Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase

Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD(+) synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme's catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, K(a), equal to 5.6 x 10(6)/M. DSC curve analysis is consistent with this finding. The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer <==> folded monomer <==> unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large K(a) down to approximately 10(6)/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented.     

cAMP-stimulated Na+ transport in H441 distal lung epithelial cells: role of PKA, phosphatidylinositol 3-kinase, and sgk1

H441 cells, a bronchiolar epithelial cell line, develop a cAMP-regulated benzamil-sensitive Na+ transport pathway on permeable supports (Itani OA, Auerbach SD, Husted RF, Volk KA, Ageloff S, Knepper MA, Stokes JB, Thomas CP. Am J Physiol Lung Cell Mol Physiol 282: L631-L641, 2002). To understand the molecular basis for the stimulation of Na+ transport, we delineated the role of specific intracellular pathways and examined the effect of cAMP on alphabetagamma-epithelial Na+ channel (ENaC) and sgk1 expression. Na+ transport increases within 5 min of cAMP stimulation and is sustained for >24 h. The sustained effect of cAMP on Na+ transport is abolished by LY-294002, an inhibitor of phosphatidylinositol 3-kinase, by H89, an inhibitor of PKA, or by SB-202190, an inhibitor of p38 MAP kinase. The sustained effect of cAMP was associated with increases in alpha-ENaC mRNA and protein but without a detectable increase in betagamma-ENaC and sgk1. The early effect of cAMP on Na+ transport is brefeldin sensitive and is mediated via PKA. These results are consistent with a model where the early effect of cAMP is to increase trafficking of Na+ channels to the apical cell surface whereas the sustained effect requires the synthesis of alpha-ENaC.     

Mac-1, but not LFA-1, uses intercellular adhesion molecule-1 to mediate slow leukocyte rolling in TNF-alpha-induced inflammation

We have previously shown that Mac-1 and LFA-1 play a cooperative role in slow leukocyte rolling in inflamed vessels, and that, although both have a role in leukocyte adhesion, the contribution from LFA-1 exceeds that of Mac-1. In this study, we used mice deficient in ICAM-1 (ICAM-1(null)) to study the function of ICAM-1 as an endothelial ligand for Mac-1 and LFA-1. The cremaster muscles of these mice were treated with TNF-alpha and prepared for intravital microscopy. We found that the average rolling velocity in venules was not different in ICAM-1(null) mice (4.7 micro m/s) compared with wild-type mice (5.1 micro m/s). Similarly, leukocyte adhesion efficiency in ICAM-1(null) mice (0.11 +/- 0.01 mm) was similar to that in Mac-1(-/-) (0.12 +/- 0.03 mm) mice but significantly increased compared with that in LFA-1(-/-) (0.08 +/- 0.01 mm) mice and significantly reduced from that in wild type (0.26 +/- 0.04 mm). When both LFA-1 and ICAM-1 were blocked, rolling velocity increased, and adhesion efficiency and arrest decreased. However, blocking both Mac-1 and ICAM-1 had no greater effect than either blockade alone. We conclude that endothelial ICAM-1 is the main ligand responsible for slow leukocyte rolling mediated by Mac-1, but not LFA-1.     

Ultra scale-down approach for the prediction of full-scale recovery of ovine polycolonal immunoglobulins used in the manufacture of snake venom-specific Fab fragment

In this article, we describe a new approach that allows the prediction of the performance of a large-scale integrated process for the primary recovery of a therapeutic antibody from an analysis of the individual unit operations and their interactions in an ultra scale-down mimic of the process. The recovery process consisted of four distinct unit operations. Using the new approach we defined the important engineering parameters in each operation that impacted the overall recovery process and in each case verified its effect by a combination of modelling and experimentation. Immunoglobulins were precipitated from large volumes of dilute blood plasma and the precipitated flocs were recovered by centrifugal separation from the liquor containing contaminating proteins, including albumin. The fluid mechanical forces acting on the precipitate and the time of exposure to these forces were used to define a time-integrated fluid stress. This was used as a scaling factor to predict the properties of the precipitated flocs at large scale. In the case of centrifugation, the performance of a full-scale disc stack centrifuge was predicted. This was achieved from a computational fluid dynamics (CFD) analysis of the flow field in the centrifuge coupled with experimental data obtained from the precipitated immunoglobulin flocs using the scale-down precipitation tank, a rotating shear device, and a standard swing-out rotor centrifuge operating under defined conditions. In this way, the performance of the individual unit operations, and their linkage, was successfully analysed from a combination of modelling and experiments. These experiments required only millilitre quantities of the process material. The overall performance of the large-scale process was predicted by tracking the changes in physical and biological properties of the key components in the system, including the size distribution of the antibody precipitates and antibody activity through the individual unit operations in the ultra scale-down process flowsheet.     

Agrobacterium tumefaciens VirB6 protein participates in formation of VirB7 and VirB9 complexes required for type IV secretion

This study characterized the contribution of Agrobacterium tumefaciens VirB6, a polytopic inner membrane protein, to the formation of outer membrane VirB7 lipoprotein and VirB9 protein multimers required for type IV secretion. VirB7 assembles as a disulfide cross-linked homodimer that associates with the T pilus and a VirB7-VirB9 heterodimer that stabilizes other VirB proteins during biogenesis of the secretion machine. Two presumptive VirB protein complexes, composed of VirB6, VirB7, and VirB9 and of VirB7, VirB9, and VirB10, were isolated by immunoprecipitation or glutathione S-transferase pulldown assays from detergent-solubilized membrane extracts of wild-type A348 and a strain producing only VirB6 through VirB10 among the VirB proteins. To examine the biological importance of VirB6 complex formation for type IV secretion, we monitored the effects of nonstoichiometric VirB6 production and the synthesis of VirB6 derivatives with 4-residue insertions (VirB6.i4) on VirB7 and VirB9 multimerization, T-pilus assembly, and substrate transfer. A virB6 gene deletion mutant accumulated VirB7 dimers at diminished steady-state levels, whereas complementation with a plasmid bearing wild-type virB6 partially restored accumulation of the dimers. VirB6 overproduction was correlated with formation of higher-order VirB9 complexes or aggregates and also blocked substrate transfer without a detectable disruption of T-pilus production; these phenotypes were displayed by cells grown at 28 degrees C, a temperature that favors VirB protein turnover, but not by cells grown at 20 degrees C. Strains producing several VirB6.i4 mutant proteins assembled novel VirB7 and VirB9 complexes detectable by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two strains producing the D60.i4 and L191.i4 mutant proteins translocated IncQ plasmid and VirE2 effector protein substrates in the absence of a detectable T pilus. Our findings support a model that VirB6 mediates formation of VirB7 and VirB9 complexes required for biogenesis of the T pilus and the secretion channel.     

Opioid agonists have different efficacy profiles for G protein activation,rapid desensitization,and endocytosis of mu-opioid receptors

The differential ability of various mu-opioid receptor (MOP) agonists to induce rapid receptor desensitization and endocytosis of MOP could arise simply from differences in their efficacy to activate G proteins or, alternatively, be due to differential capacity for activation of other signaling processes. We used AtT20 cells stably expressing a low density of FLAG-tagged MOP to compare the efficacies of a range of agonists to 1) activate G proteins using inhibition of calcium channel currents (ICa) as a reporter before and after inactivation of a fraction of receptors by beta-chlornaltrexamine, 2) produce rapid, homologous desensitization of ICa inhibition, and 3) internalize receptors. Relative efficacies determined for G protein coupling were [Tyr-D-Ala-Gly-MePhe-Glyol]enkephalin (DAMGO) (1) > or = methadone (0.98) > morphine (0.58) > pentazocine (0.15). The same rank order of efficacies for rapid desensitization of MOP was observed, but greater concentrations of agonist were required than for G protein activation. By contrast, relative efficacies for promoting endocytosis of MOP were DAMGO (1) > methadone (0.59) > morphine (0.07) > or = pentazocine (0.03). These results indicate that the efficacy of opioids to produce activation of G proteins and rapid desensitization is distinct from their capacity to internalize mu-opioid receptors but that, contrary to some previous reports, morphine can produce rapid, homologous desensitization of MOP.     

Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner.

Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes of Xenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small ( approximately 48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70(S6K) and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70(S6K) was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70(S6K).     

Asymmetrical,water-soluble phthalocyanine dyes for covalent labeling of oligonucleotides

Two new water-soluble, porphyrazine (Pz) dyes containing an isothiocyanate function for covalent linking have each been prepared by cross condensation of two different aromatic dinitriles, one containing carboxylates for solubilizing purposes and the other containing a nitro group for conversion into the labeling function. The initial mononitrotricarboxylato Pzs have been purified to homogeneity from the mixture of Pz congeners formed in the condensation reaction by anion exchange chromatography. The phthalocyanine dye 1 has an absorption maxima at 683 nm while the trinaphthoporphyrazine dye 2 has an absorption maxima at 755 nm, due to the increased size of the aromatic system. Both dyes were successfully conjugated to oligonucleotide primers, showing their potential for use in near-infrared-based DNA diagnostic applications.