Neurotoxic Organophosphate Degradation with Polyvinyl Alcohol Gel-Immobilized Microbial Cells

A genetically engineered strain of Escherichia coli that expresses organophosphorus hydrolase (OPH) was immobilized in a polyvinyl alcohol (PVA) cryogel to form a porous biocatalyst that successfully degrades organophosphorus (OP) neurotoxins. The impacts of both diffusion and reaction on biocatalyst efficiency were determined to enable prediction and optimization of the biocatalyst performance. The kinetic rate parameters and activation energies of pure OPH, free cell suspensions, and the immobilized cell biocatalyst were compared. Diffusion was a determining factor for paraoxon hydrolysis because of the very rapid OPH kinetics for its model substrate. Both the paraoxon diffusion through the PVA matrix and the diffusion associated with microbial transport of paraoxon were shown to impact the biocatalyst reaction. However, the enhancement in storage stability resulting from diffusional limitations provides an advantage to diffusion-limited operation. This research may serve as a guide to define the influence of diffusion in biological reaction systems. The broad substrate specificity and hydrolytic efficiency of OPH coupled with the ability to genetically engineer the enzyme for specific target OP neurotoxins enhance the suitability of OPH-based technologies for detoxification of these compounds. Cryoimmobilization provides a suitable vehicle as a cost-effective, efficient technology for bioremediation of environmental media contaminated with OP compounds.     

(1–3)-β-Glucan synthesis ofNeurospora crassa

(1–3)--d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.     

Design,synthesis and in vitro evaluation against human cancer cells of 5-methyl-5-styryl-2,5-dihydrofuran-2-ones,a new series of goniothalamin analogues

The present work describes the preparation of a novel series of compounds based on the structure of goniothalamin (1), a natural styryl lactone with known cytotoxic and antiproliferative activities against a variety of cancer cell lines. A focused library of 17 goniothalamin analogues displaying the 5-methyl-2,5-dihydrofuran-2-one motif were prepared, and their cytotoxicity evaluated. While the analogues bearing methoxy and/or hydroxy groups on the aromatic moiety usually were at least three times less potent than the lead compound (1), ortho and para-trifluoromethyl analogues 10 and 11 exhibited levels of cytotoxicity similar to goniothalamin (1) against most cancer cell lines evaluated. One could suggest that the electronic effect of the trifluoromethyl group activates the inhibitor’s electrophilic site via reduction of the electron density of the α,β-unsaturated ester oxygen atom. These results provide new information on the structure activity relationship of these α,β-unsaturated styryl lactones, thereby further focusing the design of novel candidates.     

Cadmium removal capacities of filamentous soil fungi isolated from industrially polluted sediments,in La Plata (Argentina)

Aspergillus terreus, Cladosporium cladosporioides, Fusarium oxysporum, Gliocladium roseum, Penicillium spp., Talaromyces helicus and Trichoderma koningii were isolated from heavily polluted streams near an industrial area in La Plata, Argentina. The fungi were obtained from sediments with 0.25–0.50 mg Cd/l and they were isolated in cadmium-basal medium. They were then cultivated to evaluate their Cd detoxification abilities. The biomass developed in static assays represented 5–53% of the yield of stirred cultures, for the different fungal species, although the cadmium absorption were similar in both cases. These soil fungi represented 50% of the total isolates and their mycelial growth was conspicuous in these polluted sediments. Although bacteria have been mentioned as active microorganisms against heavy metals, the mycelial fungi were able to develop a significantly higher mass to sequestrate more metals. Thus, they could be used in remediation biotechnology to improve the Cd detoxification of chronically contaminated habitats.     

Glutaredoxin 2 knockout increases sensitivity to oxidative stress in mouse lens epithelial cells

Glutaredoxin belongs to the oxidoreductase family, with cytosolic glutaredoxin 1 (Grx1) and mitochondrial glutaredoxin 2 (Grx2) isoforms. Of the two isozymes, the function of Grx2 is not well understood. This paper describes the effects of Grx2 deletion on cellular function using primary lens epithelial cell cultures isolated from Grx2 gene knockout (KO) and wild-type (WT) mice. We found that both cell types showed similar growth patterns and morphology and comparable mitochondrial glutathione pool and complex I activity. Cells with deleted Grx2 did not show affected Grx1 or thioredoxin expression but exhibited high sensitivity to oxidative stress. Under treatment with H(2)O(2), the KO cells showed less viability, higher membrane leakage, enhanced ATP loss and complex I inactivation, and weakened ability to detoxify H(2)O(2) in comparison with the WT cells. The KO cells had higher glutathionylation in the mitochondrial proteins, particularly the 75-kDa subunit of complex I. Recombinant Grx2 deglutathionylated complex I and restored most of its activity. We conclude that Grx2 has a function that protects cells against H(2)O(2)-induced injury via its peroxidase and dethiolase activities; particularly, Grx2 prevents complex I inactivation and preserves mitochondrial function.