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.     

Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.     

A model for light distribution and average solar irradiance inside outdoor tubular photobioreactors for the microalgal mass culture

A mathematical model to estimate the solar irradiance profile and average light intensity inside a tubular photobioreactor under outdoor conditions is proposed, requiring only geographic, geometric, and solar position parameters. First, the length of the path into the culture traveled by any direct or disperse ray of light was calculated as the function of three variables: day of year, solar hour, and geographic latitude. Then, the phenomenon of light attenuation by biomass was studied considering Lambert-Beer's law (only considering absorption) and the monodimensional model of Cornet et al. (1900) (considering absorption and scattering phenomena). Due to the existence of differential wavelength absorption, none of the literature models are useful for explaining light attenuation by the biomass. Therefore, an empirical hyperbolic expression is proposed. The equations to calculate light path length were substituted in the proposed hyperbolic expression, reproducing light intensity data obtained in the center of the loop tubes. The proposed model was also likely to estimate the irradiance accurately at any point inside the culture. Calculation of the local intensity was thus extended to the full culture volume in order to obtain the average irradiance, showing how the higher biomass productivities in a Phaeodactylum tricornutum UTEX 640 outdoor chemostat culture could be maintained by delaying light limitation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 701-714, 1997.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

Genome Size: A Novel Genomic Signature in Support of Afrotheria

Molecular phylogenetic analyses suggest an emerging phylogeny for the extant Placentalia (eutherian) that radically departs from morphologically based constructions of the past. Placental mammals are partitioned into four supraordinal clades: Afrotheria, Xenarthra, Laurasiatheria, and Euarchontoglires. Afrotheria form an endemic African clade that includes elephant shrews, golden moles, tenrecs, aardvarks, hyraxes, elephants, dugongs, and manatees. Datamining databases of genome size (GS) shows that till today just one afrotherian GS has been evaluated, that of the aardvark Orycteropus afer. We show that the GSs of six selected representatives across the Afrotheria supraordinal group are among the highest for the extant Placentalia, providing a novel genomic signature of this enigmatic group. The mean GS value of Afrotheria, 5.3 ± 0.7 pg, is the highest reported for the extant Placentalia. This should assist in planning new genome sequencing initiatives. [Reviewing Editor: Dmitri Petrov]     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

New cationic exchanger support for reversible immobilization of proteins

New tailor-made cationic exchange resins have been prepared by covalently binding aspartic-dextran polymers (e.g. MW 15 000-20 000) to porous supports (aminated agarose and Sepabeads). More than 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans have been strongly adsorbed on these porous materials at pH 5. This interaction was stronger than in conventional carboxymethyl cellulose (e.g., at pH 7 and 25 degrees C, all proteins previously adsorbed at pH 5 were released from carboxymethyl cellulose, whereas no protein was released from the new supports under similar conditions). Ionic exchange properties of such composites were strongly dependent on the size of the aspartic-dextran polymers as well as on the exact conditions of the covalent coating of the solids with the polymer (optimal conditions: 100 mg aspartic-dextran 20 000/(mL of support); room temperature). Finally, some industrially relevant enzymes (Kluyveromices lactis, Aspergillus oryzae, and Thermus sp. beta-galactosidases, Candida antarctica B lipase, and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recovery and immobilization rates. After enzyme inactivation, the enzyme can be fully desorbed from the support and the support could be reused for several cycles.     

Targeted tandem affinity purification of PSD‐95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K⁺ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K⁺ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.     

Photobioreactors for the production of microalgae

Microalgae are produced today for human and animal markets, as food-feed and source of active compounds. Microalgae can be also used in wastewater treatment and they has been proposed as biofuels source to reduce global warming problem. Whatever the final application of microalgae its production is based on the same principles as light availability, enough mass and heat transfer and adequate control of culture parameters. In this paper these principals are revised. Moreover, the production must be carried out at adequate scale using photobioreactors. Design of photobioreactor is determined by the final use of biomass and quality required. Different designs today used are revised, including last designs proposed, identifying his characteristics parameters and applications. In addition, the obligation of adequate control strategies is discussed. Finally, the bottlenecks for the scale-up of the different technologies and thus of microalgae production are summarized.