Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells

7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI-TOF, ES-TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 14,9297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 15,2878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 15,4459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing.     

Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography-mass spectrometry

5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography-electrospray ionization mass spectrometry (LC-MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-(2)H(4)) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C(18) cartridge prior to injection onto a gradient eluted Hypurity C(18) reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6-8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n=10) and <6.0% (n=9), respectively. The new LC-MS method was highly correlated with an established GC-MS method for urinary 5-HTOL (r(2)=0.99, n=70; mean 5-HTOL/GTOL ratio=1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.     

Cellular and molecular mechanisms of regeneration in Xenopus

We have employed transgenic methods combined with embryonic grafting to analyse the mechanisms of regeneration in Xenopus tadpoles. The Xenopus tadpole tail contains a spinal cord, notochord and segmented muscles, and all tissues are replaced when the tail regenerates after amputation. We show that there is a refractory period of very low regenerative ability in the early tadpole stage. Tracing of cell lineage with the use of single tissue transgenic grafts labelled with green fluorescent protein (GFP) shows that there is no de-differentiation and no metaplasia during regeneration. The spinal cord, notochord and muscle all regenerate from the corresponding tissue in the stump; in the case of the muscle the satellite cells provide the material for regeneration. By using constitutive or dominant negative gene products, induced under the control of a heat shock promoter, we show that the bone morphogenetic protein (BMP) and Notch signalling pathways are both essential for regeneration. BMP is upstream of Notch and has an independent effect on regeneration of muscle. The Xenopus limb bud will regenerate completely at the early stages but regenerative ability falls during digit differentiation. We have developed a procedure for making tadpoles in which one hindlimb is transgenic and the remainder wild-type. This has been used to introduce various gene products expected to prolong the period of regenerative capacity, but none has so far been successful.     

Methods for molecular dynamics simulations of protein folding/unfolding in solution

All atom molecular dynamics simulations have become a standard method for mapping equilibrium protein dynamics and non-equilibrium events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and analysis derived from previous studies are also presented. As a measure of validation, our water model is compared with experiment. An example of current applications of these methods, simulations of the ultrafast folding protein Engrailed Homeodomain are presented including the experimental evidence used to verify their results. Ultrafast folders are an invaluable tool for studying protein behavior as folding and unfolding events measured by experiment occur on timescales accessible with the high-resolution molecular dynamics methods we describe. Finally, to demonstrate the prospect of these methods for folding proteins, a temperature quench simulation of a thermal unfolding intermediate of the Engrailed Homeodomain is described.     

Paradoxical roles of different nitric oxide synthase isoforms in colonic injury

Nitric oxide (NO) is a free radical that is largely produced by three isoforms of NO synthase (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). NO regulates numerous processes in the gastrointestinal tract; however, the overall role that NO plays in intestinal inflammation is unclear. NO is upregulated in both ulcerative colitis and Crohn's disease as well as in animal models of colitis. There have been conflicting reports on whether NO protects or exacerbates injury in colitis or is simply a marker of inflammation. To determine whether the site, timing, and level of NO production modulate the effect on the inflammatory responses, the dextran sodium sulfate model of colitis was assessed in murine lines rendered deficient in iNOS, nNOS, eNOS, or e/nNOS by targeted gene disruption. The loss of nNOS resulted in more severe disease and increased mortality, whereas the loss of eNOS or iNOS was protective. Furthermore, concomitant loss of eNOS reversed the susceptibility found in nNOS-/- mice. Deficiencies in specific NOS isoforms led to distinctive alterations of inflammatory responses, including changes in leukocyte recruitment and alterations in colonic lymphocyte populations. The present studies indicate that NO produced by individual NOS isoforms plays different roles in modulating an inflammatory process.     

kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila

Drosophila myoblast fusion proceeds in two steps. The first one gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. We have identified Kette as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterised by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, we show that kette interacts genetically with blown fuse (blow) which is known to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. We propose a model in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion.     

Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function

Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.