Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients

Charge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp-41 of the HIV-1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (pI) spectrum is maintained, but there is marked redistribution of the relative isoform abundance towards the lower pI components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.     

Proteomic profiling of pancreatic ductal carcinoma cell lines treated with trichostatin-A

A pancreatic adenocarcinoma cell line (Paca44) was treated with trichostatin-A (TSA), a potent inhibitor of histone deacetylases, in order to evaluate the effect of this drug on protein expression. Master maps of control and treated Paca44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 51 polypeptide chains, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry analysis enabled the identification of 22 of these spots. Among these proteins, of particular interest are the two downregulated proteins nucleophosmin and translationally controlled tumor protein, as well as the upregulated proteins programmed cell death protein 5 (also designated as TFAR19) and stathmin (oncoprotein 18). The modulation of these four proteins is consistent with our observation that TSA is able to inhibit cell growth of Paca44 by causing cell cycle arrest at the G2 phase and apoptotic cell death.     

Fractionation of carrier ampholytes for isoelectric focusing.

A simple method for fractionating synthetic carrier ampholytes is reported, based on the principle of continuous-flow isoelectric focusing in gel-stabilized layers. An 8% ampholyte solution, encompassing the pH range 3-9.5, is separated into 12 fractions in a chamber filled with Sephadex G-100 by a continuous-flow technique. We are thus able to obtain ampholytes of narrow pH range, encompassing approximately 2 pH units, whose resolving power is comparable with that obtained with commercial Ampholine covering similar pH ranges.     

Recent developments in electrophoretic methods

13. Given the recent extended review by Vesterberg [J. Chromatogr., 480 (1989) 3-19] of electrokinetic methods, this survey has been restricted to the last decade, which has seen tremendous progress in several fields. DNA electrophoresis has experienced strong developments, both in the sequencing strategies (which have been largely automated with the use of fluorescent probes) and in pulsed field analysis of mega-DNA fragments, which has seen such developments as inverse-field, contour-clamped and rotating gel platforms, all allowing for straight band migration in each lane. Chromosome size mapping has now become a reality. Two-dimensional (2D) maps have also shown a dramatic improvement in performance, largely through the development of immobilized pH gradients, giving highly reproducible protein spots in the 2D plane and allowing the exploration of very narrow pH regions. Blotting techniques, combined with 2D mapping, allow sequence analysis and fingerprinting of a single polypeptide spot in a complex sample without resorting to lengthy chromatographic purification steps. Chromatophoresis generates a novel type of 2D mapping, based on hydrophobicity vs. size, rather than on charge vs. size, by direct coupling of a reversed-phase high-performance liquid chromatographic (HPLC) eluate to sodium dodecyl sulphate electrophoresis. The new rising star, capillary zone electrophoresis, offers speed, a large number of theoretical plates, selectivity and small sample requirements in a highly automated equipment.     

Inorganic species in water and sediments of the Lamone and Marzeno rivers ecosystem

Analytical results are reported for the determination of inorganic species in water and sediments sampled in the Lamone and Marzeno rivers ecosystem. The species determined are Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn, Hg, F-, Cl- Br-, NO3-, SO4--, Na+, K+, Mg++, Ca++, NH4+ in superficial water, and Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn, Hg in sediments. For all the elements, in addition to detection limits, precision and accuracy are given: the former, expressed as relative standard deviation (s(r)), and the latter, expressed as relative error (e), were good, being in all cases lower than 6%.     

Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of commercial bovine milk

Proteins in commercial bovine milk have been separated by two-dimensional gel electrophoresis and examined by matrix-assisted laser desorption/ionisation mass spectrometry. Gel separation was conducted in two different pH gradients, 3-10 and 6-11; the latter range resulted in a higher spot resolution and favoured the basic proteins. We have limited the time-of-flight mass spectrometry analysis to the linear mode to examine the capability of reliable relative molecular masses of the intact proteins in their characterisation. The present study draws attention to the difficulty of identifying basic proteins with low molecular masses (below 12000 Da) that are commonly encountered in milk samples.     

Recent progress in DNA analysis by capillary electrophoresis

A number of recent developments in DNA analysis by capillary electrophoresis are here reviewed. They include capillary arrays for fast, parallel DNA sequencing as well as microfabricated capillary arrays. Microfluidic chips for DNA sizing and quantitation are also covered, as well as microdevices containing arrays of regular obstacles acting as size-separators during DNA migration. Screening of DNA point mutations by two much improved techniques is also reported: in one case, such mutations are detected (but only on relative short, ca. 60-70 base-long fragments) by free electrophoresis in rather acidic (pH ca. 3) buffers; in the case of single-strand chain polymorphism, an improved technique is described based on near-neutral pH buffers with mixtures of Tris/MES cations/zwitterions. When studying the behavior of inorganic and organic cations in the Debye-Hückel layer of DNA, it was found that the latter (especially a large number of Good's buffers and other zwitterions, such as His) would bind to the DNA filament not only via charge interaction, but also via additional bonds, notably hydrogen bonds, thus altering the electrophoretic (and possibly the biological) behavior of DNA molecules. However, whether or not borate ions would bind to DNA remains still an unsettled question. Finally, capillary electrophoresis was found to be instrumental in measuring fine physicochemical parameters pertaining to DNA polyelectrolytes, such as their free mobility and their translational diffusion coefficients.     

High-order divergence-free velocity reconstruction for free surface flows on unstructured Voronoi meshes

In this paper, we present an efficient semi-implicit scheme for the solution of the Reynolds-averaged Navier-Stokes equations for the simulation of hydrostatic and nonhydrostatic free surface flow problems. A staggered unstructured mesh composed by Voronoi polygons is used to pave the horizontal domain, whereas parallel layers are adopted along the vertical direction. Pressure, velocity, and vertical viscosity terms are taken implicitly, whereas the nonlinear convective terms as well as the horizontal viscous terms are discretized explicitly by using a semi-Lagrangian approach, which requires an interpolation of the three-dimensional velocity field to integrate the flow trajectories backward in time. To this purpose, a high-order reconstruction technique is proposed, which is based on a constrained least squares operator that guarantees a globally and pointwise divergence-free velocity field. A comparison with an analogous reconstruction, which is not divergence-free preserving, is also presented to give evidence of the new strategy. This allows the continuity equation to be satisfied up to machine precision even for high-order spatial discretizations. The reconstructed velocity field is then used for evaluating high-order terms of a Taylor method that is here adopted as ODE integrator for the flow trajectories. The proposed semi-implicit scheme is validated against a set of academic test problems, and proof of convergence up to fourth-order of accuracy in space is shown.     

Isoelectric Focusing with Immobilized pH Gradients

Abstract

The technique of isoelectric focusing (IEF) via immobilized pH gradients (IPG) was first announced to the scientific community at a meeting of the International Electrophoresis Society in Athens, April 1982, as the result of an intensive collaborative effort [1]. In five years, the technique has been extensively developed in three fundamental aspects: analytical, preparative and as a first dimension of two-dimensional (2-D) maps. The merits and, recently, the flaws of the IPG technique have been evaluated and recognized, so that at the present writing we feel it is ready for successful introduction in most life-science laboratories.