Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane

Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.     

Selective recognition of acetate ion based on fluorescence enhancement chemosensor

Fluorescence study of the complexation between uranyl salophen (L) and some common anions in acetonitrile–water (90:10, v/v) solution showed a tendency of L toward acetate ion (AcO^?). The fluorescence enhancement of L is attributed to a 1:1 complex formation between L and acetate ion which was utilized as the basis for the selective detection of AcO^?. The association constant of the 1:1 complex formation of L–AcO^? was calculated as 6.60 × 10⁶. The linear response range of the fluorescent chemosensor covers a AcO^? concentration range of 1.6 × 10^?7 to 2.5 × 10^?5 mol/L, with a detection limit of 2.5 × 10^?8 mol/L. L showed a selective and sensitive fluorescence enhancement response toward acetate ion over I₃^?, NO₃^?, CN^?, CO₃^2?, Br^?, Cl^?, F^?, H₂PO4^? and SO₄^2?, which was attributed to the higher stability of inorganic complex between acetate and L. Copyright © 2012 John Wiley & Sons, Ltd.     

Proline dehydrogenase from Pseudomonas fluorescence: gene cloning, purification, characterization and homology modeling

The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescence was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K(m) and V(max) values of the P. fluorescence ProDH were estimated to be 35 mM and 116 micromol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30 degrees C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescence ProDH.     

Effect of salt stress on some physio-biochemical traits and antioxidative enzymes of two Brassica species under callus culture

The changes in lipid peroxidation, H₂O₂, proline, protein, involvement of different antioxidant systems (catalase, guaiacol peroxidase, ascorbate peroxidase) and callus-related traits were investigated under salt stress in the callus of two different ploidy levels of Brassica including B. juncea and B. oleracea. The calluses of B. juncea genotypes were less sensitive to NaCl stress in comparison with those of B. oleracea while increasing the concentrations of NaCl from 0 to 200 mM. Tetraploid genotype (B. juncea cr3356) showed a significant increase in the contents of protein and proline, and guaiacol peroxidase activity and catalase enzymes at higher salinity levels. In addition, a significant decrease occurred in the amount of H₂O₂ and malondialdehyde along with increasing the salinity intensity. Diploid cultivar (B. oleracea bra 2828) had the lowest enzymatic activities and the highest content of H₂O₂ and malondialdehyde along with an increase in the salinity level. Therefore, this genotype was identified as the most sensitive cultivar under the salinity stress. The salinity resistance difference between diploid and amphidiploid species could be attributed to the differences in the ploidy level of these species. This result underlines the fact that the tetraploid genome of B. oleracea could be considered as a suitable candidate for production under salinity conditions through maintaining higher activities of antioxidant enzymes.

     

Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

Voltage-gated sodium channels (Na_v) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Na_v α subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Na_vs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Na_vs.     

Population Growth of Six Iranian Brachionus Rotifer Strains in Response to Salinity and Food Type

Intrinsic rates of population increase (r) were evaluated as a measure of population dynamics of four strains of Brachionus plicatilis and two strains of B. urceolaris from Iran in response to different salinities and feeding algae. Each rotifer strain was cultured at four salinities: 5, 20, 25 and 30‰ and fed with two microalgal species: Chlorella vulgaris and Nannochloropsis oculata. Salinity of 5‰ was critical for all the examined strains, at which r was at minimum and was different from the other salinities (P < 0.05). For B. plicatilis strains, the maximum r was observed in those fed on Chlorella at salinities of 10 and 30‰ (64 ± 0.01 day⁻¹). While, in B. urceolaris, maximum r was for Nannochloropsis fed rotifers at salinity of 20‰ (0.69 ± 0.01 day⁻¹). Maximum final population density (FD) was obtained for a strain of B. urceolaris fed on Nannochloropsis at 20‰ (329.3 ± 10.9 ind.mL⁻¹). FD was relatively lower in B. plicatilis strains among all examined salinities. ANOVA showed the significant effect of salinity and rotifer strain, and algae × rotifer strain on both r and FD, and salinity × rotifer × algae on FD (P < 0.05). (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Extended Latanoprost Release from Commercial Contact Lenses: In Vitro Studies Using Corneal Models

In this study, we compared, for the first time, the release of a 432 kDa prostaglandin analogue drug, Latanoprost, from commercially available contact lenses using in vitro models with corneal epithelial cells. Conventional polyHEMA-based and silicone hydrogel soft contact lenses were soaked in drug solution ( solution in phosphate buffered saline). The drug release from the contact lens material and its diffusion through three in vitro models was studied. The three in vitro models consisted of a polyethylene terephthalate (PET) membrane without corneal epithelial cells, a PET membrane with a monolayer of human corneal epithelial cells (HCEC), and a PET membrane with stratified HCEC. In the cell-based in vitro corneal epithelium models, a zero order release was obtained with the silicone hydrogel materials (linear for the duration of the experiment) whereby, after 48 hours, between 4 to 6 of latanoprost (an amount well within the range of the prescribed daily dose for glaucoma patients) was released. In the absence of cells, a significantly lower amount of drug, between 0.3 to 0.5 , was released, (). The difference observed in release from the hydrogel lens materials in the presence and absence of cells emphasizes the importance of using an in vitro corneal model that is more representative of the physiological conditions in the eye to more adequately characterize ophthalmic drug delivery materials. Our results demonstrate how in vitro models with corneal epithelial cells may allow better prediction of in vivo release. It also highlights the potential of drug-soaked silicone hydrogel contact lens materials for drug delivery purposes.