Cell immobilization induces changes in the protein response of Escherichia coli K-12 to a cold shock.

We have compared the protein expression of gel-entrapped Escherichia coli cells submitted to a cold shock at 4 degrees C with those of exponential- and stationary-phase free-floating counterparts. Autoradiograms of two-dimensional gel electrophoresis patterns of proteins radiolabeled with L-[35S]methionine were compared using computing scanning densitometry. The levels of 203 proteins synthesized during the temperature shift were significantly and reproducibly higher than those corresponding to synthesis at 37 degrees C. A principal component analysis (PCA) was performed on the synthesis levels of these 203 proteins in the different incubation conditions tested. This study showed that the protein response of immobilized cells after the cold shock was significantly different from those of exponential- and stationary-phase free-floating organisms. For instance, protein SSB was specifically overexpressed by shocked immobilized organisms. Such induction of specific molecular mechanisms in immobilized bacteria might explain the high resistance of sessile-like organisms to stresses.

Substituting Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis buffers improves the resolution of focusing patterns.

In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry. In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa. This improvement of focusing concerns more particularly basic proteins. This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips. Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.

Identification of proteins from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using electrospray quadrupole-time-of-flight tandem mass spectrometry.

The potential of electrospray tandem mass spectrometry using a quadrupole-time-of-flight tandem mass spectrometer was evaluated for the identification of two unknown proteins from one-dimensional polyacrylamide gel electrophoresis (1-D-PAGE). Two proteins from cellular cultures of mammary epithelia were purified by 1D-PAGE. Their identification was achieved using peptide sequence tags generated by LC/Q-TOF-MS/MS, whereas MALDI-TOF mass mapping failed for these proteins obtained from simple 1D-PAGE separation.

Two-dimensional electrophoresis and mass spectrometry identification of proteins bound by a murine monoclonal anti-cardiolipin antibody: a powerful technique to characterize the cross-reactivity of a single autoantibody.

Antigenic cross-reactivity, i.e., the capacity of a single antibody to react with apparently dissimilar structures, is a common characteristic of autoantibodies produced during systemic lupus erythematosus (SLE), an autoimmune disease developed by humans and certain strains of mice. Characterization of the extent of cross-reactivity of SLE-related autoantibodies may help identify the immunogenic stimulus, or stimuli, of autoantibody-secreting B-lymphocytes. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was combined with mass spectrometry (MS) to identify cell proteins recognized by a single monoclonal autoantibody (mAb 4B7), derived from an (NZW x BXSB)F1 mouse and selected based on its capacity to react with cardiolipin, that binds to elements in the cytoplasm and nucleoli of HEp-2 cells as assessed by indirect immunofluorescence assay. Proteins from HL-60 extract were separated by 1-D and 2-D PAGE. Western blotting with mAb 4B7 after SDS-PAGE revealed four bands, two intensely labeled at 35 and 32 kDa, and two weaker ones at 20 and 60 kDa; three spots were detected after 2-D PAGE. After trypsin in-gel digestion of the three protein spots, MS yielded representative matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) Reflector or quadrupole-time of flight (Q-TOF) spectra. The three corresponding proteins were identified as the nucleolar phosphoprotein B23 (nucleophosmin), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 60 kDa Ro/SS-A RNP. Thus, these results showed that 2-D PAGE combined with MS constitutes a sensitive and powerful technique to characterize the full extent of cross-reactivity of a single mAb and may constitute a new approach to further characterize the immunogenic cellular components involved in the breakage of B-cell tolerance observed in SLE.

Protein patterns of gel-entrapped Escherichia coli cells differ from those of free-floating organisms.

The two-dimensional electrophoretic patterns of cellular proteins from gel-entrapped Escherichia coli cells were compared to those of exponential- and stationary-phase free-floating organisms. The amounts of several proteins in immobilized cells were significantly different from those in free bacteria. Immobilized organisms rapidly produced a high level of dipeptide permease and a single-strand binding protein, and progressively accumulated an aldehyde dehydrogenase. Immobilization also induced a decrease in the levels of two proteins, i.e., the YFID protein and a DNA-binding, stationary-phase protein. The possible role of these proteins in the high resistance of immobilized bacteria to stresses is discussed.

Oriented macroporous polyacrylamide gels.

Macroporous gels with huge cavities and partition walls result from controlled microsyneresis during gelation. In this report we show that the microsyneresis process can be further controlled: it is possible to orient the partition walls of macropores by the use of an electric field throughout polymerization. Our oriented macroporous polyacrylamide gels offer a specific structure: a set of parallel channels of about 2 microm inner diameter, with polyacrylamide walls including acidic groups linked to the aggregated matter.

Eliciting macroporosity in polyacrylamide and agarose gels with polyethylene glycol.

Preparation of highly porous polyacrylamide has recently been described (Righetti et al., Electrophoresis, 1992, 13, 587-595). In this report we add new observations on the conditions of promoting macroporosity in polyacrylamide gels and extend the possibility of eliciting this phenomenon to agarose matrices by the combined use of polyethylene glycol and glycerol. The process of cluster formation in hot agarose solutions was studied and gel structures were examined by scanning electron microscopy. A definition of macroporosity in gel, related to controlled microsyneresis during gelation, is tentatively proposed. The unexpected influence of acrylamide and agarose concentrations upon the size of macroporous structures in the corresponding gels is revealed.

Multiphasic electrophoresis with diversified spacers.

The principle and some applications of multiphasic electrophoresis with diversified spacers are described. The method combines previously published concepts of introducing highly diversified spacers into the stack of an isotachophoretic system with the theory of snow- and telescope-electrophoresis. The link between this electrophoretic method and isotachophoresis on the one hand and monophasic zone electrophoresis on the other hand is exemplified. The importance of the nature of the spacers as well as of the pH and ions in the leading and terminating zone is illustrated. Details are given concerning the experimental set-up. This electrophoretic technique could serve as an alternative to isoelectric focusing.

Heterogeneous nature of human complement factor B: an electrophoretic approach for the analysis of its oligosaccharide chain structure and its physiological breakdown products.

Factor B is a glycoprotein which plays an essential role in the alternative pathway of complement activation. It carries the proteolytic activity of the convertases, and its physiological breakdown products Ba and Bb have some effects on the cells of the immune system. Human factor B exhibits a microheterogeneity and five isoforms are present in serum. The nature and origin of the microheterogeneity was investigated by using electrophoretic techniques. Treatments of factor B with neuraminidase and glycopeptidase F show that this microheterogeneity is mainly due to differences in its sialic acid content, varying from seven to eleven residues per molecule, and resulting in different oligosaccharide structures. However, deglycosylated factor B reveals a residual, nonallotypic variation in the Bb region of the polypeptide backbone. We confirm the presence of four asparagine-linked oligosaccharide chains of the complex type in native factor B, two of which are located in the Ba fragment, and the two others in the Bb fragment. The prevalent isoform of the native protein carries two sialic acid residues per oligosaccharide chain. Biosynthesis experiments show that the microheterogeneity of secreted factor B from HepG2 cells is acquired during the processing of its glycans. However, in vitro-secreted factor B is more heterogeneous than the serum protein. We propose a structural model for the microheterogeneity of the native protein and its physiological fragments. We discuss as well the feasibility of electrophoretic techniques to deal with microheterogeneity analysis.

Molecular basis for the microheterogeneity of human complement factor B.

The involvement of sialic acids in the microheterogeneity of human complement factor B was investigated. Desialylation kinetics revealed all the charge intermediates from a complex native to a homogeneous form. The relation between this heterogeneity and posttranslational events was explored in cultured hepatoma cells. Intracellular factor B exhibited the same isoelectric focusing pattern as the desialylated purified protein, whereas a highly heterogeneous form was secreted. In contrast, when N-glycosylation was prevented by tunicamycin, both intracellular and secreted forms focused like intracellular factor B from control cultures. These data lead to the conclusion that the microheterogeneity of human factor B results from different degrees of sialylation of its N-glycans.

Genetic variants of factor B in a population of Jordan.

BF phenotyping was performed in a population of Jordan. The observed allele frequencies were as follows: BF*S = 0.5457, BF*F = 0.3744, BF*SO7 = 0.0763, BF*F1 = 0.0075. These values are in agreement with the geographic position and the ethnic composition of Jordan.

Alpha-1-antitrypsin phenotypes in a population of Jordan.

Frequencies of the three common subtypes of PI M were studied in a Jordanian population. In comparison with other populations, PI*M3 was found to be low (0.038) and PI*M2 rather high (0.155).

Inulin-induced activation of factor B in whole serum: description of structural modifications in the Ba fragment.

The investigation of inulin-induced conversion of human factor B in serum by isoelectrofocusing revealed physiological modifications in the primary structure of the Ba fragment. Evidence has been obtained that a nascent Ba, generated by the hydrolytic action of the D component on B in serum, was a short-lived product and that a fast release of carboxy-terminal arginine and lysine residues occurred involving a serum carboxypeptidase B enzyme.

Human alpha 1-antitrypsin genetic polymorphism: PI N subtypes.

Three new genetic variants (PI types) of alpha 1-antitrypsin are described. They have been compared to previously described phenotypes by several techniques including narrow pH range isoelectric focusing in ultrathin polyacrylamide gels. In this system, the relevant alpha 1-antitrypsin gel bands, identified by crossed immunoelectrophoresis, focused between PI M2, the most cathodal PI M subtype, and PI P BUD, the most anodal PI P subtype. They were therefore considered to be PI N subtypes. Two of them, PI N GRO and PI N YER, could not be separated by isoelectric focusing, but gave a different pattern in agarose gel electrophoresis. None of the new alleles seemed to be associated with disease. The high resolving power of isoelectric focusing is emphasized with respect to the information it may provide concerning amino acid substitutions, while the use of other techniques proved to be of utmost importance in the differentiation of other variants showing similar isoelectric points.

Genetic variants of human B component (BF system) and alpha-1-antitrypsin (PI system) in a population from Sardinia.

BF- and PI-type determinations have been performed in a population from Sardinia. The corresponding allele frequencies are as follows: BF*S = 0.5783, BF*F = 0.2189, BF*SO7 = 0.0046, BF*F1 = 0.1982 and PI*M1 = 0.5872, PI*M2 = 0.2041, PI*M3 = 0.0459, PI*M4 = 0.0940, PI*S = 0.0619, PI*Z = 0.0046, PI*N = 0.0023. Whereas the BF system shows the originality of the Sardinian population with a very high BF*F1 allele frequency, the PI system does not reveal any characteristic features.

Genetic variations of serum alpha-1-antitrypsin (Pi types) in Normans. Common Pi M subtypes and new phenotypes.

The results of Pi typing by high resolution isoelectric focusing on 1,030 Normans from the area of Rouen (France) is reported. The frequency of the three common subtypes of Pi M is given and new variants partly described. Pi gene frequencies are compared to previously obtained data and results from other countries. The application of high resolution isoelectric focusing to the Pi system is discussed together with the advantages it provides in the knowledge of alpha-1-antitrypsin genetic polymorphism.

Synthesis of highly diversified carrier ampholytes. Evaluation of the resolving power of isoelectric focusing in the Pi system (alpha-1-antitrypsin genetic polymorphism).

The use of condensing reagents such as epoxypropanol, diepoxyoctane, acrylamide and N,N'-methylenebisacrylamide in the synthesis of carrier ampholytes increased the diversity of amphoteric components. The quality of these synthetic carrier ampholytes has been tested in the separation of variants of alpha-1-anti-trypsin, a genetic polymorphism called the Pi system. A resolving power of the order of 0.005 pH unit was obtained.