NMR spectroscopic characterization of a beta-(1,4)-glycosidase along its reaction pathway: stabilization upon formation of the glycosyl-enzyme intermediate

NMR spectroscopy was used to search for mechanistically significant differences between the thermodynamic and dynamic properties of the 34 kDa (alpha/beta)8-barrel catalytic domain of beta-(1,4)-glycosidase Cex (or CfXyn10A) in its free (apo-CexCD) and trapped glycosyl-enzyme intermediate (2FCb-CexCD) states. The main chain chemical shift perturbations due to the covalent modification of CexCD with the mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside are limited to residues within its active site. Thus, consistent with previous crystallographic studies, formation of the glycosyl-enzyme intermediate leads to only localized structural changes. Furthermore, 15N relaxation methods demonstrated that the backbone amide and tryptophan side chains of apo-CexCD are very well ordered on both the nanosecond to picosecond and millisecond to microsecond time scales and that these dynamic features also do not change significantly upon formation of the trapped intermediate. However, covalent modification of CexCD led to the increased protection of many amides and indoles, clustered around the active site of the enzyme, against fluctuations leading to hydrogen exchange. Similarly, thermal denaturation studies demonstrated that 2FCb-CexCD has a significantly higher midpoint unfolding temperature than apo-CexCD. The covalently modified protein also exhibited markedly increased resistance to proteolytic degradation by thermolysin relative to apo-CexCD. Thus, the local and global stability of CexCD increase along its reaction pathway upon formation of the glycosyl-enzyme intermediate, while its structure and fast time scale dynamics remain relatively unperturbed. This may reflect thermodynamically favorable interactions with the relatively rigid active site of Cex necessary to bind, distort, and subsequently hydrolyze glycoside substrates.     

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.     

Effect of pedal cadence on the accumulated oxygen deficit, maximal aerobic power and blood lactate transition thresholds of high-performance junior endurance cyclists

In this study we investigated the effect of pedal cadence on the cycling economy, accumulated oxygen deficit (AOD), maximal oxygen consumption (VO2max) and blood lactate transition thresholds of ten high-performance junior endurance cyclists [mean (SD): 17.4 (0.4) years; 183.8 (3.5) cm, 71.56 (3.75) kg]. Cycling economy was measured on three ergometers with the specific cadence requirements of: 90-100 rpm for the road dual chain ring (RDCR90-100 rpm) ergometer, 120-130 rpm for the track dual chain ring (TDCR120-130 rpm) ergometer, and 90-130 rpm for the track single chain ring (TSCR90-130 rpm) ergometer. AODs were then estimated using the regression of oxygen consumption (VO2) on power output for each of these ergometers, in conjunction with the data from a 2-min supramaximal paced effort on the TSCR90-130 rpm ergometer. A regression of VO2 on power output for each ergometer resulted in significant differences (P<0.001) between the slopes and intercepts that produced a lower AOD for the RDCR90-100 rpm [2.79 (0.43) l] compared with those for the TDCR120-130 rpm [4.11 (0.78) l] and TSCR90-130 rpm [4.06 (0.84) l]. While there were no statistically significant VO2max differences (P = 0.153) between the three treatments [RDCR90-100 rpm: 5.31 (0.24) l x min(-1); TDCR120-130 rpm; 5.33 (0.25) 1 x min(-1); TSCR90-130 rpm: 5.44 (0.27) l x min(-1)], all pairwise comparisons of the power output at which VO2max occurred were significantly different (P<0.001). Statistically significant differences were identified between the RDCR90-100 rpm and TDCR120-130 rpm tests for power output (P = 0.003) and blood lactate (P = 0.003) at the lactate threshold (Thla-), and for power output (P = 0.005) at the individual anaerobic threshold (Thiat). Our findings emphasise that pedal cadence specificity is essential when assessing the cycling economy, AOD and blood lactate transition thresholds of high-performance junior endurance cyclists.     

A new type of pharmacological chaperone for GM1-gangliosidosis related human lysosomal β-galactosidase: N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols

N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of β-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for G_M1-gangliosidosis-associated lysosomal acid β-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.     

Assessing biodiversity with species accumulation curves; inventories of small reptiles by pit‐trapping in Western Australia

Abstract We examined 11 non‐linear regression models to determine which of them best fitted curvilinear species accumulation curves based on pit‐trapping data for reptiles in a range of heterogeneous and homogenous sites in mesic, semi‐arid and arid regions of Western Australia. A well‐defined plateau in a species accumulation curve is required for any of the models accurately to estimate species richness. Two different measures of effort (pit‐trapping days and number of individuals caught) were used to determine if the measure of effort influenced the choice of the best model(s). We used species accumulation curves to predict species richness, determined the trapping effort required to catch a nominated percentage (e.g. 95%) of the predicted number of species in an area, and examined the relationship between species accumulation curves with diversity and rarity. Species richness, diversity and the proportion of rare species in a community influenced the shape of species accumulation curves. The Beta‐P model provided the best overall fit (highest r²) for heterogeneous and homogeneous sites. For heterogeneous sites, Hill, Rational, Clench, Exponential and Weibull models were the next best. For homogeneous habitats, Hill, Weibull and Chapman–Richards were the next best models. There was very little difference between Beta‐P and Hill models in fitting the data to accumulation curves, although the Hill model generally over‐estimated species richness. Most models worked equally well for both measures of trapping effort. Because the number of individuals caught was influenced by both pit‐trapping effort and the abundance of individuals, both measures of effort must be considered if species accumulation curves are to be used as a planning tool. Trapping effort to catch a nominated percentage of the total predicted species in homogeneous and heterogeneous habitats varied among sites, but even for only 75% of the predicted number of species it was generally much higher than the typical effort currently being used for terrestrial vertebrate fauna surveys in Australia. It was not possible to provide a general indication of the effort required to predict species richness for a site, or to capture a nominated proportion of species at a site, because species accumulation curves are heavily influenced by the characteristics of particular sites.     

Fluorescent glycosidase inhibiting 1,5-dideoxy-1,5-iminoalditols

1,5-Dideoxy-1,5-iminoalditols of various configurations as well as isofagomine were N-alkylated with non-polar straight chain spacer-arms by a set of simple standard procedures. The spacer-arms' terminal functional groups, primary amines, were employed to introduce fluorescent tags such as dansyl and dapoxyl moieties. Resulting derivatives in the D-xylo, D-gluco, D-galacto as well as GlcNAc series showed distinctly improved glycosidase inhibitory activities compared to parent compounds and are designed to be useful analytical tools.     

Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection

Using a novel cDNA microarray prepared from sources of actively responding immune system cells, we have investigated the changes in gene expression in the target tissue during the early stages of infection of neonatal chickens with infectious bursal disease virus. Infections of two lines of chickens previously documented as genetically resistant and sensitive to infection were compared in order to ascertain early differences in the response to infection that might provide clues to the mechanism of differential genetic resistance. In addition to major changes that could be explained by previously described changes in infected tissue, some differences in gene expression on infection, and differences between the two chicken lines, were observed that led to a model for resistance in which a more rapid inflammatory response and more-extensive p53-related induction of apoptosis in the target B cells might limit viral replication and consequent pathology. Ironically, the effect in the asymptomatic neonatal infection is that more-severe B-cell depletion is seen in the more genetically resistant chicken. Changes of expression of many chicken genes of unknown function, indicating possible roles in the response to infection, may aid in the functional annotation of these genes.