Assembly and disassembly kinetics of anthrax toxin complexes

Proteolytic activation of the protective antigen (PA) component of anthrax toxin allows it to self-associate into a ring-shaped homoheptamer, [PA(63)](7), which can bind the enzymatic components lethal factor (LF) and edema factor (EF). [PA(63)](7) is a pore-precursor (prepore), and under the low-pH conditions of the endosome, it forms a transmembrane pore that allows LF and EF to enter the cytosol. PA was labeled with donor and acceptor fluorescent dyes, and F?rster resonance energy transfer was used to measure the assembly and disassembly kinetics of the prepore complex in solution. The dissociation rate constant for [PA(63)](7) was 1 x 10(-)(6) s(-)(1) (t(1/2) approximately 7 days). In contrast, a ternary complex containing the PA-binding domain of LF (LF(N)) bound to a PA(63) dimer composed of two nonoligomerizing mutants dissociated rapidly (t(1/2) approximately 1 min). Thus, the substantial decrease in the rate of disassembly of [PA(63)](7) relative to the ternary complex is due to the cooperative interactions among neighboring subunits in the heptameric ring. Low concentrations of LF(N) promoted assembly of the prepore from proteolytically activated PA, whereas high concentrations inhibited assembly of both the prepore and the ternary complex. A self-assembly scheme of anthrax toxin complexes is proposed.     

Identification of structural and catalytic classes of highly conserved amino acid residues in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4 catalyzes the interconversion of (S)-lysine and (S)-beta-lysine by a radical mechanism involving coenzymatic actions of S-adenosylmethionine (SAM), a [4Fe-4S] cluster, and pyridoxal 5'-phosphate (PLP). The enzyme contains a number of conserved acidic residues and a cysteine- and arginine-rich motif, which binds iron and sulfide in the [4Fe-4S] cluster. The results of activity and iron, sulfide, and PLP analysis of variants resulting from site-specific mutations of the conserved acidic residues and the arginine residues in the iron-sulfide binding motif indicate two classes of conserved residues of each type. Mutation of the conserved residues Arg134, Asp293, and Asp330 abolishes all enzymatic activity. On the basis of the X-ray crystal structure, these residues bind the epsilon-aminium and alpha-carboxylate groups of (S)-lysine. However, among these residues, only Asp293 appears to be important for stabilizing the [4Fe-4S] cluster. Members of a second group of conserved residues appear to stabilize the structure of LAM. Mutations of arginine 130, 135, and 136 and acidic residues Glu86, Asp165, Glu236, and Asp172 dramatically decrease iron and sulfide contents in the purified variants. Mutation of Asp96 significantly decreases iron and sulfide content. Arg130 or Asp172 variants display no detectable activity, whereas variants mutated at the other positions display low to very low activities. Structural roles are assigned to this latter class of conserved amino acids. In particular, a network of hydrogen bonded interactions of Arg130, Glu86, Arg135, and the main chain carbonyl groups of Cys132 and Leu55 appears to stabilize the [4Fe-4S] cluster.     

Genetic overexpression of eNOS attenuates hepatic ischemia-reperfusion injury

Previous studies have shown that endothelial nitric oxide (NO) synthase (eNOS)-derived NO is an important signaling molecule in ischemia-reperfusion (I-R) injury. Deficiency of eNOS-derived NO has been shown to exacerbate injury in hepatic and myocardial models of I-R. We hypothesized that transgenic overexpression of eNOS (eNOS-TG) would reduce hepatic I-R injury. We subjected two strains of eNOS-TG mice to 45 min of hepatic ischemia and 5 h of reperfusion. Both strains were protected from hepatic I-R injury compared with wild-type littermates. Because the mechanism for this protection is still unclear, additional studies were performed by using inhibitors and activators of both soluble guanylyl cyclase (sGC) and heme oxygenase-1 (HO-1) enzymes. Blocking sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and HO-1 with zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG) in wild-type mice increased hepatic I-R injury, whereas pharmacologically activating these enzymes significantly attenuated I-R injury in wild-type mice. Interestingly, ODQ abolished the protective effects of eNOS overexpression, whereas ZnDPBG had no effect. These results suggest that hepatic protection in eNOS-TG mice may be mediated in part by NO signaling via the sGC-cGMP pathway and is independent of HO-1 signal transduction pathways.     

Distinct roles for spontaneous and visual activity in remodeling of the retinogeniculate synapse

Sensory experience and spontaneous activity play important roles in development of sensory circuits; however, their relative contributions are unclear. Here, we test the role of different forms of activity on remodeling of the mouse retinogeniculate synapse. We found that the bulk of maturation occurs without patterned sensory activity over 4 days spanning eye opening. During this early developmental period, blockade of spontaneous retinal activity by tetrodotoxin, but not visual deprivation, retarded synaptic strengthening and inhibited pruning of excess retinal afferents. Later in development, synaptic remodeling becomes sensitive to changes in visually evoked activity, but only if there has been previous visual experience. Synaptic strengthening and pruning were disrupted by visual deprivation following 1 week of vision, but not by chronic deprivation from birth. Thus, spontaneous activity is necessary to drive the bulk of synaptic refinement around the time of eye opening, while sensory experience is important for the subsequent maintenance of connections.     

The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans enlarges by distal growth and is rearranged during budding

The capsule of Cryptococcus neoformans can undergo dramatic enlargement, a phenomenon associated with virulence. A prior study that used Ab to the capsule as a marker for older capsular material concluded that capsule growth involved the intermixing of new and old capsular material with displacement of older capsular polysaccharide towards the surface. Here we have revisited that question using complement (C), which binds to capsular polysaccharide covalently, and cannot redistribute by dissociation and binding at different sites. The experimental approach involved binding of C to cells with small capsules, inducing capsule growth, and following the location of C relative to the cell wall as the capsule enlarged. C remained close to the cell wall during capsule growth, indicating that capsule enlargement occurred by addition of new polysaccharide near the capsule edge. This conclusion was confirmed by an independent method that employed radioactive metabolic labelling of newly synthesized capsule with 3H-mannose followed by gradual capsular stripping with gamma-radiation. Capsule growth proceeded to a certain size, which was a function of cell size, and was not degraded when the cells were transferred to a non-inducing medium. During budding, an opening appeared in the capsule of the mother cell that permitted the nascent bud to separate. Scanning EM suggested that a physical separation formed between the capsules of the mother and daughter cells during budding, which may avoid mixture between both capsules. Our results indicate that C. neoformans capsular enlargement also occurs by apical growth and that budding results in capsular rearrangements.     

Histone modifications: signalling receptors and potential elements of a heritable epigenetic code

The genetic code epitomises simplicity, near universality and absolute predictive power. By contrast, epigenetic information, in the form of histone modifications, is characterised by complexity, diversity and an overall tendency to respond to changes in genomic function rather than to predict them. Perhaps the transient changes in histone modifications involved in intranuclear signalling and ongoing chromatin functions mask stable, predictive modifications that lie beneath. The current rapid progress in unravelling the diversity and complexity of epigenetic information might eventually reveal an underlying histone or epigenetic code. But whether it does or not, it will certainly provide unprecedented opportunities, both for understanding how the genome responds to environmental and metabolic change and for manipulating its activities for experimental and therapeutic benefit.     

Discovery of potent and selective inhibitors of 11beta-HSD1 for the treatment of metabolic syndrome

High throughput screening efforts have identified a novel class of dichloroaniline amide 11β-HSD1 inhibitors. SAR studies initiated from dichloroaniline 4 focused on retaining the potency and selectivity profile of the lead.     

Binding of methoxy-substituted N1-benzenesulfonylindole analogs at human 5-HT6 serotonin receptors

Comparison of several amine-substituted and methoxy-substituted analogs of N1-(4-aminobenzene)sulfonylindole suggests that these substituents might contribute to the 5-HT6 serotonin receptor affinity of these agents via their electronic effect on the indolic nucleus. Their 1,2,3,4-tetrahydrocarbazole counterparts behave differently.     

Frequent fire promotes diversity and cover of biological soil crusts in a derived temperate grassland

The intermediate disturbance hypothesis (IDH) predicts that species diversity is maximized at moderate disturbance levels. This model is often applied to grassy ecosystems, where disturbance can be important for maintaining vascular plant composition and diversity. However, effects of disturbance type and frequency on cover and diversity of non-vascular plants comprising biological soil crusts are poorly known, despite their potentially important role in ecosystem function. We established replicated disturbance regimes of different type (fire vs. mowing) and frequency (2, 4, 8 yearly and unburnt) in a high-quality, representative Themeda australis–Poa sieberiana derived grassland in south-eastern Australia. Effects on soil crust bryophytes and lichens (hereafter cryptogams) were measured after 12 years. Consistent with expectations under IDH, cryptogam richness and abundance declined under no disturbance, likely due to competitive exclusion by vascular plants as well as high soil turnover by soil invertebrates beneath thick grass. Disturbance type was also significant, with burning enhancing richness and abundance more than mowing. Contrary to expectations, however, cryptogam richness increased most dramatically under our most frequent and recent (2 year) burning regime, even when changes in abundance were accounted for by rarefaction analysis. Thus, from the perspective of cryptogams, 2-year burning was not an adequately severe disturbance regime to reduce diversity, highlighting the difficulty associated with expression of disturbance gradients in the application of IDH. Indeed, significant correlations with grassland structure suggest that cryptogam abundance and diversity in this relatively mesic (600 mm annual rainfall) grassland is maximised by frequent fires that reduce vegetation and litter cover, providing light, open areas and stable soil surfaces for colonisation. This contrasts with detrimental effects of 2-year burning on native perennial grasses, indicating that this proliferation of cryptogams has potentially high functional significance for situations where vegetation cover is depleted, particularly for reducing soil erosion.