improve our understanding of the cellular mechanisms affected by media supplementation and suggest approaches for modifying antibody glycosylation in antibody production processes.     

Specificity of extrafloral nectar induction by herbivores differs among native and invasive populations of tallow tree

Background and Aims

Invasive plants can be released from specialist herbivores and encounter novel generalists in their introduced ranges, leading to variation in defence among native and invasive populations. However, few studies have examined how constitutive and induced indirect defences change during plant invasion, especially during the juvenile stage.

Methods

Constitutive extrafloral nectar (EFN) production of native and invasive populations of juvenile tallow tree (Triadica sebifera) were compared, and leaf clipping, and damage by a native specialist (Noctuid) and two native generalist caterpillars (Noctuid and Limacodid) were used to examine inducible EFN production.

Key results

Plants from introduced populations had more leaves producing constitutive EFN than did native populations, but the content of soluble solids of EFN did not differ. Herbivores induced EFN production more than simulated herbivory. The specialist (Noctuid) induced more EFN than either generalist for native populations. The content of soluble solids in EFN was higher (2·1 times), with the specialist vs. the generalists causing the stronger response for native populations, but the specialist response was always comparable with the generalist responses for invasive populations.

Conclusions

These results suggest that constitutive and induced indirect defences are retained in juvenile plants of invasive populations even during plant establishment, perhaps due to generalist herbivory in the introduced range. However, responses specific to a specialist herbivore may be reduced in the introduced range where specialists are absent. This decreased defence may benefit specialist insects that are introduced for classical biological control of invasive plants.     

Mesenchymal stem cells contribute to endogenous FVIII:c production

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative‐RT‐PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord‐derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Unique gene alterations are induced in FACS‐purified Fos‐positive neurons activated during cue‐induced relapse to heroin seeking

Cue‐induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non‐activated neurons during cue‐induced heroin seeking after prolonged withdrawal. We trained rats to self‐administer heroin for 10 days (6 h/day) and assessed cue‐induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent‐activated cell sorting (FACS) to purify Fos‐positive and Fos‐negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos‐immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos‐positive, but not Fos‐negative, neurons. In support of these findings, double‐label immunohistochemistry indicated substantial coexpression of neuropeptide Y (NPY)‐ and Arc‐immunoreactivity in Fos‐positive neurons. Our data indicate that cue‐induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non‐activated neurons.     

Effects of dietary fibre and protein on urea transport across the cecal mucosa of piglets

In ruminants, gastrointestinal recycling of urea is acutely enhanced by fibre-rich diets that lead to high ruminal concentration of short chain fatty acids (SCFA), while high ammonia has inhibitory effects. This study attempted to clarify if urea flux to the porcine cecum is similarly regulated. Thirty-two weaned piglets were fed diets containing protein (P) of poor prececal digestibility and fibre (F) at high (H) or low levels (L) in a 2 × 2 factorial design. After slaughter, cecal content was analyzed and the cecal mucosa incubated in Ussing chambers to measure the effect of pH, SCFA and NH₄ ⁺ on the flux rates of urea, short-circuit current (I _sc) and tissue conductance (G _t). NH₄ ⁺ significantly enhanced I _sc (from 0.5 ± 0.2 to 1.2 ± 0.1 μEq cm^?2 h^?1). No acute effects of SCFA or ammonia on urea flux were observed. Tissue conductance was significantly lower in the high dietary fibre groups irrespective of the protein content. Only the HP-LF group emerged as different from all others in terms of urea flux (74 ± 6 versus 53 ± 3 nmol cm^?2 h^?1), associated with higher cecal ammonia concentration and reduced fecal consistency. The data suggest that as in the rumen, uptake of ammonia by the cecum may involve electrogenic transport of the ionic form (NH₄ ⁺). In contrast to findings in the rumen, neither a high fibre diet nor acute addition of SCFA enhanced urea transport across the pig cecum. Instead, a HP-LF diet had stimulatory effects. A potential role for urea recycling in stabilizing luminal pH is discussed.     

Wavelength dependence of patman equilibration dynamics in phosphatidylcholine bilayers

Assessment of the equilibration kinetics of Patman at the edges of its emission spectra provided additional insights about membrane properties beyond those obtained from end-point fluorescence measurements. Upon introduction of the probe to aqueous suspensions of liposomes, the emission intensity slowly increased about 10-fold (t_½ = ~ 100 s). The rate of equilibration depended on emission wavelength, and was usually faster at 500 than at 435 nm. However, this trend was reversed for equilibration with lipids at their phase transition temperature. The apparent rotational motion of the dye also differed between the long and short emission wavelengths but did not display the slow equilibration time dependence observed with intensity measurements. These results suggested that slow equilibration reflects relaxation of the immediate membrane microenvironment around the probe rather than slow insertion into the membrane. The data were rationalized with a model that allows two membrane/probe configurations with distinct microenvironments. The analysis suggests that by monitoring the equilibration pattern of Patman, inferences can be made regarding the polarity of two microenvironments occupied by the probe, the distribution of the probe among those microenvironments, and the kinetics with which they relax to equilibrium.     

Interpreting skeletal growth in the past from a functional and physiological perspective

The study of juvenile skeletal remains can yield important insights into the health, behavior, and biological relationships of past populations. However, most studies of past skeletal growth have been limited to relatively simple metrics. Considering additional skeletal parameters and taking a broader physiological perspective can provide a more complete assessment of growth patterns and environmental and genetic effects on those patterns. We review here some alternative approaches to ontogenetic studies of archaeological and paleontological skeletal material, including analyses of body size (stature and body mass) and cortical bone structure of long bone diaphyses and the mandibular corpus. Together such analyses can shed new light on both systemic and localized influences on bone growth, and the metabolic and mechanical factors underlying variation in growth. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

Genomic Pathology of SLE-Associated Copy-Number Variation at the FCGR2C/FCGR3B/FCGR2B Locus

Reduced FCGR3B copy number is associated with increased risk of systemic lupus erythematosus (SLE). The five FCGR2/FCGR3 genes are arranged across two highly paralogous genomic segments on chromosome 1q23. Previous studies have suggested mechanisms for structural rearrangements at the FCGR2/FCGR3 locus and have proposed mechanisms whereby altered FCGR3B copy number predisposes to autoimmunity, but the high degree of sequence similarity between paralogous segments has prevented precise definition of the molecular events and their functional consequences. To pursue the genomic pathology associated with FCGR3B copy-number variation, we integrated sequencing data from fosmid and bacterial artificial chromosome clones and sequence-captured DNA from FCGR3B-deleted genomes to establish a detailed map of allelic and paralogous sequence variation across the FCGR2/FCGR3 locus. This analysis identified two highly paralogous 24.5 kb blocks within the FCGR2C/FCGR3B/FCGR2B locus that are devoid of nonpolymorphic paralogous sequence variations and that define the limits of the genomic regions in which nonallelic homologous recombination leads to FCGR2C/FCGR3B copy-number variation. Further, the data showed evidence of swapping of haplotype blocks between these highly paralogous blocks that most likely arose from sequential ancestral recombination events across the region. Functionally, we found by flow cytometry, immunoblotting and cDNA sequencing that individuals with FCGR3B-deleted alleles show ectopic presence of FcγRIIb on natural killer (NK) cells. We conclude that FCGR3B deletion juxtaposes the 5′-regulatory sequences of FCGR2C with the coding sequence of FCGR2B, creating a chimeric gene that results in an ectopic accumulation of FcγRIIb on NK cells and provides an explanation for SLE risk associated with reduced FCGR3B gene copy number.     

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.     

Two-component covalent inhibitor

Inhibitors that covalently damage proteins or nucleic acids offer great potency, but are difficult to rationally design and suffer from poor specificity. Here we outline a general concept for constructing covalent inhibitors, called the two-component covalent inhibitor (TCCI). The approach takes advantage of two ligand analogs equipped with pre-reactive groups. Binding of the analogs to the adjacent sites of a target biopolymer brings the pre-reactive groups in close proximity and causes their interaction followed by covalent damage of the target. In the present study we used light-activated pre-reactive groups to inactivate a DNA polymerase. It was found that the efficiency of a traditional single-component inhibitor was greatly reduced in the presence of a non-target protein, while the TCCI was not significantly affected. Our findings suggest that TCCI approach has advantages in inactivation of biopolymers in complex multi-component systems.