Effect of dimethyl sulfoxide on the structure and the functional properties of horseradish peroxidase as observed by spectroscopy and cyclic voltammetry

Electrochemical biosensors have found wide application in food and clinical areas, as well as in environmental field. A large number of articles focused on horseradish peroxidase (HRP)-based biosensors have been published in the last decade, due to the capability of HRP to quantitatively detect the presence of hydrogen peroxide produced in a reaction. At present a large body of multi-enzymatic amperometric biosensors are realized by entrapping HRP together with other enzymes into a polymeric matrix; these systems represent a promising example of simple, low-cost electrochemical tools for the analysis of bioanalytes in solution, such as glucose, choline and cholesterol. Due to the fact that polymers used for HRP entrapping are soluble in organic solvents and that many solvents are strong denaturants of aquo-soluble proteins, in this paper we investigate (in particular, by circular dichroism and electron paramagnetic spectroscopies) the effect of dimethyl sulfoxide, one of the organic solvents employed for polymer solubilization, on the structure and the functionality of HRP, in order to determine the effect induced by the solvent concentration on the structure and activity of the hemoprotein. This is relevant for basic and applied biochemistry, HRP being largely employed in bioinorganic chemistry and sensor area.     

Bovine fetal microchimerism in normal and embryo transfer pregnancies and its implications for biotechnology applications in cattle

Fetal cells and DNA have been detected in the maternal circulation during and after pregnancy in a few mammalian species. The incidence of similar microchimerism in cattle could have repercussion for the application of modern biotechnologies such as the transfer of transgenic embryos. To determine if feto-maternal leakage can occur in pregnant cows, we have analyzed maternal blood samples for the presence of fetal DNA during gestation and post-partum periods. Y chromosome-specific DNA was detected in up to 73% of blood samples from naturally mated heifers carrying conventional bull calves and a transgene-specific sequence in up to 50% of recipient cows carrying transgenic fetuses. These findings document for the first time that transplacental leakage of fetal DNA into the maternal circulation can occur in cattle despite the epitheliochorial placenta of ruminants, with potential implications for the utilization of recipient cows in the food chain.     

Amber force field implementation, molecular modelling study, synthesis and MMP-1/MMP-2 inhibition profile of (R)- and (S)-N-hydroxy-2-(N-isopropoxybiphenyl-4-ylsulfonamido)-3-methylbutanamides

Ab initio calculations (B3LYP/Lanl2DZ level of theory) were performed in this study to determine all the structural and catalytic zinc parameters required in order to study MMPs and their complexes with hydroxamate inhibitors by means of the AMBER force field. The parameters thus obtained were used in order to study the docking of some known MMPi (Batimastat, CGS 27023A and Prinomastat) and our previously described inhibitor a which had shown an inhibitory activity for MMP-1, and -2, with the aim of explaining the different selectivity. On this basis the two enantiomers (R)-b and (S)-b were designed and synthesized, as more potent MMP-2 inhibitors than our previously described inhibitor a. Between these two enantiomers the eutomer (R)-b proved to be 24.7 times and 15.3 times more potent than CGS 27023A and the parent compound a on MMP-2, maintaining a higher index of MMP-2/MMP-1 selectivity compared with CGS 27023A and the more potent inhibitor Prinomastat. The hydroxamate (R)-b can be considered as a progenitor of a new class of biphenylsulfonamido-based inhibitors that differ from compound a in the presence of an alkyl side chain on the C alpha atom, and show different potency and selectivity profiles on the two MMPs considered.     

Differential biodiversity responses between kingdoms (plants,fungi, bacteria and metazoa) along an Alpine succession gradient

Biological diversities of multiple kingdoms potentially respond in similar ways to environmental changes. However, studies either compare details of microbial diversity across general vegetation or land use classes or relate details of plant community diversity with the extent of microbially governed soil processes, via physiological profiling. Here, we test the hypothesis of shared responses of plant and rhizosphere bacterial, fungal and metazoan biodiversities (especially across‐habitat β‐diversity patterns) along a disturbance gradient encompassing grazed to abandoned Alpine pasture, on acid soil in the European Central Alps. Rhizosphere biological diversity was inferred from eDNA fractions specific to bacteria, fungi and metazoans from contrasting plant habitats indicative of different disturbance levels. We found that soil β‐diversity patterns were weakly correlated with plant diversity measures and similarly ordinated along an evident edaphic (pH, C:N, assimilable P) and disturbance gradient but, contrary to our hypothesis, did not demonstrate the same diversity patterns. While plant communities were well separated along the disturbance gradient, correlating with fungal diversity, the majority of bacterial taxa were shared between disturbance levels (75% of bacteria were ubiquitous, cf. 29% plant species). Metazoa exhibited an intermediate response, with communities at the lowest levels of disturbance partially overlapping. Thus, plant and soil biological diversities were only loosely dependent and did not exhibit strictly linked environmental responses. This probably reflects the different spatial scales of organisms (and their habitats) and capacity to invest resources in persistent multicellular tissues, suggesting that vegetation responses to environmental change are unreliable indicators of below‐ground biodiversity responses.     

Antioxidant activity of oligomeric acylphloroglucinols from Myrtus communis L

The use of myrtle (Myrtus communis L.) as a culinary spice and as a flavoring agent for alcoholic beverages is widespread in the Mediterranean area, and especially in Sardinia. Myrtle contains unique oligomeric non-prenylated acylphloroglucinols, whose antioxidant activity was investigated in various systems. Both semimyrtucommulone (1) and myrtucommulone A (2) showed powerful antioxidant properties, protecting linoleic acid against free radical attack in simple in vitro systems, inhibiting its autoxidation and its FeCl3- and EDTA-mediated oxidation. While both compounds lacked pro-oxidant activity, semimyrtucommulone was more powerful than myrtucommulone A, and was further evaluated in rat liver homogenates for activity against lipid peroxidation induced by ferric-nitrilotriacetate, and in cell cultures for cytotoxicity and the inhibition of TBH- or FeCl3-induced oxidation. The results of these studies established semimyrtucommulone as a novel dietary antioxidant lead.     

454 Genome sequencing of Pseudoperonospora cubensis reveals effector proteins with a QXLR translocation motif

Pseudoperonospora cubensis is a biotrophic oomycete pathogen that causes downy mildew of cucurbits, a devastating foliar disease threatening cucurbit production worldwide. We sequenced P. cubensis genomic DNA using 454 pyrosequencing and obtained random genomic sequences covering approximately 14% of the genome, thus providing the first set of useful genomic sequence information for P. cubensis. Using bioinformatics approaches, we identified 32 putative RXLR effector proteins. Interestingly, we also identified 29 secreted peptides with high similarity to RXLR effectors at the N-terminal translocation domain, yet containing an R-to-Q substitution in the first residue of the translocation motif. Among these, a family of QXLR-containing proteins, designated as PcQNE, was confirmed to have a functional signal peptide and was further characterized as being localized in the plant nucleus. Internalization of secreted PcQNE into plant cells requires the QXLR-EER motif. This family has a large number of near-identical copies within the P. cubensis genome, is under diversifying selection at the C-terminal domain, and is upregulated during infection of plants, all of which are common characteristics of characterized oomycete effectors. Taken together, the data suggest that PcQNE are bona fide effector proteins with a QXLR translocation motif, and QXLR effectors are prevalent in P. cubensis. Furthermore, the massive duplication of PcQNE suggests that they might play pivotal roles in pathogen fitness and pathogenicity.     

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.     

Intracellular Ionic Variations in the Apoptotic Death of L Cells by Inhibitors of Cell Cycle Progression

Treatment with VP-16 (1-50 μM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light seattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca²⁺ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K⁺ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.