A study of immunoglobulin G glycosylation in monoclonal and polyclonal species by electrospray and matrix-assisted laser desorption/ionization mass spectrometry

N-linked oligosaccharides were released from human and bovine polyclonal immunoglobulin G (IgG) obtained from commercial sources and also from a monoclonal IgG(1) secreted by murine B-lymphocyte hybridoma cells (CC9C10) grown under different serum-free conditions. These conditions differed according to their steady-state dissolved oxygen concentrations. This work is based on a previous quantitative study where released glycans were characterized by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (J. P. Kunkel, D. C. H. Jan, J. C. Jamieson, and M. Butler, 1998, J. Biotechnol. 62, 55-71). In the present article, peptide-N-glycosidase F-released glycans from different species of polyclonal IgG and murine monoclonal IgG were characterized qualitatively by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). The glycans were also analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The MALDI mass spectrometer used allowed acquisition of MS and tandem MS data, which were useful in structural investigations at a more detailed level than allowed by FACE and HPAEC-PAD. Predominant N-linked structures, as determined by all techniques, were core-fucosyl asialyl biantennary chains with varying galactosylation. Minor amounts of afucosyl, bisected, and monosialyl oligosaccharides were also detected. In contrast to FACE and HPAEC-PAD, MALDI-double quadrupole/time-of-flight MS and HPLC/ESI-MS also detected low-abundance high-mannose and hybrid structures in some of the species under investigation.     

Association of rasGAPSH3 binding protein 1, G3BP1, and rasGap120 with integrin containing complexes induced by an adhesion blocking antibody

The adhesion blocking antibody 3S3 was used to probe the regulation of alpha5beta1 integrin mediated adhesion in K562 cells. This antibody prevented cellular adherence but it did not interfere with ligand binding by cells or purified integrin. Interaction with 3S3 induced change in the cytoskeletal organization resulting in extensive filopodia formation. The antibody also prevented ligand and anti-integrin antibody induced phosphorylation of FAK in a trans acting fashion. MS based analysis of 3S3 induced integrin containing complexes identified rasGAP SH3 binding protein 1, G3BP1, as a component of these structures. The G3BP1 binding molecule, rasGap120, was also identified in the complexes. Microscopic examination confirmed the recruitment of a component of cellular G3BP1 and rasGap120 pools to sites of integrin cross-linking. G3BP1 was also observed in the 3S3 induced filopodia. In untreated cells, G3BP1 was shown to associate with submembranous regions involved in cellular polarization. Collectively, these results suggest that G3BP1 and rasGap120 can be recruited to sites of integrin ligation where they may play a role in cytoskeletal reorganization. Such changes may result in reduced adhesive potential and account for the 3S3 effects on cellular adhesion. It should be emphasized that these results do not necessarily indicate a direct interaction of integrin with G3BP1 and rasGap120.     

Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins

The aim was to determine the proportion of membrane glycoproteins captured using concanavalin A or wheat germ agglutinin lectin affinity chromatography. Digests of the isolated proteins were separated by reversed-phase liquid chromatography and analyzed by matrix-assisted laser desorption tandem mass spectrometry. The two lectins identified different groups of proteins with a broad range of molecular mass and p/ values, including a number of proteins that overlapped the two groups. Approximately 30% of the proteins were positively identified as containing domains that were predicted using standard bioinformatics methods to be characteristic of integral membrane proteins. This approach represents an effective method of surveying the membrane protein pool of mammalian cells for subsequent proteomic analysis.     

Mass spectrometric based mapping of the disulfide bonding patterns of integrin alpha chains

Integrins are one of the major mediators of cellular adherence. Structurally the component alpha and beta chains are characterized by extensive intrachain disulfide bonding. The assignment of these bonds is currently based on homology with the chains of the integrin alphaIIbbeta3. However, recent crystallographic analysis of the soluble alphaVbeta3 construct indicates that the alphaV chain displays bonding patterns different from those predicted for alphaIIb. In an effort to define the disulfide bonding patterns in integrins, we have used mass spectrometric based approaches to map the human alpha3, alpha5, alphaV, and alphaIIb. The results indicate that there are differences in the disulfide patterns of the alpha chains. These do not correlate with the integrin capacity to bind ligands as all integrins used in the present study displayed functional activity. The differences were observed in the bonding patterns linking the heavy (H) and light (L) components of the of the alpha chains. It was also possible to assign the location in alpha5 of an additional disulfide bond involving a pair of cysteines not present in alphaV or alphaIIb. This second bond between the H and L chains of alpha5 has not been previously described. These results indicate that not all integrin species display the same disulfide bonding patterns. They also highlight the need for caution in the use of assignments based on sequence homology.     

Multiple equilibria of the Escherichia coli chaperonin GroES revealed by mass spectrometry

Nanospray time-of-flight mass spectrometry has been used to study the assembly of the heptamer of the Escherichia coli cochaperonin protein GroES, a system previously described as a monomer-heptamer equilibrium. In addition to the monomers and heptamers, we have found measurable amounts of dimers and hexamers, the presence of which suggests the following mechanism for heptamer assembly: 2 Monomers <--> Dimer; 3 Dimers <--> Hexamer; Hexamer + Monomer <--> Heptamer. Equilibrium constants for each of these steps, and an overall constant for the Monomer <--> Heptamer equilibrium, have been estimated from the data. These constants imply a standard free-energy change, DeltaG(0), of about 9 kcal/mol for each contact surface formed between GroES subunits, except for the addition of the last subunit, where DeltaG(0) = 6 kcal/mol. This lower value probably reflects the loss of entropy when the heptamer ring is formed. These experiments illustrate the advantages of electrospray mass spectrometry as a method of measuring all components of a multiple equilibrium system.     

On the theory of blood-tissue exchanges: I. Fundamental equations

A mathematical analysis of the absorption of an inert gas by a heterogeneous system ofn phases, e.g., a limb consisting ofn tissues, is presented. The total uptake of gas, ϕ(t), up to timet is given in terms of arterial concentration, cardiac delivery, blood volume, and the volume, permeability, and partition coefficient of each tissue. The theory predicts how the uptake curve should change in shape under a variety of physiological conditions, and how from the numerical values of the constants the values of certain tissue constants, e.g. permeabilities, may be obtained. The material in this article should be construed only as the personal opinion of the writers and not as representing the opinion of the Navy Department officially.     

Arctic Geese Tune Migration to a Warming Climate but Still Suffer from a Phenological Mismatch

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Termite cohabitation: the relative effect of biotic and abiotic factors on mound biodiversity

1. Termites are important ecosystem engineers that improve primary productivity in trees and animal diversity outside their mounds. However, their ecological relationship with the species nesting inside their mounds is poorly understood. 2. The presence of termite cohabitant colonies inside 145 Cornitermes cumulans mounds of known size and location was recorded. Using network‐theoretical methods in conjunction with a suite of statistical analyses, the relative influence of biotic and abiotic drivers of termite within‐mound diversity on the composition and species richness of the termite community was investigated, specifically builder presence and physical aspects of the mound. 3. We found that richness inside the mound increases with mound size, and the species similarity between mounds decreases with distance. The physical attributes (abiotic drivers) of termite mounds (size and relative distance to other mounds) are the strongest predictors of termite species richness and composition. The biotic driver (presence of a builder colony) has an important, though smaller, negative effect on within‐mound termite species richness. 4. The findings suggest that the termites' physical manipulation of their environment is an important driver of within‐mound community diversity. More generally, the approach taken here, using a combination of statistical and network‐theoretical methods, can be used to determine the relative importance of abiotic and biotic drivers of diversity in a wide range of communities of interacting species.     

The Changing Face of Natural Resources Students,Education, and the Profession

ABSTRACT There are many challenges facing natural resources programs in North American higher education today. Pressures exerted by a new generation of students, changing workplace requirements (including undergraduate core-knowledge requirements), and an increasingly specialized professoriate are great but not insurmountable. We discuss each of these issues and pose potential solutions to address each including adopting new pedagogical techniques for content delivery (e.g., adapting courses to be inclusive of new technologies), revising curriculum to meet the needs of a new suite of learners (e.g., developing curricula that allow structured flexibility of choices, designing a core curriculum that is a mix of single-discipline courses and courses that integrate across disciplines), and new strategies for faculty engagement in discipline-specific survey courses. By remaining deliberate and effective in our pursuit of quality higher education we have the opportunity to ensure we are delivering the best possible education to the future professionals of our disciplines.