BcrC from Bacillus subtilis acts as an undecaprenyl pyrophosphate phosphatase in bacitracin resistance

Overexpression of the BcrC(Bs) protein, formerly called YwoA, in Escherichia coli or in Bacillus subtilis allows these bacteria to stand higher concentrations of bacitracin. It was suggested that BcrC(Bs) was a membrane-spanning domain of an ATP binding cassette (ABC) transporter involved in bacitracin resistance. However, we hypothesized that this protein has an undecaprenyl pyrophosphate (UPP) phosphatase activity able to compete with bacitracin for UPP. We found that overexpression of a recombinant His6-BcrC(Bs) protein in E. coli (i) increased the resistance of the cells to bacitracin and (ii) increased UPP phosphatase activity in membrane preparations by 600-fold. We solubilized and prepared an electrophoretically pure protein exhibiting a strong UPP phosphatase activity. BcrC(Bs), which belongs to the type 2 phosphatidic acid phosphatase (PAP2) phosphatase superfamily (PF01569), differs totally from the already known BacA UPP phosphatase from E. coli, a member of the PF02673 family of the Protein family (Pfam) database. Thus, BcrC(Bs) and its orthologs form a new class of proteins within the PAP2 phosphatase superfamily, and likely all of them share a UPP phosphatase activity.     

Practical and Reliable Synthesis of 1,2-Dideoxy-d-ribofuranose and its Application in RNAi Studies

We developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-d-ribofuranose (dR^H) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dR^H by the standard phosphoramidite solid-phase method, dR^H was converted to the corresponding phosphoramidite derivative and linked to a solid support (controlled pore glass resin). Chemically modified small interfering RNAs (siRNAs) possessing dR^H at their 3′-overhang regions were synthesized. Introducing dR^H to the 3′-end of the antisense strand of siRNA reduced its knockdown effect.     

Photoinduced pH and membrane potential changes in the algaPithophora pragensis

Biologia Plantarum - Glass microelectrodes filled with antimony were constructed, the electrical potential of which linearly depends on pH and is not influenced by light. Recording at the surface...     

A Single Mutation in Framework 2 of the Heavy Variable Domain Improves the Properties of a Diabody and a Related Single-Chain Antibody

Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single‐chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy‐chain variable domain. Diabody D4 displayed an increase in T_m (thermal transition midpoint temperature) of 6.3 °C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem. 286, 6143–6151]. The new variant, scFv LER, displayed an increase in T_m of 3.4 °C and was capable of neutralizing 2 LD₅₀ of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.     

Inhibition of mitochondrial respiration mediates apoptosis induced by the anti-tumoral alkaloid lamellarin D

Lamellarin D (Lam D), a marine alkaloid, exhibits a potent cytotoxicity against many different tumors. The pro-apoptotic function of Lam D has been attributed to its direct induction of mitochondrial permeability transition (MPT). This study was undertaken to explore the mechanisms through which Lam D promotes changes in mitochondrial function and as a result apoptosis. The use of eight Lam derivatives provides useful structure-apoptosis relationships. We demonstrate that Lam D and structural analogues induce apoptosis of cancer cells by acting directly on mitochondria inducing reduction of mitochondrial membrane potential, swelling and cytochrome c release. Cyclosporin A, a well-known inhibitor of MPT, completely prevents mitochondrial signs of apoptosis. The drug decreases calcium uptake by mitochondria but not by microsomes indicating that Lam D-dependent permeability is specific to mitochondrial membranes. In addition, upon Lam D exposure, a rapid decline of mitochondrial respiration and ATP synthesis occurs in isolated mitochondria as well as in intact cells. Evaluation of the site of action of Lam D on the electron-transport chain revealed that the activity of respiratory chain complex III is reduced by a half. To determine whether Lam D could induce MPT-dependent apoptosis by inhibiting mitochondrial respiration, we generated respiration-deficient cells (ρ0) derived from human melanoma cells. In comparison to parental cells, ρ0 cells are totally resistant to the induction of MPT-dependent apoptosis by Lam D. Our results indicate that functional mitochondria are required for Lam D-induced apoptosis. Inhibition of mitochondrial respiration is responsible for MPT-dependent apoptosis of cancer cells induced by Lam-D.     

Characterization and visualization of cholecystokinin receptors in rat brain using [3H]pentagastrin

[³H]Pentagastrin binds specifically to an apparent single class of CCK receptors on slide-mounted sections of rat brain (K_D=5.6 nM; B_max=36.6 fmol/mg protein). This specific binding is temperature-dependant and regulated by ions and nucleotides. The relative potencies of C-terminal fragments of CCK-8(SO₃H), benzotript and proglumide in inhibiting specific [³H]pentagastrin binding to CCK brain receptors reinforce the concept of different brain and pancreas CCK receptors. CCK receptors were visualized by using tritium-sensitive LKB film analyzed by computerized densitometry. CCK receptors are highly concentrated in the cortex, dentate gyrus, granular and external plexiform layers of the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, claustrum, accumbens nucleus, some nuclei of the amygdala, thalamus and hypothalamus.     

The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression

The formation of continental Europe in the Neogene was due to the regression of the Tethys Ocean and of the Paratethys Sea. The dynamic geology of the area and repetitious transitions between marine and freshwater conditions presented opportunities for the colonization of newly emerging hydrological networks and diversification of aquatic biota. Implementing mitochondrial and nuclear markers in conjunction with a large‐scale sampling strategy, we investigated the impact of this spatiotemporal framework on the evolutionary history of a freshwater crustacean morphospecies. The Gammarus balcanicus species complex is widely distributed in the area previously occupied by the Paratethys Sea. Our results revealed its high diversification and polyphyly in relation to a number of other morphospecies. The distribution of the studied amphipod is generally characterized by very high local endemism and divergence. The Bayesian time‐calibrated reconstruction of phylogeny and geographical distribution of ancestral nodes indicates that this species complex started to diversify in the Early Miocene in the central Balkans, partially in the shallow epicontinental sea. It is possible that there were several episodes of inland water colonization by local brackish water lineages. Subsequent diversification within clades and spread to new areas could have been induced by Alpine orogeny in the Miocene/Pliocene and, finally, by Pleistocene glaciations. The present distribution of clades, in many cases, still reflects Miocene palaeogeography of the area. Our results point out that investigations of the historical aspect of cryptic diversity in other taxa may help in a general understanding of the origins of freshwater invertebrate fauna of Europe.