Preclinical Assessment of Adjunctive tPA and DNase for Peritoneal Dialysis Associated Peritonitis

A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.     

Deuterium NMR of 2HCO-Val1...gramicidin A and 2HCO-Val1-D-Leu2...gramicidin A in oriented DMPC bilayers.

Deuterium NMR is used to study the structure and dynamics of the formyl C-2H bond in selectively deuterated gramicidin molecules. Specifically, the functionally different analogues 2HCO-Val1...gramicidin A and 2HCO-Val1-D-Leu2...gramicidin A are studied by 2H NMR so that any conformational or dynamical differences between the two analogues can be correlated with their difference in lifetime. These analogues are first synthesized, purified, and characterized and then incorporated into oriented bilayers of dimyristoylphosphatidylcholine sandwiched between glass coverslips. Phosphorous NMR line shapes obtained from these samples are consistent with the presence of the bilayer phase and indicate that the disorder exhibited by the lipid matrix is approximately of the same type and degree for both analogues. Deuterium NMR line shapes obtained from these samples indicate that the motional axis of the formyl group of gramicidin is parallel to the coverslip normal, that the distribution of motional axis orientations has a width of 7-9 degrees, and that a similar, major conformational and dynamical state exists for the formyl C-2H bond of both analogues. In this state, if the only motion present is fast axial rotation, then the experimentally derived angle between the formyl C-2H bond and the motional axis is consistent with the presence of a right-handed, single-stranded, beta 6.3 helical dimer but is not consistent with the presence of a left-handed, single-stranded, beta 6.3 helical dimer. However, if fast axial rotation is not the only motion present, then the left-handed, single-stranded, beta 6.3 helical dimer cannot be absolutely excluded as a possibility. Also, a second, minor conformational and dynamical state appears to be present in the spectrum of 2HCO-Val1-D-Leu2...gramicidin A but is not observed in the spectrum of 2HCO-Val1...gramicidin A. This minor conformational and dynamical state may reflect the presence of monomers, while the major conformational and dynamical state may reflect the presence of dimers.     

Amyloid-beta peptide, substance P, and bombesin bind to the serpin-enzyme complex receptor.

During the formation of an inhibitory complex with neutrophil elastase, alpha 1 antitrypsin (alpha 1 AT) undergoes a structural rearrangement and the resulting alpha 1 AT-elastase complex becomes endowed with chemoattractant activities, mediates an increase in synthesis of alpha 1 AT, and is rapidly cleared from the circulation. In previous studies we have provided evidence that these biological activities involve the recognition of a conformation-specific domain in the alpha 1 AT molecule by a cell surface receptor on human hepatoma HepG2 cells and human monocytes. The receptor has been termed the serpin-enzyme complex (SEC) receptor because it also recognizes complex of serpins antithrombin III, alpha 1 anti-chymotrypsin, and C1 inhibitor with their cognate enzymes. Because a pentapeptide domain of alpha 1 AT (amino acids 370-374, Phe-Val-Phe-Leu-Met) is sufficient for binding to the SEC receptor and the sequence of this domain is remarkably similar to those of substance P, several other tachykinins, bombesin, and the amyloid-beta peptide, we have examined the possibility that these other ligands bind to the SEC receptor. The results indicate that substance P, several other tachykinins, and bombesin compete for binding to, and cross-linking of, the SEC receptor. The SEC receptor is distinct from the substance P receptor by several criteria. There is no substance P receptor mRNA in HepG2 cells; the SEC receptor is present in much higher density on receptor-bearing cells and binds its ligands at lower affinity than the substance P receptor; the SEC receptor is much less restricted in the specificity with which it recognizes ligand; ligands for the SEC receptor including peptide 105Y (based on alpha 1 AT sequence 359-374), alpha 1 AT-protease complexes, and bombesin do not compete for binding of substance P to a stable transfected cell line expressing the substance P receptor. Finally, we show here that the amyloid-beta peptide competes for binding to the SEC receptor but does not bind to the substance P receptor, therein raising the possibility that the SEC receptor is involved in certain biological activities, including the recently described neurotrophic and neurotoxic effects ascribed to the amyloid-beta peptide.     

Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.     

Regulation of endomembrane biogenesis in arabidopsis by phospatidic acid hydrolase

Coordination of membrane lipid biosynthesis is important for cell function during plant growth and development. Here we summarize our recent work on PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) which suggests that this enzyme is a key regulator of phosphaticylcholine (PC) biosynthesis in Arabidopsis thaliana. Disruption of PAH activity elevates phosphatidic acid (PA) levels and stimulates PC biosynthesis and biogenesis of the endoplasmic reticulum (ER). Furthermore, the activity of PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT), which is the key enzyme controlling the rate of PC biosynthesis, is directly stimulated by PA and expression of a constitutively active version of CCT replicates the effects of PAH disruption. Hence PAH activity can control the abundance of PA, which in turn can modulate CCT activity to govern the rate of PC biosynthesis. Crucially it is not yet clear how PAH activity is regulated in Arabidopsis but there is evidence that PAH1 and PAH2 are both phosphorylated and further work will be required to investigate whether this is functionally significant.     

Spatial distribution of fine root biomass in a remnant Eucalyptus tereticornis woodland in Eastern Australia

Plant Ecology - In forests, the majority of fine roots are located within the upper soil horizons, and fine root biomass decreases with depth. We evaluated spatial patterns in the distribution of...     

Site-specific modification of the bovine genome using Cre recombinase-mediated gene targeting.

Cre recombinase (Cre)-mediated targeted insertion of a transgene is a powerful technique that can be used to tailor genomes. When combined with somatic cell nuclear transfer it could offer an efficient way to generate transgenic livestock with site-specific genetic modifications that are free of antibiotic selection markers. We have engineered primary bovine fibroblasts to contain a chromosomal acceptor site with incompatible loxP/lox2272 sites for Cre-mediated cassette exchange and show for the first time that Cre-mediated targeting can be applied in these acceptor cells. Molecular characterization of the resulting cell clones revealed Cre-mediated transgene insertion efficiencies of up to 98% when antibiotic selection was used to identify transgene containing cell clones. Most clonal lines also contained random insertions of the targeting and Cre expression plasmids with only about 10% of the clones being exclusively modified by the intended targeted insertion. This targeting efficiency was sufficient to enable the isolation of correctly targeted clones without the help of antibiotic selection. Therefore, this recombinase-mediated insertion strategy has the potential to produce transgenic cattle from antibiotic selection marker-free somatic cells with transgenes inserted into proven genomic loci ensuring reliable expression levels.     

Interaction between the light quality and flowering time pathways in Arabidopsis

Flowering in Arabidopsis is accelerated by a reduced ratio of red light to far-red light (R/FR), which indicates the proximity of competitive vegetation. By exploiting the natural genetic variation in flowering time responses to low R/FR, we obtained further insight into the complex pathways that fine-tune the transition to flowering in Arabidopsis. The Bla-6 ecotype does not flower significantly earlier in response to low R/FR, but is still able to display other features of shade avoidance, suggesting branching of low R/FR signalling. Here we show that the muted flowering response of Bla-6 is due to high levels of the floral repressor FLOWERING LOCUS C (FLC), conferred by a combination of functional FLC and FRIGIDA ( FRI ) alleles with a 'weak' FY allele. The Bla-6 FY allele encodes a protein with a corrupted WW binding domain, and we provide evidence that this locus plays a key role in the natural variation in light quality-induced flowering in Arabidopsis. In Bla-6, FLC blocks promotion to flowering by reduced R/FR by inhibiting expression of the floral integrator FLOWERING LOCUS T ( FT ) in a dose-dependent manner. Reduction of FLC removes this obstruction, and Bla6 plants then exhibit strong induction of FT and flower early in response to a low R/FR signal. This paper illustrates the intricate interaction of environmental signals and genetic factors to regulate flowering in Arabidopsis.     

Identification of neural circuits by imaging coherent electrical activity with FRET-based dyes.

We show that neurons that underlie rhythmic patterns of electrical output may be identified by optical imaging and frequency-domain analysis. Our contrast agent is a two-component dye system in which changes in membrane potential modulate the relative emission between a pair of fluorophores. We demonstrate our methods with the circuit responsible for fictive swimming in the isolated leech nerve cord. The output of a motor neuron provides a reference signal for the phase-sensitive detection of changes in fluorescence from individual neurons in a ganglion. We identify known and possibly novel neurons that participate in the swim rhythm and determine their phases within a cycle. A variant of this approach is used to identify the postsynaptic followers of intracellularly stimulated neurons.