Characterization of the biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene in direct micellar systems

The whole cell biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by the E. coli JM109(pPS1778) recombinant strain carrying the naphthalene dioxygenase and regulatory genes cloned from Pseudomonas fluorescens N3 in micellar systems has been investigated using biochemical and chemico-physical techniques. Reverse and direct micellar systems have been tested. Non-ionic surfactants (Tween and Triton X series) were found not to inhibit either the growth of the bacteria and the expression of the hydroxylating dioxygenase enzyme in such systems and were utilized in order to speed up the naphthalene conversion by increasing its solubility and also its bioavailability. The phase behavior of the direct micellar system was characterized through light scattering and other chemico-physical techniques. Further addition of isopropyl-palmitate 1–2% v/v to the micellar systems resulted in an increase of the apparent substrate concentration in solution and particularly its bioavailability thus allowing faster catalytic conversions resulting in an increase in productivity for the process. Since the cis-dihydrodiols are acquiring considerable potential as chiral pool synthons in asymmetric synthesis for a variety of industrial processes, possible applications for efficient small and large-scale production of such compounds are discussed.     

In vitro evidence for the participation of Drosophila melanogaster sperm β‐N‐acetylglucosaminidases in the interactions with glycans carrying terminal N‐acetylglucosamine residues on the egg's envelopes

Fertilization is a complex and multiphasic process, consisting of several steps, where egg‐coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm‐associated β‐N‐acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid‐phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well‐defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β‐N‐acetylglucosamine, but not core β‐N‐acetylglucosamine residues. The capacity of sperm β‐N‐acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β‐N‐acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β‐N‐acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm–egg interactions in Drosophila.     

The flower biology of Daphne gnidium L. (Thymelaeaceae)

Daphne gnidium is a circum-Mediterranean evergreen shrub. Flower and pollen features related to pollination mechanism were analyzed, and the seasonal presence of insects visiting the plant was monitored. The morphology and functionality of flowers are compatible with entomophily, as are pollen viability, which is maintained for a few hours only after anthesis, and the very short stigmatic receptivity. The reproductive fitness of the species is low and the ripening process often fails (35% collapsed embryos). While male organ functionality is fairly regular, the female organ often shows developmental arrest. The complex or transient evolutionary traits displayed by D. gnidium are comparable to those of Thymelaea hirsuta and other members of the Thymelaeaceae.     

The role of diet in the metabolism of daidzein by human faecal microbiota sampled from Italian volunteers

The intestinal microbial transformation of daidzein into equol is subject to a wide inter-individual variability. The aim of this study was to investigate in vitro this transformation and to evaluate possible correlations between individual diet and equol production. The transformation of daidzein was investigated in anaerobic batch cultures inoculated with mixed fecal bacteria from 90 volunteers. The daidzein metabolism was monitored by liquid chromatography-mass spectrometry, and a chiral column was used to distinguish equol and dihydrodaidzein enantiomers. The obtained results show that daidzein was unchanged (≈27%) or degraded to equol (≈28%), O-desmethylangolensin (≈12%) or dihydrodaidzein (≈31%). Furthermore, some subjects (≈2%) are able to produce both equol and O-desmethylangolensin. Bacteria represent sub-dominant populations (10⁵–10⁹ cell/g wet faeces) in “slow” equol producers, while higher counts of equol-producing microorganisms (10¹⁰–10¹¹ cell/g wet faeces) were found in “quick” equol producers. The in vitro test to evaluate equol-producing status is quick and not invasive, and the obtained results are comparable with those reported in vivo. Indeed, the only enantiomer present in the batch cultures containing equol was the S-form. No significant correlations between equol production, BMI, age and sex were found. It seems that the equol-producer group consumed less fibre, vegetables and cereals, and more lipids from animal sources.     

Modulation of mitochondrial K permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K⁺ current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K⁺ in the assay medium. The effects of p13 on ROS are mimicked by the K⁺ ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K⁺ vs. H⁺ currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K⁺ permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.     

Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17(+)] and from the same cell line transfected with the corresponding empty vector [GPR17(−)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)-IAM and GPR17(−)-IAM stationary phases. Frontal chromatography experiments on both GPR17(+)-IAM and GPR17(−)-IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)-IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants (K_d) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y₁₂/P2Y₁₃ antagonist), MRS2179 (a P2Y₁ receptor antagonist), and the agonist UDP—all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K_d values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)-IAM in ranking affinity studies for the selection of new potential candidates.     

Transmembrane Signaling in the Maltose ABC Transporter MalFGK2-E: PERIPLASMIC MalF-P2 LOOP COMMUNICATES SUBSTRATE AVAILABILITY TO THE ATP-BOUND MalK DIMER*

ABC transporters are ubiquitous membrane proteins that translocate solutes across biological membranes at the expense of ATP. In prokaryotic ABC importers, the extracytoplasmic anchoring of the substrate-binding protein (receptor) is emerging as a key determinant for the structural rearrangements in the cytoplasmically exposed ATP-binding cassette domains and in the transmembrane gates during the nucleotide cycle. Here the molecular mechanism of such signaling events was addressed by electron paramagnetic resonance spectroscopy of spin-labeled ATP-binding cassette maltose transporter variants (MalFGK₂-E). A series of doubly spin-labeled mutants in the MalF-P2 domain involving positions 92, 205, 239, 252, and 273 and one triple mutant labeled at positions 205/252 in P2 and 83 in the Q-loop of MalK were assayed. The EPR data revealed that the substrate-binding protein MalE is bound to the transporter throughout the transport cycle. Concomitantly with the three conformations of the ATP-binding cassette MalK₂, three functionally relevant conformations are found also in the periplasmic MalF-P2 loop, strictly dependent on cytoplasmic nucleotide binding and periplasmic docking of liganded MalE to MalFG. The reciprocal communication across the membrane unveiled here gives first insights into the stimulatory effect of MalE on the ATPase activity, and it is suggested to be an important mechanistic feature of receptor-coupled ABC transporters.     

Multiple species‐specific molecular markers using nanofluidic array as a tool to detect prey DNA from carnivore scats

Large carnivore feeding ecology plays a crucial role for management and conservation for predators and their prey. One of the keys to this kind of research is to identify the species composition in the predator diet, for example, prey determination from scat content. DNA‐based methods applied to detect prey in predators’ scats are viable alternatives to traditional macroscopic approaches, showing an increased reliability and higher prey detection rate. Here, we developed a molecular method for prey species identification in wolf (Canis lupus) scats using multiple species‐specific marker loci on the cytochrome b gene for 18 target species. The final panel consisted of 80 assays, with a minimum of four markers per target species, and that amplified specifically when using a high‐throughput Nanofluidic array technology (Fluidigm Inc.). As a practical example, we applied the method to identify target prey species DNA in 80 wolf scats collected in Sweden. Depending on the number of amplifying markers required to obtain a positive species call in a scat, the success in determining at least one prey species from the scats ranged from 44% to 92%. Although we highlight the need to evaluate the optimal number of markers for sensitive target species detection, the developed method is a fast and cost‐efficient tool for prey identification in wolf scats and it also has the potential to be further developed and applied to other areas and large carnivores as well.