Controlling chronic Pseudomonas aeruginosa infections by strategically interfering with the sensory function of SagS

The hybrid sensor SagS plays a central role in the formation of Pseudomonas aeruginosa biofilms, by enabling the switch from the planktonic to the biofilm mode of growth and by facilitating the transition of biofilm cells to a highly tolerant state. In this study, we examined the importance of the SagS key amino acid residues associated with biofilm formation (L154) and antibiotic tolerance (D105) in P. aeruginosa virulence. Recombinant P. aeruginosa ΔsagS and ΔsagS chromosomally expressing wild‐type sagS, or its two variants D105A and L154A, were tested for their potential to form biofilms and cause virulence in plants and mouse models of acute and chronic pneumonia. Although mutation of sagS did not alter P. aeruginosa virulence during acute infections, a significant difference in pathogenicity of sagS mutants was observed during chronic infections, with the L154A variant showing reduced bacterial loads in the chronic pneumonia model, while interference with the D105 residue enhanced the susceptibility of P. aeruginosa biofilms during tobramycin treatment. Our findings suggest that interference with the biofilm or tolerance regulatory circuits of SagS affects P. aeruginosa pathogenicity in chronic but not acute infections, and reveal SagS to be a promising new target to treat P. aeruginosa biofilm infections.     

Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. Regulation of nuclear localization by PDZ interactions

Syntrophins are modular adapter proteins that link ion channels and signaling proteins to dystrophin and its homologues. A yeast two-hybrid screen of a human brain cDNA library using the PDZ domain of gamma 1- syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts diacylglycerol into phosphatidic acid. In biochemical assays, the C terminus of DGK-zeta, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with gamma 1-syntrophin. When coexpressed in HeLa cells, DGK-zeta and gamma 1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus. DGK-zeta translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation. We found that DGK-zeta recruits gamma 1-syntrophin into the nucleus and that the PDZ-binding motif is required. Disrupting the interaction altered the intracellular localization of both proteins; DGK-zeta accumulated in the nucleus, whereas gamma 1-syntrophin remained in the cytoplasm. The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-zeta. In the brain, DGK-zeta and gamma 1-syntrophin were colocalized in cell bodies and dendrites of cerebellar Purkinjie neurons and other neuronal cell types, suggesting that their interaction is physiologically relevant. Moreover, coimmunoprecipitation and pull-down experiments from brain extracts and cells suggest that DGK-zeta, gamma 1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that gamma 1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling.     

Simultaneous monitoring of Zn2+ secretion and intracellular Ca2+ from islets and islet cells by fluorescence microscopy

A method for simultaneously imaging Zn2+ secretion and intracellular Ca2+ at beta-cell clusters and single islets of Langerhans was developed. Cells were loaded with the Ca2+ indicator Fura Red, incubated in buffer containing the Zn2+ indicator FluoZin-3, and imaged via laser scanning fluorescence confocal microscopy. FluoZin-3 and Fura Red are excited at 488 nm and emit at 515 and 665 nm, respectively. Zn2+, which is co-released with insulin, reacts with extracellular FluoZin-3 to form a fluorescent product. Stimulation of cell clusters with glucose evoked increases and oscillations in intracellular Ca2+ and Zn2+ secretion that were correlated with each other and were synchronized among cells. In single islets, spatially resolved dynamics of secretion including detection of first phase, second phase, and synchronized oscillations around the islet were observed. Fura Red did not yield detectable Ca2+ signals at islets. For islet measurements, cells were loaded with Fura-2 and incubated in FluoZin-3 while sequentially illuminating the islets with 340, 380, and 470 nm light and acquiring epi-fluorescence images with a charge-coupled device (CCD) camera. This allowed Ca2+ and secretion to be observed with approximately 2 s temporal resolution. This method should be useful for studying Ca2+ secretion coupling and any application, requiring rapid assays of secretion.     

Fluorescent sodium ion indicators based on the 1,7-diaza-15-crown-5 system

A series of novel sodium ion-sensitive fluorescent reagents suitable for biological applications is described. The chelator nitrogen atom substituents affect the selectivity and affinity of cation binding, while the nature of the fluorophore determines the type of fluorescent response to metal ion chelation.     

Use of reversed phase HP liquid chromatography to assay conversion of N-acylglycines to primary fatty acid amides by peptidylglycine-alpha-amidating monooxygenase

Primary fatty acid amides (R-CO-NH2) and N-acylglycines (R-CO-NH-CH2-COOH) are classes of compounds that have only recently been isolated and characterized from biological sources. Key questions remain regarding how these lipid amides are produced and degraded in biological systems. Relative to the fatty acids, little has been done to develop methods to separate and quantify the fatty acid amides and N-acylglycines. We describe reversed phase HPLC methods for the separation of C2-C12 primary fatty acid amides and N-acylglycines and also C12-C22 fatty acid amides. Separation within each class occurs primarily on the basis of simple interactions between the acyl chain and the chromatographic stationary phase, but the polar headgroups on these and related fatty acids and N-acylethanolamides modulate the absolute retention in reversed phase mode. We use these methods to measure the enzyme-mediated, two-step conversion of N-octanoylglycine to octanoamide.     

Protein transfer through polyacrylamide hydrogel membranes polymerized in lyotropic phases

A way to control the average pore size in cross-linked polyacrylamide-based membranes is by altering the ratio of cross-linker to acylamide monomer. Larger pore sizes are prepared with a minimum amount of cross-linker, resulting in membranes that are mechanically weak and have short lifetimes. The aim of this study was to prepare cross-linked polyacrylamide membranes with large pore sizes and with good mechanical integrity. The methodology was to carry out the polymerization in a template, formed from the self-aggregation of surfactant. Two surfactant templates were used, and their pore size was examined with proteins of different sizes. The surfactants chosen for this study were sodium dodecyl sulfate (SDS, ionic surfactant) and TERIC BL8 (nonionic surfactant), both of which have very different aggregation properties. The data showed that at 10% and greater of TERIC BL8, a very different and open gel structure is formed, in which the pore size was significantly increased. SDS seemed to have little effect on the pore size. The data suggests that the gel structures for both surfactants up to 4% (w/v) are similar and micellular, because SDS is known to favor a micelle structure. Above 4% (w/v), TERIC BL8 then goes through a change in its lyotropic phase, thus, producing membranes of a large pore size. In conclusion, the pore size and gel structure of polyacrylamide hydrogel membranes can be significantly increased using TERIC BL8 (nonionic) surfactant. This allows large-pore-size membranes with a high cross-link density and consequently high mechanical strength to be prepared for the separation of large biomolecules.     

Genetic consequences of white-tailed deer (Odocoileus virginianus) restoration in Mississippi

White-tailed deer (Odocoileus virginianus) were nearly extirpated from the southeastern USA during the late 19th and early 20th centuries. Recovery programmes, including protection of remnant native stocks and transplants from other parts of the species' range, were initiated in the early 1900's. The recovery programmes were highly successful and deer are presently numerous and continuously distributed throughout the southeastern USA. However, the impact of the recovery programmes on the present genetic structure of white-tailed deer remains to be thoroughly investigated. We used 17 microsatellite DNA loci to assess genetic differentiation and diversity for 543 white-tailed deer representing 16 populations in Mississippi and three extra-state reference populations. There was significant genetic differentiation among all populations and the majority of genetic variation (> or = 93%) was contained within populations. Patterns of genetic structure, genetic similarity and isolation by distance within Mississippi were not concordant with geographical proximity of populations or subspecies delineations. We detected evidence of past genetic bottlenecks in nine of the 19 populations examined. However, despite experiencing genetic bottlenecks or founder events, allelic diversity and heterozygosity were uniformly high in all populations. These exceeded reported values for other cervid species that experienced similar population declines within the past century. The recovery programme was successful in that deer were restored to their former range while maintaining high and uniform genetic variability. Our results seem to confirm the importance of rapid population expansion and habitat continuity in retaining genetic variation in restored populations. However, the use of diverse transplant stocks and the varied demographic histories of populations resulted in fine-scale genetic structuring.     

Tumor necrosis factor-alpha induces functionally active hyaluronan-adhesive CD44 by activating sialidase through p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated human monocytic cells

Interaction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-alpha and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-alpha-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-alpha. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-alpha-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-alpha production. Blocking TNF-alpha production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-alpha-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-alpha-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells.