Distinct and enhanced hygienic responses of a leaf‐cutting ant toward repeated fungi exposures

Leaf‐cutting ants and their fungal crops are a textbook example of a long‐term obligatory mutualism. Many microbes continuously enter their nest containing the fungal cultivars, destabilizing the symbiosis and, in some cases, outcompeting the mutualistic partners. Preferably, the ant workers should distinguish between different microorganisms to respond according to their threat level and recurrence in the colony. To address these assumptions, we investigated how workers of Atta sexdens sanitize their fungal crop toward five different fungi commonly isolated from the fungus gardens: Escovopsis sp., Fusarium oxysporum, Metarhizium anisopliae, Trichoderma spirale, and Syncephalastrum sp. Also, to investigate the plasticity of these responses toward recurrences of these fungi, we exposed the colonies with each fungus three times fourteen days apart. As expected, intensities in sanitization differed according to the fungal species. Ants significantly groom their fungal crop more toward F. oxysporum, M. anisopliae, and Syncephalastrum sp. than toward Escovopsis sp. and T. spirale. Weeding, self‐, and allogrooming were observed in less frequency than fungus grooming in all cases. Moreover, we detected a significant increase in the overall responses after repeated exposures for each fungus, except for Escovopsis sp. Our results indicate that A. sexdens workers are able to distinguish between different fungi and apply distinct responses to remove these from the fungus gardens. Our findings also suggest that successive exposures to the same antagonist increase hygiene, indicating plasticity of ant colonies'' defenses to previously encountered pathogens.     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

Human Embryonic Stem Cell Technology: Large Scale Cell Amplification and Differentiation

Embryonic stem cells (ESC) hold the promise of overcoming many diseases as potential sources of, for example, dopaminergic neural cells for Parkinson’s Disease to pancreatic islets to relieve diabetic patients of their daily insulin injections. While an embryo has the innate capacity to develop fully functional differentiated tissues; biologists are finding that it is much more complex to derive singular, pure populations of primary cells from the highly versatile ESC from this embryonic parent. Thus, a substantial investment in developing the technologies to expand and differentiate these cells is required in the next decade to move this promise into reality. In this review we document the current standard assays for characterising human ESC (hESC), the status of ‘defined’ feeder-free culture conditions for undifferentiated hESC growth, examine the quality controls that will be required to be established for monitoring their growth, review current methods for expansion and differentiation, and speculate on the possible routes of scaling up the differentiation of hESC to therapeutic quantities.     

Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.     

In Vivo Evaluation of Blood Based and Reference Tissue Based PET Quantifications of [11C]DASB in the Canine Brain

This first-in-dog study evaluates the use of the PET-radioligand [¹¹C]DASB to image the density and availability of the serotonin transporter (SERT) in the canine brain. Imaging the serotonergic system could improve diagnosis and therapy of multiple canine behavioural disorders. Furthermore, as many similarities are reported between several human neuropsychiatric conditions and naturally occurring canine behavioural disorders, making this tracer available for use in dogs also provide researchers an interesting non-primate animal model to investigate human disorders. Five adult beagles underwent a 90 minutes dynamic PET scan and arterial whole blood was sampled throughout the scan. For each ROI, the distribution volume (V_T), obtained via the one- and two- tissue compartment model (1-TC, 2-TC) and the Logan Plot, was calculated and the goodness-of-fit was evaluated by the Akaike Information Criterion (AIC). For the preferred compartmental model BP_ND values were estimated and compared with those derived by four reference tissue models: 4-parameter RTM, SRTM2, MRTM2 and the Logan reference tissue model. The 2-TC model indicated in 61% of the ROIs a better fit compared to the 1-TC model. The Logan plot produced almost identical V_T values and can be used as an alternative. Compared with the 2-TC model, all investigated reference tissue models showed high correlations but small underestimations of the BP_ND-parameter. The highest correlation was achieved with the Logan reference tissue model (Y = 0.9266 x + 0.0257; R² = 0.9722). Therefore, this model can be put forward as a non-invasive standard model for future PET-experiments with [¹¹C]DASB in dogs.     

Determination of stereochemistry in the fatty acid hydroperoxide products of lipoxygenase catalysis

High-performance liquid chromatography has been found to be an effective method for the determination of absolute configuration in the products of the lipoxygenase-catalyzed oxygenation of polyunsaturated fatty acids. Methyl esters of fatty acid hydroperoxides that had been reduced to the corresponding alcohols were converted into (+)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid esters. Enantiomeric alcohols were converted into diastereomeric esters that were readily resolved by normal-phase HPLC. The instrumental requirements for the technique are an isocratic HPLC and a uv absorbance monitor. The method was found to be effective in the determination of stereochemistry in the products derived from the action of plant lipoxygenases on linoleic acid. In addition, the chromatography of the derivatives obtained from lipoxygenase catalysis on arachidonic acid was found to be effective for the assignment of stereochemistry in those products. A comparison of the chromatography of these derivatives with that for the corresponding menthyloxycarbonyl derivatives demonstrated the superiority of this approach for the resolution of the diastereomeric pairs. The technique was applied to the determination of stereochemistry in the products derived from soybean lipoxygenase isoenzymes under a variety of experimental conditions.     

Biochemical and proton NMR characterization of the isolated functional beta-subunit of coupling factor one from spinach chloroplasts

Beta subunits have been dissociated from CF1 of spinach chloroplasts, purified by HPLC and characterized by two-dimensional electrophoresis and fluorescence emission. The solutions of isolated beta subunits are able to hydrolyze MgATP; this ATPase activity is an intrinsic property of the beta molecule. From proton NMR at 300 and 500 MHz, it is shown that the preparations are fully reproducible and that beta subunits remain monomeric with 75% aliphatic protons associated with rigid parts of the molecule. The other 25% give rise to separate resonances and belong to mobile side-chains and/or to flexible regions. The measurement of the transverse relaxation times T2 has permitted a detailed characterization of the molecular dynamics of the isolated beta subunits.     

Chemiluminescence of enterococci isolates from freshwater

All Enterococcus spp., isolated from environmental water samples (n=81), emitted a high chemiluminescence signal in the presence of luminol (10(-2) M). Kinetic studies of chemiluminescence show a close correlation between chemiluminescence and growth curves during the exponential phase, with a maximum chemiluminescence reached just before bacterial growth entered in the stationary phase. On the other hand, genera closely related to Enterococcus such as Streptococcus or Lactococcus produced a very weak chemiluminescent signal. Chemiluminescence of enterococci could therefore offer a rapid test, in aiding the identification of the genus Enterococcus and in the survey of the microbiological quality of water supplies.