Use of SPR to Study the Interaction of G7-18NATE Peptide with the Grb7-SH2 Domain

Surface plasmon resonance (SPR) is a useful biosensor technique for the study of biomolecular interactions, with the potential for high-throughput screening of ligand interactions with drug targets. The key to its successful use, however, is in the appropriate design of the experiment, including the mode of immobilization to the biosensor chip. We report an investigation of the use of SPR for measuring the affinity of the G7-18NATE peptide ligand for its Grb7-SH2 domain target involved in the migratory and proliferative potential of cancer cells. Previous studies have shown that the cyclic non-phosphorylated peptide, G7-18NATE, inhibits Grb7 interactions with upstream binding partners and is able to inhibit both cell migration and proliferation of cancer cells. We report the synthesis of a biotinylated G7-18NATE covalently attached to a linker (G7-18NATE-ASASASK-Biotin) and compare its interaction with the Grb7-SH2 domain by SPR using three different immobilization strategies; immobilisation of the peptide via streptavidin, immobilization of glutathione S-transferase (GST)-Grb7-SH2 domain via anti-GST antibody, and immobilization of biotinylated Grb7-SH2 domain via streptavidin. This revealed that sensorgrams free from non-specific binding and displaying simple kinetics were most readily achieved by immobilising the protein rather than the peptide, in spite of the lower response associated with this method. K _D values of ~300 μM were determined for both strategies at pH 7.4. This compared with a K _D value of 4.4 μM at pH 6 demonstrating the importance of pH on this interaction. Overall, the immobilised protein systems are most suitable for future comparative screening efforts using SPR.     

Differential effects of trichostatin A on gelatinase A expression in 3T3 fibroblasts and HT-1080 fibrosarcoma cells: implications for use of TSA in cancer therapy

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor with potential in cancer therapeutics. In a recent communication, we demonstrated that TSA is a selective, potent inhibitor of gelatinase A in 3T3 fibroblasts. In the present study, we extend these observations and examine the effects of TSA in 3T3 fibroblasts compared to HT-1080 fibrosarcoma cells with respect to gelatinase A expression, cell viability, and apoptosis. We find that while expression of gelatinase A in 3T3 fibroblasts is exquisitely sensitive to inhibition by TSA, expression of this enzyme in HT-1080 cells is minimally affected by this compound. Moreover, we show that TSA is pro-apoptotic in HT-1080 cells, but is anti-apoptotic in 3T3 cells. We propose a two-pronged model for the therapeutic action of TSA. On the one hand TSA selectively decreases cancer cell viability, while enhancing the viability of stromal cells. On the other hand, by selectively decreasing gelatinase A expression in stromal but not cancer cells, TSA acts to control metastatic potential by reducing the ability of metastatic cells to recruit stromal cells to secrete gelatinase A.     

Regulation of Staphylococcus epidermidis-induced IFN-gamma in whole human blood: the role of endogenous IL-18, IL-12, IL-1, and TNF

Interleukin 12 (IL-12) and IL-18 act synergistically to stimulate interferon gamma (IFN-gamma) production; moreover, IL-1 and tumor necrosis factor (TNF) may also augment IFN-gamma synthesis. We have investigated the relative contributions of these cytokines in the production of IFN-gamma and TNF by the Gram-positive bacterium Staphylococcus epidermidis, using the specific cytokine inhibitors IL-18 binding protein (IL-18BP), IL-1 receptor antagonist (IL-1Ra), anti-IL-12 antibodies (anti-IL-12 Ab), and TNF binding protein. Inhibition of caspase-1 reduced IFN-gamma and IL-1beta levels (by 80 and 67%, respectively) when heat-killed S. epidermidis was added to whole human blood cultures. IL-18BP reduced S. epidermidis-induced IFN-gamma (77% maximal suppression). In contrast, blocking IL-1 receptors by IL-1Ra had no effect on IFN-gamma production. Blocking endogenous IL-12 and TNF reduced IFN-gamma production by 69 and 36%. S. epidermidis-induced TNF-alpha was inhibited by IL-18BP and IL-1Ra, but not anti-IL-12 Ab, whereas IL-8 production was unaffected by any of the specific cytokine blocking agents. In conclusion, S. epidermidis stimulates IFN-gamma which is IL-18, IL-12 and TNF-dependent, but IL-1 independent.     

Gauging of the PhoE channel by a single freely diffusing proton

In the present study we combined a continuum approximation with a detailed mapping of the electrostatic potential inside an ionic channel to define the most probable trajectory for proton propagation through the channel (propagation along a structure-supported trajectory (PSST)). The conversion of the three-dimensional diffusion space into propagation along a one-dimensional pathway permits reconstruction of an ion motion by a short calculation (a few seconds on a state-of-the-art workstation) rather than a laborious, time-consuming random walk simulations. The experimental system selected for testing the accuracy of this concept was the reversible dissociation of a proton from a single pyranine molecule (8-hydroxypyrene-1,2,3-trisulfonate) bound by electrostatic forces inside the PhoE ionic channel of the Escherichia coli outer membrane. The crystal structure coordinates were used for calculation of the intra-cavity electrostatic potential, and the reconstruction of the observed fluorescence decay curve was carried out using the dielectric constant of the intra-cavity space as an adjustable parameter. The fitting of past experimental observations (Shimoni, E., Y. Tsfadia, E. Nachliel, and M. Gutman. 1993. Biophys. J. 64:472-479) was carried out by a modified version of the Agmon geminate recombination program (Krissinel, E. B., and N. Agmon. 1996. J. Comp. Chem. 17:1085-1098), where the gradient of the electrostatic potential and the entropic terms were calculated by the PSST program. The best-fitted reconstruction of the observed dynamics was attained when the water in the cavity was assigned epsilon 相似文献   

A fast in silico simulation of ion flux through the large-pore channel proteins

The PSST program (see accompanying article) utilizes the detailed structure of a large-pore channel protein as the sole input for selection of trajectories along which negative and positive ions propagate. In the present study we applied this program to reconstruct the ion flux through five large-pore channel proteins (PhoE, OmpF, the WT R. blastica general diffusion porin and two of its mutants). The conducting trajectories, one for positive and one for negative particles, are contorted pathways that run close to arrays of charged residues on the inner surface of the channel. In silico propagation of the charged particles yielded passage time values that are compatible with the measured average passage time of ions. The calculated ionic mobilities are close to those of the electrolyte solution of comparable concentrations. Inspection of the transition probabilities along the channel revealed no region that could impose a rate-limiting step. It is concluded that the ion flux is a function of the whole array of local barriers. Thus, the conductance of the large-pore channel protein is determined by the channel's shape and charge distribution, while the selectivity also reflects the features of the channel's vestibule.     

Identification and nucleotide sequence of Brucella melitensis L7/L12 ribosomal protein

Abstract DNA sequencing of the gene encoding a Brucella melitensis 12-kDa protein revealed that this protein was the ribosomal protein L7/L12. The B. melitensis L7/L12 DNA sequence was identical to that of the corresponding B. abortus gene, showing the near identity of these two organisms. When comparing the sequence of this protein to that of other organisms some domains were highly conserved, especially the C-terminus, which contrasted with the lack of conservation of the sequences at the N-terminus. The finding that the ribosomal protein L7/L12 of Brucella is an immunodominant antigen provides a new rationale to explain the activity of ribosomal vaccines.     

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig

Glial cells of the myenteric plexus from guinea pig small intestine were intracellulary filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid, somata (85±0.7 m equivalent diameter, i.e., about 330 m³ volume), and send up to 20 thin and short processes (less than 26 to about 110 m in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly, branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of holes in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of 2000 m², almost 90% of which are contributed by the cell processes. This geometry may enable an intense exchange of metabolites and ions between neurons, glial cells, and the capillaries and/or environment of enteric ganglia.     

Interactions between plasma membrane aquaporins modulate their water channel activity

Plant plasma membrane intrinsic proteins (PIPs) cluster in two evolutionary subgroups, PIP1 and PIP2, with different aquaporin activities when expressed in Xenopus oocytes. Maize ZmPIP1;1 and ZmPIP1;2 do not increase the osmotic water permeability coefficient (Pf), whereas ZmPIP2;1, ZmPIP2;4, and ZmPIP2;5 do. Here, we show that coexpression of the nonfunctional ZmPIP1;2 and the functional ZmPIP2;1, ZmPIP2;4, or ZmPIP2;5 resulted in an increase in Pf that was dependent on the amount of injected ZmPIP1;2 complementary RNA. Confocal analysis of oocytes expressing ZmPIP1;2-green fluorescent protein (GFP) alone or ZmPIP1;2-GFP plus ZmPIP2;5 showed that the amount of ZmPIP1;2-GFP present in the plasma membrane was significantly greater in coexpressing cells. Nickel affinity chromatography purification of ZmPIP2;1 fused to a His tag coeluted with ZmPIP1;2-GFP demonstrated physical interaction and heteromerization of both isoforms. Interestingly, coexpression of ZmPIP1;1 and ZmPIP2;5 did not result in a greater increase in Pf than did the expression of ZmPIP2;5 alone, but coexpression of the ZmPIP1;1 and ZmPIP1;2 isoforms induced a Pf increase, indicating that PIP1 isoform heteromerization is required for both of them to act as functional water channels. Mutational analysis demonstrated the important role of the C-terminal part of loop E in PIP interaction and water channel activity induction. This study has revealed a new mechanism of plant aquaporin regulation that might be important in plant water relations.     

Morphological and electrophysiological changes in mouse dorsal root ganglia after partial colonic obstruction

There is evidence that sensitization of neurons in dorsal root ganglia (DRG) may contribute to pain induced by intestinal injury. We hypothesized that obstruction-induced pain is related to changes in DRG neurons and satellite glial cells (SGCs). In this study, partial colonic obstruction was induced by ligation. The neurons projecting to the colon were traced by an injection of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate into the colon wall. The electrophysiological properties of DRG neurons were determined using intracellular electrodes. Dye coupling was examined with an intracellular injection of Lucifer yellow (LY). Morphological changes in the colon and DRG were examined. Pain was assessed with von Frey hairs. Partial colonic obstruction caused the following changes. First, coupling between SGCs enveloping different neurons increased 18-fold when LY was injected into SGCs near neurons projecting to the colon. Second, neurons were not coupled to other neurons or SGCs. Third, the firing threshold of neurons projecting to the colon decreased by more than 40% (P < 0.01), and the resting potential was more positive by 4-6 mV (P < 0.05). Finally, the number of neurons displaying spontaneous spikes increased eightfold, and the number of neurons with subthreshold voltage oscillations increased over threefold. These changes are consistent with augmented neuronal excitability. The pain threshold to abdominal stimulation decreased by 70.2%. Inflammatory responses were found in the colon wall. We conclude that obstruction increased neuronal excitability, which is likely to be a major factor in the pain behavior observed. The augmented dye coupling between glial cells may contribute to the neuronal hyperexcitability.     

Influence of growth phase on adhesion kinetics of Escherichia coli D21g

The influence of bacterial growth stage and the evolution of surface macromolecules on cell adhesion have been examined by using a mutant of Escherichia coli K-12. To better understand the adhesion kinetics of bacteria in the mid-exponential and stationary growth phases under flow conditions, deposition experiments were conducted in a well-controlled radial stagnation point flow (RSPF) system. Complementary cell characterization techniques were conducted in combination with the RSPF experiments to evaluate the hydrophobicity, electrophoretic mobility, size, and titratable surface charge of the cells in the two growth phases considered. It was observed that cells in stationary phase were notably more adhesive than those in mid-exponential phase. This behavior is attributed to the high degree of local charge heterogeneity on the outer membranes of stationary-phase cells, which results in decreased electrostatic repulsion between the cells and a quartz surface. The mid-exponential-phase cells, on the other hand, have a more uniform charge distribution on the outer membrane, resulting in greater electrostatic repulsion and, subsequently, less adhesion. Our results suggest that the macromolecules responsible for this phenomenon are outer membrane-bound proteins and lipopolysaccharide-associated functional groups.