Crystal structure of Hyp-1, a St. John’s wort protein implicated in the biosynthesis of hypericin

Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, widely known as St. John’s wort. Hypericin has been attracting a growing attention of the pharmaceutical industry because of its potential application in various therapies, including the treatment of depression. In vivo, hypericin is synthesized by dimerization of emodin in a complicated multistep reaction that is reportedly catalyzed by a small (17.8 kDa) protein, Hyp-1. Based on relatively low sequence similarity (～50%), Hyp-1 has been tentatively classified as a plant PR-10 (pathogenesis-related class 10) protein. Members of the PR-10 family are ubiquitous plant proteins associated with stress control and tissue differentiation but with no clearly understood molecular mechanism. They have, however, a well-defined folding canon, consisting of an extended antiparallel β-sheet wrapped around a C-terminal α-helix, enclosing in the protein interior a huge cavity, in which various hydrophobic ligands can be bound. Apart from Hyp-1, only two other PR-10 members have been found to possess enzymatic activity (S-norcoclaurine synthase and TcmN aromatase/cyclase). In this paper, we report a high-resolution crystal structure of Hyp-1, confirming that it indeed has a PR-10 fold. The protein binds multiple polyethylene glycol molecules, some of which occupy the hydrophobic cavity. The crystallographic model illustrates a high degree of conformational adaptability of both interacting partners for efficient binding. We have been unable, however, to dimerize emodin to hypericin using Hyp-1 as biocatalyst. This puzzling result does not have a clear explanation at this time.     

Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium

Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au).     

Osteogenic protein 1 in synovial fluid from patients with rheumatoid arthritis or osteoarthritis: relationship with disease and levels of hyaluronan and antigenic keratan sulfate

The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1 – mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.     

Theoretical studies of the interaction between enflurane and water

Increase of the atmospheric concentration of halogenated organic compounds is partially responsible for a change of the global climate. In this work we have investigated the interaction between halogenated ether and water, which is one of the most important constituent of the atmosphere. The structures of the complexes formed by the two most stable conformers of enflurane (a volatile anaesthetic) with one and two water molecules were calculated by means of the counterpoise CP-corrected gradient optimization at the MP2/6–311++G(d,p) level. In these complexes the CH…O_w hydrogen bonds are formed, with the H…O_w distances varying between 2.23 and 2.32 Å. A small contraction of the CH bonds and the blue shifts of the ν(CH) stretching vibrations are predicted. There is also a weak interaction between one of the F atoms and the H atom of water, with the H_w…F distances between 2.41 and 2.87 Å. The CCSD(T)/CBS calculated stabilization energies in these complexes are between ?5.89 and ?4.66 kcal?mol^?1, while the enthalpies of formation are between ?4.35 and ?3.22 kcal?mol^?1. The Cl halogen bonding between enflurane and water has been found in two complexes. The intermolecular (Cl···O) distance is smaller than the sum of the corresponding van der Waals radii. The CCSD(T)/CBS stabilization energies for these complexes are about ?2 kcal?mol^?1.

The effect of the presence of quiescent female nymphs, males and their spermatophores on spermatophore placement in two species of eriophyoid mites

Under sex dissociated sperm transfer, females seek spermatophores and pick up sperm without male assistance. In several species males adjust spermatophore deposition rate to the presence of conspecifics. It is not known, however, which factors could favor such elasticity in non-pairing males. In this paper, we compare male response towards conspecifics between the sex dissociated eriophyoid mites Aculus fockeui (Nalepa and Trouessart) and Aculops allotrichus (Nalepa). The species differ significantly in male reproductive strategies and, consequently, the intensity of male–male-competition. Aculus fockeui males deposit spematophores all over the leaves and occasionally leave single spermatophores beside quiescent female nymphs (QFNs). In contrast, A. allotrichus males guard QFNs and encircle them with spermatophores. In this study, males of both species deposited spermatophores close to and apart from the rival spermatophores. Aculops allotrichus males had similar spermatophore output whether they were kept alone or in a group of seven males. They did not change spermatophore output in the presence of five rival spermatophores, a QFN or a QFN and varying number of rivals, either. In contrast, A. fockeui males increased spermatophore output in the presence of rival spermatophores or when on the arena with a QFN the male number increased to eight males. They did not respond, however, to the presence of a QFN and one rival or a QFN alone. The possible effect of the species-specific intensity of male–male competition, population density, the availability of receptive females and the rate of spermatophore output on the flexibility of eriophyoid spermatophore deposition is discussed.     

High mitochondrial DNA diversity of an introduced alien carnivore: comparison of feral and ranch American mink Neovison vison in Poland

Aim Invasive alien species usually exhibit very high adaptation and rapid evolution in a new environment, but they often have low levels of genetic diversity (invasive species paradox). Genetic variation and population genetic structure of feral American mink, Neovison vison, in Poland was investigated to explain the invasion paradox and to assess current gene flow. Furthermore, the influence of mink farming on adaptation of the feral population was evaluated by comparing the genetic structure of feral and ranch mink. Location Samples from feral mink were collected in 11 study areas in northern and central Poland and from ranch mink at 10 farms distributed throughout the country. Methods A 373‐bp‐long mtDNA control region fragment was amplified from 276 feral and 166 ranch mink. Results Overall, 31 haplotypes, belonging to two groups from genetically diverse sources, were detected: 11 only in feral mink, 12 only in ranch mink and eight in both. The genetic differentiation of feral mink from the trapping sites was high, while that among ranch mink from various farms was moderate. There was no significant relationship between genetic and geographic distance. The number of trapping sites where given haplotypes occurred correlated with the number of farms with these haplotypes. The mink from two sites were the most divergent, both from all other feral mink and from ranch mink. Comparison of mtDNA and microsatellite differentiation suggests male‐biased dispersal in this species. Main conclusions American mink in Poland exhibit high genetic diversity and originate from different source populations of their native range. The process of colonization was triggered by numerous escapees from various farms and by immigrants from Belarus. The genetic structure of local feral mink populations was shaped by the founder effect and multiple introductions. The genomic admixture that occurred during mixing of different populations might have increased the fitness of individuals and accelerated the invasiveness of this species.     

Prooxidant action of carazolol in the Fenton‐like reaction

Carazolol [4‐(2‐hydroxy‐3‐isopropyl‐amino‐propoxy)‐carbazole], a β₃‐adrenoceptor agonist, is clinically used in the treatment of hypertension, cardiac arrhythmias and angina pectoris. Despite the beneficial effect of the drug, its high dose may contribute to cardiotoxicity. This study was conducted to examine whether carazolol can influence hydroxyl radical formation by a Fenton‐like reaction [Co(II) + H₂O₂ + HO^‐] in the presence of ethylenediaminetetraacetic acid. The oxygen free radicals and singlet oxygen (¹O₂) formation was traced by three different assay methods: chemiluminescence (CL), an electron spin resonance (ESR) spin trapping with 2,2,6,6‐tetramethyl‐4‐piperidine and 5,5‐dimethyl‐1‐pyrroline‐1‐oxide, and spectrophotometric determination of ¹O₂ based on bleaching of p‐nitrosodimethylaniline. The effect of hydroxyl radical inhibitors and ¹O₂ quenchers on peroxidation of carazolol was also examined. The results indicated that carazolol enhanced the HO radical and ¹O₂ formation in a Fenton‐like reaction. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins

In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein-ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure-function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino‐acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequence-based methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.     

Evaluation of the antioxidant activity of tetracycline antibiotics in vitro

Tetracyclines are the second most common antibiotic family in medicine usage. These antibiotics exhibit antioxidant potential; however, the exact mechanism remains unclear. The antiradical activity of the seven tetracyclines (TCs; tetracycline, chlortetracycline, oxytetracycline, doxocycline, methacycline, demeclocycline, minocycline) was determined using the free radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) and hydroxyl radicals (HO^?) generated in a Fenton reaction. Electron spin resonance (ESR), ESR spin‐trapping, chemiluminescence and spectrophotometry techniques were applied. It was found that the TCs showed high DPPH antiradical activity in the range 26–96% at 2.5 mmol/L concentration. The second‐order rate constants for the reaction between HO^? and TCs were calculated, in the range (3.6–9.6) × 10⁹ L/mol/s. The tetracycline compounds also exhibited a strong decrease in light emission (range 61–85% at concentration of 1 mmol/L). This study also showed that TCs promote the generation of singlet oxygen in the presence of and Fe(II)/Fe(III) ions. Our findings suggest direct scavenging activity of the examined tetracyclines towards free radicals, and may be relevant to therapeutic strategy. Copyright © 2011 John Wiley & Sons, Ltd.     

Towards an Optimization of Stimulus Parameters for Brain-Computer Interfaces Based on Steady State Visual Evoked Potentials

Efforts to construct an effective brain-computer interface (BCI) system based on Steady State Visual Evoked Potentials (SSVEP) commonly focus on sophisticated mathematical methods for data analysis. The role of different stimulus features in evoking strong SSVEP is less often considered and the knowledge on the optimal stimulus properties is still fragmentary. The goal of this study was to provide insight into the influence of stimulus characteristics on the magnitude of SSVEP response. Five stimuli parameters were tested: size, distance, colour, shape, and presence of a fixation point in the middle of each flickering field. The stimuli were presented on four squares on LCD screen, with each square highlighted by LEDs flickering with different frequencies. Brighter colours and larger dimensions of flickering fields resulted in a significantly stronger SSVEP response. The distance between stimulation fields and the presence or absence of the fixation point had no significant effect on the response. Contrary to a popular belief, these results suggest that absence of the fixation point does not reduce the magnitude of SSVEP response. However, some parameters of the stimuli such as colour and the size of the flickering field play an important role in evoking SSVEP response, which indicates that stimuli rendering is an important factor in building effective SSVEP based BCI systems.