Kinetic mechanism of the GCN5-related chromosomal aminoglycoside acetyltransferase AAC(6')-Ii from Enterococcus faecium: evidence of dimer subunit cooperativity

The aminoglycoside 6'-N-acetyltransferase AAC(6')-Ii from Enterococcus faecium is an important microbial resistance determinant and a member of the GCN5-related N-acetyltransferase (GNAT) superfamily. We report here the further characterization of this enzyme in terms of the kinetic mechanism of acetyl transfer and identification of rate-contributing step(s) in catalysis, as well as investigations into the binding of both acetyl-CoA and aminoglycoside substrates to the AAC(6')-Ii dimer. Product and dead-end inhibition studies revealed that AAC(6')-Ii follows an ordered bi-bi ternary complex mechanism with acetyl-CoA binding first followed by antibiotic. Solvent viscosity studies demonstrated that aminoglycoside binding and product release govern the rate of acetyl transfer, as evidenced by changes in both the k(cat)/K(b) for aminoglycoside and k(cat), respectively, with increasing solvent viscosity. Solvent isotope effects were consistent with our viscosity studies that diffusion-controlled processes and not the chemical step were rate-limiting in drug modification. The patterns of partial and mixed inhibition observed during our mechanistic studies were followed up by investigating the possibility of subunit cooperativity in the AAC(6')-Ii dimer. Through the use of AAC-Trp(164) --> Ala, an active mutant which exists as a monomer in solution, the partial nature of the competitive inhibition observed in wild-type dead-end inhibition studies was alleviated. Isothermal titration calorimetry studies also indicated two nonequivalent antibiotic binding sites for the AAC(6')-Ii dimer but only one binding site for the Trp(164) --> Ala mutant. Taken together, these results demonstrate subunit cooperativity in the AAC(6')-Ii dimer, with possible relevance to other oligomeric members of the GNAT superfamily.     

Functional diversity and community structure of microorganisms in rhizosphere and non-rhizosphere Canadian arctic soils

Functional diversities of microorganisms in arctic soil samples at three incubation temperatures were assessed using sole-carbon-source-utilization (SCSU). Soil samples from four sites were collected from the rhizosphere and non-rhizosphere soils. Microorganisms were extracted from samples and inoculated into ECO-Biolog plates and incubated at 4, 10 and 28 °C. Calculations of Shannon–Weaver diversity and Shannon–Weaver evenness were based on the substrate utilization in the Biolog plates. Shannon–Weaver diversities (H) in rhizosphere samples were significantly greater ( _H = 3.023 ± 0.197; P < 0.005) than in non-rhizosphere samples ( _H = 2.770 ± 0.154). Similarly, the evenness (E) of the inoculated microbial cells exhibited significant differences (P < 0.005) between the rhizosphere and non-rhizosphere soil samples ( _E = 0.880 ± 0.057 for soils with rhizosphere; _E = 0.807 ± 0.044 for non-rhizosphere samples). Higher microbial diversity and evenness were observed in samples incubated at 4 °C than at 28 °C [least significant difference (lsd) = 0.29], and evenness indices were higher in rhizosphere samples than in non-rhizosphere soils incubated at all three temperatures (lsd = 0.02). Principal component analysis (PCA) of the multivariate data set differentiated the soil samples on the relatively gross scale of microbial communities isolated from rhizosphere and non-rhizosphere soils at all three temperatures.     

Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form

Transforming growth factor-beta (TGF-beta) is a key modulator of epidermal development and homeostasis, and has been shown to potently regulate keratinocyte migration and function during wound repair. There are three cloned TGF-beta receptors termed type I, type II, and type III that are found on most cell types. The types I and II are the signaling receptors, while the type III is believed to facilitate TGF-beta binding to the types I and II receptors. Recently, we reported that in addition to these receptors, human keratinocytes express a 150 kDa TGF-beta 1 binding protein (r150) which forms a heteromeric complex with the TGF-beta signaling receptors. This accessory receptor was described as glycosyl phosphatidylinositol-specific anchored based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). In the present study, we demonstrate that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta 1 with an affinity similar to those of the types I and II TGF-beta signaling receptors. Furthermore, the PIPLC released (soluble) form of this protein is capable of binding TGF-beta 1 independently from the signaling receptors. In addition, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Our observation that r150 interacts with the TGF-beta signaling receptors, together with the finding that the soluble r150 binds TGF-beta 1 suggest that r150 in either its membrane anchored or soluble form may potentiate or antagonize TGF-beta signaling. Elucidating the mechanism by which r150 functions as an accessory molecule in TGF-beta signaling may be critical to understanding the molecular mechanisms underlying the regulation of TGF-beta action in keratinocytes.     

Effect of NOS inhibition on central response to atrial distension during pregnancy

Atrial distension increases c-fos expression in the paraventricular nucleus of virgin, but not pregnant, rats. We proposed that nitric oxide (NO), biosynthesis of which increases during pregnancy, blunts this reflex and that blocking NO biosynthesis would restore the response. Female rats were implanted with indwelling intracardiac balloons. On day 14 of pregnancy, osmotic minipumps containing either D- or N(G)-nitro-L-arginine methyl ester (L-NAME) (120 mg/2 ml at 10 microg/min) were implanted. On day 20, the rats were infused with saline (3 ml/h) with or without atrial balloon inflation (1 h). The brains were then processed for quantitation of c-fos expression. In the virgin rats, and in the pregnant rats treated with L-NAME, atrial distension significantly increased hypothalamic c-fos expression. In the pregnant animals treated with D-NAME, the response was greatly attenuated. NO had no effect on the increase in atrial receptor afferent discharge (single-fiber recordings) elicited by atrial distension. We conclude that, during pregnancy, NO attenuates central processing of the reflex response to atrial distension but does not alter the transducer properties of the volume receptors.     

Twist function is required for the morphogenesis of the cephalic neural tube and the differentiation of the cranial neural crest cells in the mouse embryo

Loss of Twist function in the cranial mesenchyme of the mouse embryo causes failure of closure of the cephalic neural tube and malformation of the branchial arches. In the Twist(-/-) embryo, the expression of molecular markers that signify dorsal forebrain tissues is either absent or reduced, but those associated with ventral tissues display expanded domains of expression. Dorsoventral organization of the mid- and hindbrain and the anterior-posterior pattern of the neural tube are not affected. In the Twist(-/-) embryo, neural crest cells stray from the subectodermal migratory path and the late-migrating subpopulation invades the cell-free zone separating streams of cells going to the first and second branchial arches. Cell transplantation studies reveal that Twist activity is required in the cranial mesenchyme for directing the migration of the neural crest cells, as well as in the neural crest cells within the first branchial arch to achieve correct localization. Twist is also required for the proper differentiation of the first arch tissues into bone, muscle, and teeth.     

Correlations of cationic charges with salt sensitivity and microbial specificity of cystine-stabilized beta -strand antimicrobial peptides

The electrostatic interaction of the charge cluster of an amphipathic peptide antibiotic with microbial membranes is a salt-sensitive step that often determines organism specificity. We have examined the correlation between charge clusters and salt insensitivity and microbial specificity in linear, cyclic, and retro-isomeric cystine-stabilized beta-strand (CSbeta) tachyplesin (TP) in a panel of 10 test organisms. Cyclic tachyplesins consisting of 14 and 18 amino acids are constrained by an end-to-end peptide backbone and two or three disulfide bonds to cross-brace the anti-parallel beta-strand that approximates a "beta-tile" structure. Circular dichroism measurements of beta-tile TPs showed that they displayed ordered structures. Control peptides containing the same number of basic amino acids as TP but lacking disulfide constraints were highly salt sensitive. Cyclic TP analogues with six cationic charges were more broadly active and salt-insensitive than those with fewer cationic charges. Reducing their proximity or number of cationic charges, particularly those with three or fewer basic amino acids, led to a significant decrease in potency and salt insensitivity, but an increased selectivity to certain Gram-positive bacteria. An end-group effect of the dibasic N-terminal Lys of TP in the open-chain TP and its retroisomer was observed in certain Gram-negative bacteria under high-salt conditions, an effect that was not found in the cyclic analogs. These results suggest that a stable folded structure together with three or more basic amino acids closely packed in a charged region in CSbeta peptides is important for salt insensitivity and organism specificity.