Novel isosorbide di-ester compounds as inhibitors of acetylcholinesterase

We report herein that a variety of isosorbide di-esters, previously reported to be novel substrates for butyrylcholinesterase (BuChE, EC 3.1.1.8), are in fact inhibitors of the homologous enzyme acetylcholinesterase (AChE), with IC(50) values in the micromolar range. In vitro studies show that they are mixed inhibitors of the enzyme, and thus the ternary enzyme-inhibitor-substrate complex can form in acetylcholinesterase. This is rationalised by molecular modelling which shows that the compounds bind in the mid-gorge area. In this position, simultaneous substrate binding might be possible, but the hydrolysis of this substrate is prevented. The di-esters dock within the butyrylcholinesterase gorge in a very different manner, with the ester sidechain at the 5-position occupying the acyl pocket at residues Leu286 and Val288, and the 2-ester binding to Trp82. The carbonyl group of the 2-ester is susceptible to nucleophilic attack by Ser198 of the catalytic triad. The larger residues of the acyl pocket in acetylcholinesterase prevent binding in this manner. The results complement each other and explain the differing behaviours of the esters in the cholinesterase enzymes. These findings may prove very significant for future work.     

Protein expression profiles distinguish between experimental invasive pulmonary aspergillosis and Pseudomonas pneumonia

We hypothesized that invasive pulmonary aspergillosis (IPA) may generate a distinctive proteomic signature in plasma and bronchoalveolar lavage (BAL). Proteins in plasma and BAL from two neutropenic rabbit models of IPA and Pseudomonas pneumonia were analyzed by SELDI-TOF MS. Hierarchical clustering analysis of plasma time course spectra demonstrated two clusters of peaks that were differentially regulated between IPA and Pseudomonas pneumonia (57 and 34 peaks, respectively, p<0.001). PCA of plasma proteins demonstrated a time-dependent separation of the two infections. A random forest analysis that ranked the top 30 spectral points distinguished between late Aspergillus and Pseudomonas pneumonias with 100% sensitivity and specificity. Based on spectral data analysis, three proteins were identified using SDS-PAGE and LC/MS and quantified using reverse phase arrays. Differences in the temporal sequence of plasma haptoglobin (p<0.001), apolipoprotein A1 (p<0.001) and transthyretin (p<0.038) were observed between IPA and Pseudomonas pneumonia, as was C-reactive protein (p<0.001). In summary, proteomic analysis of plasma and BAL proteins of experimental Aspergillus and Pseudomonas pneumonias demonstrates unique protein profiles with principal components and spectral regions that are shared in early infection and diverge at later stages of infection. Haptoglobin, apolipoprotein A1, transthyretin, and C-reactive protein are differentially expressed in these infections suggesting important contributions to host defense against IPA.     

Urban Stormwater Runoff Drives Denitrifying Community Composition Through Changes in Sediment Texture and Carbon Content

The export of nitrogen from urban catchments is a global problem, and denitrifying bacteria in stream ecosystems are critical for reducing in-stream N. However, the environmental factors that control the composition of denitrifying communities in streams are not well understood. We determined whether denitrifying community composition in sediments of nine streams on the eastern fringe of Melbourne, Australia was correlated with two measures of catchment urban impact: effective imperviousness (EI, the proportion of a catchment covered by impervious surfaces with direct connection to streams) or septic tank density (which affects stream water chemistry, particularly stream N concentrations). Denitrifying community structure was examined by comparing terminal restriction fragment length polymorphisms of nosZ genes in the sediments, as the nosZ gene codes for nitrous oxide reductase, the last step in the denitrification pathway. We also determined the chemical and physical characteristics of the streams that were best correlated with denitrifying community composition. EI was strongly correlated with community composition and sediment physical and chemical properties, while septic tank density was not. Sites with high EI were sandier, with less fine sediment and lower organic carbon content, higher sediment cations (calcium, sodium and magnesium) and water filterable reactive phosphorus concentrations. These were also the best small-scale environmental variables that explained denitrifying community composition. Among our study streams, which differed in the degree of urban stormwater impact, sediment grain size and carbon content are the most likely drivers of change in community composition. Denitrifying community composition is another in a long list of ecological indicators that suggest the profound degradation of streams is caused by urban stormwater runoff. While the relationships between denitrifying community composition and denitrification rates are yet to be unequivocally established, landscape-scale indices of environmental impact such as EI may prove to be useful indicators of change in microbial communities.     

Factor XI binding to activated platelets is mediated by residues R(250), K(255), F(260), and Q(263) within the apple 3 domain

To localize the platelet binding site on factor XI, rationally designed, conformationally constrained synthetic peptides were used to compete with [(125)I]factor XI binding to activated platelets. The major platelet binding energy resided within the sequence of amino acids T(249)-F(260). Homology scanning, using prekallikrein amino acid substitutions within the synthetic peptide T(249)-F(260), identified a major role for R(250) in platelet binding. Inhibition of [(125)I]factor XI binding to activated platelets by the recombinant Apple 3 domain of factor XI and inhibition by unlabeled factor XI were identical, whereas the recombinant Apple 3 domain of prekallikrein had little effect. A "gain-of-function" chimera in which the C-terminal amino acid sequence of the Apple 3 domain of prekallikrein was replaced with that of factor XI was as effective as the recombinant Apple 3 domain of factor XI and unlabeled factor XI in inhibiting [(125)I]factor XI binding to activated platelets. Alanine scanning mutagenic analysis of the recombinant Apple 3 domain of factor XI indicated that amino acids R(250), K(255), F(260), and Q(263) (but not K(252) or K(253)) are important for platelet binding. Thus, the binding energy mediating the interaction of factor XI with platelets is contained within the C-terminal amino acid sequence of the Apple 3 domain (T(249)-V(271)) and is mediated in part by amino acid residues R(250), K(255), F(260), and Q(263).     

A subpopulation of platelets responds to thrombin- or SFLLRN-stimulation with binding sites for factor IXa

Strong agonists cause platelets to expose a procoagulant surface supporting the assembly of two important coagulation enzyme complexes. Equilibrium binding has determined the density of high affinity saturable factor IXa binding sites to be 500-600 sites/platelet. We have now used flow cytometry to visualize the binding of factor IX and IXa to thrombin- or SFLLRN-activated platelets. Concentrations of these agonists that are half-maximal or maximal in kinetic studies resulted in only a small subpopulation (4-20%) of platelets binding factor IX or IXa with the density of binding sites for factor IX being about half of that for factor IXa, consistent with previous equilibrium binding studies. A small subpopulation (5 +/- 1.5%) of platelets stimulated with either agonist also exposed annexin V binding sites, and this subpopulation of platelets also bound factor IXa. Annexin V decreased factor IXa binding in the presence or absence of factor VIIIa, and factor IXa could also decrease annexin V binding on some platelets indicating a common binding site in agreement with previous studies. All platelets binding factor IXa were positive for glycoprotein IX, at the same glycoprotein IX surface density as seen in platelets negative for factor IXa binding. These studies refine the results from equilibrium binding studies and suggest that, on average, only a small subpopulation (approximately 10%) of PAR 1-stimulated platelets expose approximately 6000 factor IXa binding sites/platelet.     

Steroid sulfatase activity and expression in mammary myoepithelial cells

PURPOSE: This investigation examined mRNA expression and enzymatic activity of steroid sulfatase (STS) in human mammary myoepithelial cells (MMECs) and MCF-7 cells and assessed the effects of 17-beta estradiol on the activity of STS. METHODS: The mRNA level of STS in MMECs was determined by RT-PCR analysis using specific primers for STS. STS enzymatic activity prior to and after treatment with 17-beta estradiol was determined by measuring 3H-metabolites formed after exposure to [3H]estrone 3-sulfate (E1S) and [3H]dehydroepiandrosterone-sulfate (DHEA-S). RESULTS: Our data demonstrate the presence of STS in the MMECs. Based on RT-PCR analysis, MMECs had slightly lower levels of STS compared to MCF-7 cells. However, sulfatase activity was about 120 times greater in the MMECs than the MCF-7 cells (E1S V(max)=2640nmol/(mg DNAh) compared to 20.9nmol/(mg DNAh)). Exposure to 17-beta estradiol was associated with 70% reduction in E1S sulfatase activity in the MCF-7 cells and 9% increase in the MMECs after 6 days. DISCUSSION: Our studies indicate for the first time the presence of STS in MMECs. This is suggestive of a previously undetermined role for MMECs in converting precursor hormones into active steroid hormones within mammary tissue. In addition, differential response of the MMECs and the MCF-7 cells to estrogen demonstrates differences in hormone metabolism between these two cell types, perhaps related to the absence of estrogen receptors in the MMECs and their presence in the MCF-7 cells. The MMECs may have an important role in hormonal regulation within mammary tissue.     

New Arsenite-Oxidizing Bacteria Isolated from Australian Gold Mining Environments--Phylogenetic Relationships

Nine novel arsenite-oxidizing bacteria have been isolated from two different gold mine environments in Australia. Four of these organisms grow chemolithoautotrophically with oxygen as the terminal electron acceptor, arsenite as the electron donor, and carbon dioxide-bicarbonate as the sole carbon source. Five heterotrophic arsenite-oxidizing bacteria were also isolated, one of which was found to be both phylogenetically and physiologically identical to the previously described heterotrophic arsenite oxidizer misidentified as Alcaligenes faecalis . The results showed that this strain belongs to the genus Achromobacter . Phylogenetically, the arsenite-oxidizing bacteria fall within two separate subdivisions of the Proteobacteria . Interestingly, the chemolithoautotrophic arsenite oxidizers belong to the f - Proteobacteria , whereas the heterotrophic arsenite oxidizers belong to the g - Proteobacteria .     

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.     

Effects of Varied Housing Density on a Hybrid Mouse Strain Followed for 20 Months

To evaluate the effect of increased housing density in a hybrid mouse strain, we evaluated a panel of physiological and behavioral traits in animals that were housed in groups of 3, 5, 8, or 12, using cages that provide 78.1 in² of floor space. Such groupings resulted in cage densities that ranged from half to almost twice the density recommended by the Guide for the Care and Use of Laboratory Animals. While previous studies have investigated physiological effects of increased housing density using inbred mouse strains, including C57BL/6J and 129S1/SvImJ, this study tested an F1 hybrid population of C57BL/6J x 129S1/SvImJ for changes resulting from either decreased or increased housing density. Mice were followed until they were 20 months old, a substantially longer duration than has been used in previous density studies. We evaluated mortality, growth, home cage behavior, blood pressure, body composition, clinical plasma chemistries, immune function, and organ weights (heart, kidney, adrenal glands, and testes) as endpoints of chronic stress that may arise from sub-optimal housing conditions. Few statistically different parameters were observed in this study, none of which describe chronic stress and all within normal physiological ranges for research mice, suggesting that this hybrid strain was not adversely affected by housing at twice the density currently recommended.     

Efficient transient transformation of suspension culture-derived apple protoplasts

A protocol for comparatively efficient delivery of plasmid DNA into suspension culture-derived apple protoplasts is described. Increases in efficiency of transfection are achieved by elevating the temperature during protoplast exposure to the DNA. Two methods to detect transformed protoplasts are compared and the effectiveness and ease of application of flow cytometry techniques to this high throughput method is discussed.