Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.     

Non-significance of rhizosphere degradation during phytoremediation of MTBE

Methyl tertiary butyl ether (MTBE) is a gasoline additive associated with groundwater pollution at gas station sites. Previous research on poplar trees in hydroponic systems suggests that phytovolatilization is an effective mechanism for phytoremediation of MTBE (Rubin and Ramaswami, 2001), but the potential for microbial degradation of MTBE in the rhizosphere of trees had not been assessed. MTBE had largely been considered recalcitrant to microbial processes, but recent fieldwork suggests rapid biodegradation may occur in certain cases. This paper investigates the potential for rhizosphere degradation of MTBE at time frames relevant for phytoremediation. Three experiments were conducted at different levels of aggregation to examine possible degradation of MTBE by rhizosphere microorganisms that had been acclimated to low levels of MTBE for 6 weeks. MTBE soil die-away studies, conducted with both poplar trees and fescue grass, found no significant differences between MTBE concentration in vegetated and unvegetated soils over a two-week attenuation period. Closed chamber tests comparing hydroponic and rhizospheric poplar tree systems also showed essentially complete recovery of MTBE mass in both systems, suggesting an absence of degradation. Finally, rhizosphere microbes tested in aerated bioreactors were found to be thriving and metabolizing root materials, but did not show measurable degradation of MTBE. In all tests, the MTBE degradation product, Tert Butyl Alcohol (TBA), was not detected. The insignificance of MTBE degradation by rhizosphere microorganisms suggests that plant processes be the primary focus of further research on MTBE phytoremediation.     

Identification and characterization of a unique cysteine residue proximal to the catalytic site of Arabidopsis thaliana carotenoid cleavage enzyme 1

AtCCD1 and AtNCED3 are related carotenoid cleavage enzymes from Arabidopsis thaliana that catalyze the oxidative cleavage of, respectively, the 9,10 (9',10') double bonds of carotenoid substrates such as beta-carotene, and the 11,12 double bond of 9-cis epoxycarotenoids. Although the cellular and cleavage functionalities of these enzymes have been reported, their mechanisms and related structural environments mediating these disparate specificities in homologous enzymes have not been well characterized. By relating the differences observed in UV and visible light absorption and Cu(II) electron paramagnetic signals to variations in sequence alignments and 3-D homology models of the two A. thaliana enzymes, we identified a putatively proximal cysteine residue (Cys352) in AtCCD1 that is not conserved in AtNCED3. Spectral analysis of the Cys to Ala mutant confirmed its uniqueness and proximity to the metal binding site, but precluded any role for the residue in the mediation of the observed metal binding affinity or associated steric constraint differences. Further analysis of kinetic substrate cleavage properties indicated a decrease in Vmax and a subtle increase in Km for the C352A mutant compared with those observed for the wild-type, thus confirming catalytic site proximity and suggesting possible roles for the unique cysteine in the modulation of substrate affinity and (or) the reaction rate of AtCCD1.     

Characterization of Early Disease Status in Treatment-Naive Male Paediatric Patients with Fabry Disease Enrolled in a Randomized Clinical Trial

Trial Design

This analysis characterizes the degree of early organ involvement in a cohort of oligo-symptomatic untreated young patients with Fabry disease enrolled in an ongoing randomized, open-label, parallel-group, phase 3B clinical trial.

Methods

Males aged 5–18 years with complete α-galactosidase A deficiency, without symptoms of major organ damage, were enrolled in a phase 3B trial evaluating two doses of agalsidase beta. Baseline disease characteristics of 31 eligible patients (median age 12 years) were studied, including cellular globotriaosylceramide (GL-3) accumulation in skin (n = 31) and kidney biopsy (n = 6; median age 15 years; range 13–17 years), renal function, and glycolipid levels (plasma, urine).

Results

Plasma and urinary GL-3 levels were abnormal in 25 of 30 and 31 of 31 patients, respectively. Plasma lyso-GL-3 was elevated in all patients. GL-3 accumulation was documented in superficial skin capillary endothelial cells (23/31 patients) and deep vessel endothelial cells (23/29 patients). The mean glomerular filtration rate (GFR), measured by plasma disappearance of iohexol, was 118.1 mL/min/1.73 m² (range 90.4–161.0 mL/min/1.73 m²) and the median urinary albumin/creatinine ratio was 10 mg/g (range 4.0–27.0 mg/g). On electron microscopy, renal biopsy revealed GL-3 accumulation in all glomerular cell types (podocytes and parietal, endothelial, and mesangial cells), as well as in peritubular capillary and non-capillary endothelial, interstitial, vascular smooth muscle, and distal tubules/collecting duct cells. Lesions indicative of early Fabry arteriopathy and segmental effacement of podocyte foot processes were found in all 6 patients.

Conclusions

These data reveal that in this small cohort of children with Fabry disease, histological evidence of GL-3 accumulation, and cellular and vascular injury are present in renal tissues at very early stages of the disease, and are noted before onset of microalbuminuria and development of clinically significant renal events (e.g. reduced GFR). These data give additional support to the consideration of early initiation of enzyme replacement therapy, potentially improving long-term outcome.

Trial Registration

ClinicalTrials.gov NCT00701415     

Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant loss-of-function mutations in SERCA indicates novel intra- and intermolecular interactions for SERCA in vivo, overlooked by current structural models.     

The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies

Recent results suggest that cytoplasmic mRNAs can form translationally repressed messenger ribonucleoprotein particles (mRNPs) capable of decapping and degradation, or accumulation into cytoplasmic processing bodies (P-bodies), which can function as sites of mRNA storage. The proteins that function in transitions between the translationally repressed mRNPs that accumulate in P-bodies and mRNPs engaged in translation are largely unknown. Herein, we demonstrate that the yeast translation initiation factor Ded1p can localize to P-bodies. Moreover, depletion of Ded1p leads to defects in P-body formation. Overexpression of Ded1p results in increased size and number of P-bodies and inhibition of growth in a manner partially suppressed by loss of Pat1p, Dhh1p, or Lsm1p. Mutations that inactivate the ATPase activity of Ded1p increase the overexpression growth inhibition of Ded1p and prevent Ded1p from localizing in P-bodies. Combined with earlier work showing Ded1p can have a positive effect on translation, these results suggest that Ded1p is a bifunctional protein that can affect both translation initiation and P-body formation.     

Plant-disperser mutualisms in a semi-arid habitat invaded by <Emphasis Type="Italic">Lantana camara</Emphasis> L.

Dispersal is an important ecological process that affects plant population structure and community composition. Invasive plants with fleshy fruits rapidly form associations with native and invasive dispersers, and may affect existing native plant-disperser associations. We asked whether frugivore visitation rate and fruit removal was associated with plant characteristics in a community of fleshy-fruited plants and whether an invasive plant receives more visitation and greater fruit removal than native plants in a semi-arid habitat of Andhra Pradesh, India. Tree-watches were undertaken at individuals of nine native and one invasive shrub species to assess the identity, number and fruit removal by avian frugivores. Network analyses and generalised linear mixed-effects models were used to understand species and community-level patterns. All plants received most number of visits from abundant, generalist avian frugivores. Number of frugivore visits and time spent by frugivores at individual plants was positively associated with fruit crop size, while fruit removal was positively associated with number of frugivore visits and their mean foraging time at individual plants. The invasive shrub, Lantana camara L. (Lantana), had lower average frugivore visit rate than the community of fleshy-fruited plants and received similar average frugivore visits but greater average per-hour fruit removal than two other concurrently fruiting native species. Based on the results of our study, we infer that there is little evidence of competition between native plants and Lantana for the dispersal services of native frugivores and that more data are required to assess the nature of these interactions over the long term. We speculate that plant associations with generalist frugivores may increase the functional redundancy of this frugivory network, buffering it against loss of participating species.