The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.     

Dynamic behaviour of inflorescence-bearing Triticale and Triticum stems

The mechanical response of cereal plant shoots to loads caused by wind and gravity in the field is swaying in flexure around the vertical or near vertical transient equilibrium position determined by the stationary component of the wind pressure. The aim of this work was to characterise the kinematic and dynamic attributes and their interrelations in freely swaying inflorescence-bearing stems of wheat (Triticum aestivum L.) and Triticale. The fundamental natural frequency of the stems appeared to be considerably lower than predicted from the theory of vibration using the model of a cantilever beam oscillator and assuming the spring constant to be equal to the force-deflection ratio. Because of the rate of deformation and visco-elastic behaviour of the plant material, a discrepancy of about 10% was found between the dynamic and static stem bending resistance. The presence of the tip inflorescence caused vibrating vertical stems to behave as compressed columns in which the effective spring constant was strongly biased by the apical load due to the weight of the inflorescence. At the late milk stage, in the freely swaying stems of wheat and Triticale, the resistance to dynamic lateral loads was reduced by about 30% as a result of compression exerted by the inflorescence. So the prominent effect of the tip inflorescence on the dynamic behaviour (the effective spring constant and the natural frequency) of the stem is attributed to the non-negligible magnitude of the inflorescence weight relative to the critical load producing elastic buckling in slender vertical structures. Stem softening as a consequence of increasing inflorescence weight is assumed to be one of the essential factors reducing the lodging resistance in cereal crops at the late milk stage. The feasibility of the compressed-column approach for predicting the dynamic bending performance of slender vertical plant organs is discussed. Received: 4 March 1998 / Accepted: 20 July 1998     

In vivo activated caspase-3 cleaves PARP-1 in rat liver after administration of the hepatocarcinogen N-nitrosomorpholine (NNM) generating the 85 kDa fragment

We reported previously that treatment of rats with the hepatocarcinogen N-nitrosomorpholine (NNM) caused severe hepatotoxicity associated with apoptosis of hepatocytes beginning 12 h after administration of NNM. We observed that poly(ADP-ribose) polymerase 1 (PARP-1), one of the major nuclear targets for caspases, was proteolytically degraded generating primarily 64 and 54 kDa fragments. Interestingly, at 20, 30, and 40 h post-treatment a 85 kDa cleavage product of PARP-1 resembling that generated by caspase-3 appeared additionally in hepatocytes. More detailed analysis performed in the present study revealed that the 85 kDa fragment of PARP-1 was generated in the liver in 10 of 17 (60%) animals examined between 20 and 40 h after NNM administration. The caspase-3 generated 85 kDa fragment was detected solely in hepatocytes undergoing apoptosis as evidenced by immunostaining performed with the antibody recognizing exclusively PARP-1 cleaved at position 214/215. The appearance of the 85 kDa fragment of PARP-1 in the liver nuclei coincided temporally with an significant increase of caspase-3 activity in hepatocytes. In contrast, in testis samples obtained from the same animals, no changes characteristic for apoptosis such as induction of caspases activity or degradation of nuclear PARP-1 could be detected. Our results evidence unequivocally that PARP-1 in liver is not resistant to caspases and can be processed in vivo by activated caspase-3 producing the p85 kDa fragment. Moreover, the caspase-3 induced PARP-1 fragmentation coinciding with the increase of caspase-3 activity was detected solely in the target organ and exclusively in hepatocytes undergoing apoptosis. Considering the fact that the caspase-3 mediated PARP-1 cleavage occurred only in 60% of animals tested between 20 and 40 h, it becomes obvious that the cellular response in vivo to the same trigger(s) strongly varies and may depend on a variety of intrinsic factors. It remains to elucidate which additional factors may be involved in the modulation of cellular response to the strong insults thereby activating different pathways and generating distinct outcomes.     

Microstructural characterization of cyanobacterial mats from the McMurdo Ice Shelf, Antarctica

The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.     

Impact of 1,5-disubstituted tetrazole ring on chelating ability of delta-selective opioid peptide

Complex formation between Cu(II) and three tetrazole analogues of opioid peptide-deltorphin I has been investigated. In potentiometric and spectroscopic (UV-Vis, CD and EPR) studies have been established the thermodynamic stability, speciation and structure of Cu(II) complexes with Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (L1), Tyr-Psi(CN4)-Gly-Phe-Asp-Val-Val-Gly-NH2 (L2), Tyr-Gly-Psi(CN4)-Phe-Asp-Val-Val-Gly-NH2 (L3) and Tyr-D-Ala-Psi(CN4)-Phe-Asp-Val-Val-Gly-NH2 (L4). The site of the insertion of tetrazole moiety Psi(CN4) into the peptide sequences has critical impact on their co-ordination ability. Comparison of the binding ability of the tetrazole analogues reveals that around physiological pH region the L3 and L4 are more effective ligands for copper(II) than L(1) and L(2). The peptide conformation changes achieved by Cu(II) co-ordination may be essential for binding of the tetrazole deltorphins at the opiate receptors.     

Selenium supplementation on plasma glutathione peroxidase activity in patients with end-stage chronic renal failure

Patients with chronic renal failure (CRF) usually have a lower than healthy level of selenium (Se) in whole blood and plasma. Plasma glutathione peroxidase (GSH-Px) is synthesized mostly in the kidney. In CRF patients, activity of this enzyme is significantly reduced and its reduction increases with the progress of the disease. The aim of the study was to evaluate the effect of Se supplementation to CRF patients at various stages of the disease on Se concentration in blood components and on plasma GSH-Px activity. The study group comprised 53 CRF patients at various stages of the disease supplemented with Se (200 μg/d for 3 mo as Se-enriched yeast, containing about 70% l-selenomethionine [SeMet]). The control group consisted of 20 healthy subjects. The Se concentration in blood components was measured spectrofluorometrically with 2,3-diaminonaphthalene as a complexing reagent. GSH-Px activity in red cell hemolysates and plasma was assayed by the coupled method with tert-butyl hydroperoxide as a substrate. The Se concentration in whole blood and plasma of CRF patients is significantly lower as compared with healthy subjects, but similar at all stages of the disease. In the patients’ plasma, total protein and albumin levels are also significantly lower than in healthy subjects. Plasma GSH-Px activity in patients is extremely low, and contrary to Se concentration, it decreases linearly with the increasing stage of the illness. Se-supplied patients show an increased Se concentration in all blood components and at all disease stages, whereas plasma GSH-Px activity is enhanced only at the incipient stage of the disease. Se supply has no effect on plasma GSH-Px activity in uremic patients at the end stage of the disease. Total plasma protein and albumin levels did not change after Se supplementation. Our data seem to show that in patients with CRF lower total protein and albumin levels in plasma may be the chief cause of the low blood and plasma Se concentrations. GSH-Px activity decreases along with the kidney impairment. At the end stage of the disease, Se supplementation in the form of Se-enriched yeast has no effect on the increase in plasma GSH-Px activity.     

Applications and performance of a MALDI-ToF mass spectrometer with quadratic field reflectron technology

A new matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-ToF MS), developed specifically for the identification and characterization of proteins and peptides in proteomic investigations, is described. The mass spectrometer which can be integrated with the 2-D gel electrophoresis workflow is a bench-top instrument, enabling rapid, reliable and unattended protein identification in low-, as well as high-throughput proteomics applications. To obtain precise information on peptide sequences, the instrument utilizes a timed ion gate and a unique quadratic field reflectron (Z2 technology), allowing single-run, post-source decay (PSD) of selected peptides. In this study, the performance of the instrument in reflectron, PSD and linear mode, respectively, was investigated. The results showed that the limit of detection for a single peptide in reflectron mode was 125 amol with a signal to noise ratio exceeding 20. Average mass resolution for peptides larger than 2000 u was around 13,000 full width, half maximum (FWHM). The limit for protein identification during peptide mass fingerprinting (PMF) was 500 amol with a sequence coverage of 18%. Mass error during PMF analysis was less than 15 ppm for 17 out of 25 (68%) identified peptides. In PSD mode, a complete series of y-ions of a CAF-derivatized peptide could be obtained from 3.75 fmol of material. The average mass error of PSD-generated fragments was less than 0.14 u. Finally, in linear mode, intact proteins with molecular masses greater than 300,000 u were detected with mass errors below 0.2%.     

HIV-1 Nef binds the DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.     

On conservativity and shattering for an equation of phytoplankton dynamics

A model of phytoplankton dynamics introduced by Arino describes the evolution of aggregates of phytoplankton by a kinetic-type equation composed of terms describing the growth of the aggregates and their splitting, where the latter is modelled by a singular integral operator of the same form as in the classical fragmentation theory. In this paper we shall show that despite the presence of the growth term, the model displays the typical properties of the fragmentation models, in particular, if the fragmentation rate is unbounded as the size of aggregates tends to zero, then there occurs an unaccounted for loss of the phytoplankton though formally nothing is taken out of the system.