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Pseudomonas aeruginosa strain PP4 and Acinetobaeter lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; [GDH.sub.1] was produced when cells were grown on isophthalate, while [GDH.sub.II] was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, [GDH.sub.P], when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. [GDH.sub.I] and [GDH.sub.II] were found to be homotetramers, while [GDH.sub.p] was found to be a homohexamer. [GDH.sub.II] was more sensitive to inhibition by isophthalate (2.5- and 5.5-fold more sensitive for amination and deamination reactions, respectively) than [GDH.sub.I]. Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, [GDH.sub.I] and [GDH.sub.II], in strain PP4. In strain ISP4, irrespective of the carbon source, the [GDH.sub.P] produced showed similar levels of inhibition with isophthalate. However, the specific activity of [GDH.sub.P] from isophthalate-grown cells was 2.5- to 3-fold higher than that of [GDH.sub.P] from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of [GDH.sub.P] in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy. doi: 10.1128/JB.01365-09

The adaptor proteins sodium/hydrogen exchanger regulatory factor (NHERF)-I and NHERF-2 have overlapping tissue distribution in renal cells and overlapping specificity in their binding to renal transporters and other proteins. To compare the kidney-specific differences in the function of these adaptor proteins, NHERF-1 and NHERF-2 null mice were compared with wild-type control mice. In NHERF-2 null mice, the renal proximal tubule abundance and distribution of NHERF-1 and NHERF-3 were not different from those in wild-type animals. The glomerular expression of podocalyxin and ZO-1 also did not differ. NHERF-1 null mice had increased urinary excretion of phosphate, calcium, and uric acid compared with wild-type control and NHERF-2 null mice. Because of the association between NHERF-2 and podocalyxin in glomeruli and C1C-5 in the renal proximal tubule, the urinary excretion of protein was determined. There were no differences in the urinary excretion of protein or low-molecular-weight proteins between wild-type control, NHERF-[1.sup.-/-], and NHERF-[2.sup.- /-] mice. These studies indicate that the increased urinary excretion of phosphate and uric acid are specific to NHERF-1 null mice and highlight the fact that predictions about the role of adaptor proteins such as the NHERF proteins obtained from studies of model cell systems must be confirmed in whole animals. mouse kidney; PDZ proteins; sodium/hydrogen exchanger regulatory factor

Phospholipid bilayer coatings can prevent adsorption of cationic proteins on the surface of fused silica capillaries used in capillary electrophoresis. However, the performance of such bilayer coatings is strongly dependent on solution conditions. The factors affecting the rate of formation of phospholipid bilayer coatings were investigated using the double-chained zwitterionic 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC, [C.sub.14]) as a model phospholipid. The effectiveness of these coatings for CE separations of model cationic lysozyme, cytochrome c, ribonuclease A, and [alpha]-chymotrypsinogen A was also assessed. Increasing the ionic strength of a 0.1 mM DMPC solution reduced capillary coat times from >2 hours in 2.5 mM Tris (pH 7.4) buffer to 3.4 min in 40 mM Tris and dramatically improved separation performance such that [greater than or equal to] 1.4 x [10.sup.5] plates/m were observed in capillaries coated for 5 min with 0.1 mM DMPC in 20 mM Tris-HC1 (pH 7.4). The presence of [Ca.sup.2-] in the coating solution also increases the rate of formation of the phospholipid bilayer coating. The type of vesicle strongly affects its adsorption rate onto the silica surface. The time required to coat the capillary was 7.2 min for small unilamellar vesicles (SINs) and 22.5 min for large uniiamellar vesicles and excessively long for multilamellar vesicles. Highest efficiency protein separations were achieved with bilayer coatings prepared from SUVs. The coating rate was enhanced by using greater DMPC concentrations and unaffected by pH. The type of buffer present in the DMPC coating solution affects the coating behavior, with HEPES buffer yielding a faster coat time than either Tris or phosphate buffers. Histone H1 was separated on a 0.1 mM DMPC-coated capillary.

Capillary electrophoresis (CE) with postcolumn laser-induced fluorescence detection (LIF) was used to analyze single skeletal muscle fibers from young and old rats. Due to selective labeling of mitochondria with 10-N-nonyl acridine orange, the zeptomole ([10.sup.-21] mole) sensitivity, and the high separation power, three properties of individual mitochondrial particles were revealed: the number, the distributions of cardiolipin, and their electrophoretic mobilities. Type I fibers had more mitochondrial particles and cardiolipin per particle than did type IIb fibers from rats of similar age. Individual fibers of the same fiber type from young rats contained more mitochondrial particles and cardiolipin per particle than did fibers from old rats. There were fiber typespecific and age-specific differences in the electrophoretic mobility of individual mitochondrial particles. The CE-LIF results of individual mitochondrial particles are the first of their kind in that they reveal fiber type-specific and age-specific differences that are not obviously noticed in bulk measurements of heterogeneous tissues.

Adaptation to and tolerance of bile stress are among the main limiting factors to ensure survival of bifidobacteria in the intestinal environment of humans. The effect of bile salts on protein expression patterns of Bifidobacterium longum was examined. Protein pattern comparison of strains grown with or without bile extract allowed us to identify 34 different proteins whose expression was regulated. The majority of these proteins were induced after both a minor (0.6 g [liter.sup.-1]) and a major (1.2 g [liter.sup.-1]) exposure to bile. These include general stress response chaperones, proteins involved in transcription and translation and in the metabolism of amino acids and nucleotides, and several enzymes of glycolysis and pyruvate catabolism. Remarkably, xylulose 5-phosphate/fructose 6-phosphate phosphoketolase, the key enzyme of the so-called bifidobacterial shunt, was found to be upregulated, and the activity on fructose 6-phosphate was significantly higher for protein extracts of cells grown in the presence of bile. Changes in the levels of metabolic end products (acetate and lactate) were also detected. These results suggest that bile salts, to which bifidobacteria are naturally exposed, induce a complex physiological response rather than a single event in which proteins from many different functional categories take part. This study has extended our understanding of the molecular mechanism underlying the capacity of intestinal bifidobacteria to tolerate bile.

Abstract: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis remains the core of proteomic technology because it is currently the most powerful method to analyze large collections of proteins. Advances in [...]

We have demonstrated recently that, in general, the dielectrophoretic (DEP) force experienced by a particle has two components. The first depends upon inhomogeneities in the applied electrical field strength and upon the in-phase part of the resultant dipole moment induced in the particle and can be identified with the conventional dielectrophoretic (cDEP) force. The second relates to nonuniformities in the phase distribution of the applied field and to the out-of-phase part of the induced dipole moment. A nonuniform phase distribution corresponds to a field that travels through space. This second force component gives rise to traveling-wave dielectrophoresis (twDEP). In this paper, we describe several electrode configurations designed to produce electric fields capable of inducing cDEP and twDEP forces for the purpose of manipulating particles. Using DS19 Friend murine erythroleukemia cells as test particles having well-characterized dielectric properties, we investigated the electrokinetic behaviors for these electrodes as a function of frequency and electrode excitation mode. Several characteristic cell electrokinetic behaviors were identified, including trapping, linear motion, levitation, and circulation of the cells, depending on the excitation characteristics of the electrodes and the cell dielectric properties. We describe these findings and rationalize them in terms of the field distributions produced by the electrodes, the particle dielectric properties, and generalized dielectrophoresis theory. The biotechnological applications of dielectrophoretic manipulation are then discussed. Index Terms - Cell manipulation, dielectrophoresis, electrical field simulation, microelectrode design.

A history of research studies which established the importance of glycoproteins in cell membranes was presented. There have been substantial research activities in cell adhesion molecules called selectins. Selectins are multidomain molecules which are capable of selective adhesions between cells because of their capacity to interrelate with particular cell-surface carbohydrates and a calcium-dependent carbohydrate-binding domain. Research on carbohydrates is important because carbohydrate molecules may provide cells with specific biological recognition properties.

A novel and effective methodology that uses variable field along the separation capillary at a 180 degrees centigrade angle is used to confront the problem in capillary electrophoresis in which the charged polysaccharides experience molecular stretching at constant potentials between 50 and 300 volts per centimeter, and resist the desired separation based on their molecular mass. Extremely efficient separations of polydextrans prove that molecular shape alignments of polysaccharides in relation to the separation medium are greatly affected by variables of the pumping experiment.

The potential-controlled electron-transfer reaction between cyrochrome c and 1, 1'-dimethylferrocene at a liquid-liquid interface is reported. In this approach to protein electrochemistry, the electron transfer process is apparently transport controlled, rather than adsorption limited.

Protein electrochemistry is used for interrogating the equilibrium between the oxidized and reduced conformations of the Escherichia coli enzyme (ecTrxR) and a novel, low molecular weight thioredoxin reductase (TrxR) from the thermophilic archaeon Thermoplasma acidophilum (taTrxR). The studies have shown that protein chemistry between the first and second conformations (FO and FR) have contributed to the formal potentials of the flavin and disulfide cofactors.

The scanning of bilayer over the surface of an SDS-PAGE gel has enabled the direct detection membrane proteins from gels using single-channel recording. Hydrogen scanning with water-in-oil droplet bilayers has provided a new method for discovering and characterizing ion channels with the potential for high-throughput screening.

The impact of the formation of a single advanced glycation end product (AGE) on the structure and chaperone activity of a human [alpha]B-crystallin is discussed. The findings reveal that a single AGE changes the chaperoning ability of the protein without changing its local environment.

The cleavage products of FIX resulting from activation by FIXa at several different substrates of enzyme ratios were examined. The results showed the presence of an exosite for FIX binding on the FXIa remote form its active site.