Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones.

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.     

Genome-Wide Reconstruction of Rediploidization Following Autopolyploidization across One Hundred Million Years of Salmonid Evolution

The long-term evolutionary impacts of whole-genome duplication (WGD) are strongly influenced by the ensuing rediploidization process. Following autopolyploidization, rediploidization involves a transition from tetraploid to diploid meiotic pairing, allowing duplicated genes (ohnologs) to diverge genetically and functionally. Our understanding of autopolyploid rediploidization has been informed by a WGD event ancestral to salmonid fishes, where large genomic regions are characterized by temporally delayed rediploidization, allowing lineage-specific ohnolog sequence divergence in the major salmonid clades. Here, we investigate the long-term outcomes of autopolyploid rediploidization at genome-wide resolution, exploiting a recent “explosion” of salmonid genome assemblies, including a new genome sequence for the huchen (Hucho hucho). We developed a genome alignment approach to capture duplicated regions across multiple species, allowing us to create 121,864 phylogenetic trees describing genome-wide ohnolog divergence across salmonid evolution. Using molecular clock analysis, we show that 61% of the ancestral salmonid genome experienced an initial “wave” of rediploidization in the late Cretaceous (85–106 Ma). This was followed by a period of relative genomic stasis lasting 17–39 My, where much of the genome remained tetraploid. A second rediploidization wave began in the early Eocene and proceeded alongside species diversification, generating predictable patterns of lineage-specific ohnolog divergence, scaling in complexity with the number of speciation events. Using gene set enrichment, gene expression, and codon-based selection analyses, we provide insights into potential functional outcomes of delayed rediploidization. This study enhances our understanding of delayed autopolyploid rediploidization and has broad implications for future studies of WGD events.     

Extracellular Acidic Sulfur-Containing Amino Acids and γ-Glutamyl Peptides in Global Ischemia: Postischemic Recovery of Neuronal Activity Is Paralleled by a Tetrodotoxin-Sensitive Increase in Cysteine Sulfinate in the CA1 of the Rat Hippocampus

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.     

Utilization of deletion bins to anchor and order sequences along the wheat 7B chromosome

Key message

A total of 3,671 sequence contigs and scaffolds were mapped to deletion bins on wheat chromosome 7B providing a foundation for developing high-resolution integrated physical map for this chromosome.

Abstract

Bread wheat (Triticum aestivum L.) has a large, complex and highly repetitive genome which is challenging to assemble into high quality pseudo-chromosomes. As part of the international effort to sequence the hexaploid bread wheat genome by the international wheat genome sequencing consortium (IWGSC) we are focused on assembling a reference sequence for chromosome 7B. The successful completion of the reference chromosome sequence is highly dependent on the integration of genetic and physical maps. To aid the integration of these two types of maps, we have constructed a high-density deletion bin map of chromosome 7B. Using the 270 K Nimblegen comparative genomic hybridization (CGH) array on a set of cv. Chinese spring deletion lines, a total of 3,671 sequence contigs and scaffolds (~7.8 % of chromosome 7B physical length) were mapped into nine deletion bins. Our method of genotyping deletions on chromosome 7B relied on a model-based clustering algorithm (Mclust) to accurately predict the presence or absence of a given genomic sequence in a deletion line. The bin mapping results were validated using three different approaches, viz. (a) PCR-based amplification of randomly selected bin mapped sequences (b) comparison with previously mapped ESTs and (c) comparison with a 7B genetic map developed in the present study. Validation of the bin mapping results suggested a high accuracy of the assignment of 7B sequence contigs and scaffolds to the 7B deletion bins.     

Metabolic modeling of energy balances in Mycoplasma hyopneumoniae shows that pyruvate addition increases growth rate

Mycoplasma hyopneumoniae is cultured on large‐scale to produce antigen for inactivated whole‐cell vaccines against respiratory disease in pigs. However, the fastidious nutrient requirements of this minimal bacterium and the low growth rate make it challenging to reach sufficient biomass yield for antigen production. In this study, we sequenced the genome of M. hyopneumoniae strain 11 and constructed a high quality constraint‐based genome‐scale metabolic model of 284 chemical reactions and 298 metabolites. We validated the model with time‐series data of duplicate fermentation cultures to aim for an integrated model describing the dynamic profiles measured in fermentations. The model predicted that 84% of cellular energy in a standard M. hyopneumoniae cultivation was used for non‐growth associated maintenance and only 16% of cellular energy was used for growth and growth associated maintenance. Following a cycle of model‐driven experimentation in dedicated fermentation experiments, we were able to increase the fraction of cellular energy used for growth through pyruvate addition to the medium. This increase in turn led to an increase in growth rate and a 2.3 times increase in the total biomass concentration reached after 3–4 days of fermentation, enhancing the productivity of the overall process. The model presented provides a solid basis to understand and further improve M. hyopneumoniae fermentation processes. Biotechnol. Bioeng. 2017;114: 2339–2347. © 2017 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.     

The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligand-binding domain: RU-486 induces a transconformation that leads to active antagonism

Here we describe the three-dimensional crystal structures of human glucocorticoid receptor ligand-binding domain (GR-LBD) in complex with the antagonist RU-486 at 2.3 A resolution and with the agonist dexamethasone ligand together with a coactivator peptide at 2.8 A. The RU-486 structure was solved in several different crystal forms, two with helix 12 intact (GR1 and GR3) and one with a protease-digested C terminus (GR2). In GR1, part of helix 12 is in a position that covers the co-activator pocket, whereas in the GR3, domain swapping is seen between the crystallographically identical subunits in the GR dimer. An arm consisting of the end of helix 11 and beyond stretches out from one molecule, and helix 12 binds to the other LBD, partly blocking the coactivator pocket of that molecule. This type of GR-LBD dimer has not been described before but might be an artifact from crystallization. Furthermore, the subunits of the GR3 dimers are covalently connected via a disulfide bond between the Cys-736 residues in the two molecules. All three RU-486 GR-LBD structures show that GR has a very flexible region between the end of helix 11 and the end of helix 12.     

First Ecological Study of the Bawean Warty Pig (Sus blouchi), One of the Rarest Pigs on Earth

The Bawean warty pig (Sus blouchi) is an endemic pig species confined to the 192 km² large island of Bawean in the Java Sea, Indonesia. Due to a lack of quantitative ecological research, understanding of natural history and conservation requirements have so far been based solely on anecdotal information from interviews with local people and study of captive and museum specimens. In this study we provide the first assessment of population and habitat preferences for S. blouchi by using camera trapping. From the 4th of November 2014 to January 8th 2015, we placed camera traps at 100 locations in the forested protected areas on Bawean. In 690.31 camera days (16567.45 hours) we captured 92 independent videos showing S. blouchi. Variation in S. blouchi trapping rates with cumulative trap effort stabilized after 500 camera days. An important outcome is that, in contrast to the suggestion of previous assessments, only S. blouchi was detected and no S. scrofa was found, which excludes hybridization threats. We fitted a Random Encounter Model, which does not require the identification of individual animals, to our camera-trapping data and estimated 172–377 individuals to be present on the island. Activity patterns and habitat data indicate that S. blouchi is mainly nocturnal and prefers community forests and areas near forest borders. Next to this, we found a positive relationship between S. blouchi occupancy, distance to nearest border, litter depth and tree density in the highest ranking occupancy models. Although these relationships proved non-significant based on model averaging, their presence in the top ranking models suggests that these covariables do play a role in predicting S. blouchi occurrence on Bawean. The estimated amount of sites occupied reached 58%. Based on our results, especially the estimation of the population size and area of occupancy, we determine that the species is Endangered according to the IUCN/SSC Red List criteria.     

Enriched environment increases neurogenesis in the adult rat dentate gyrus and improves spatial memory

The fetal and even the young brain possesses a considerable degree of plasticity. The plasticity and rate of neurogenesis in the adult brain is much less pronounced. The present study was conducted to investigate whether housing conditions affect neurogenesis, learning, and memory in adult rats. Three‐month‐old rats housed either in isolation or in an enriched environment were injected intraperitoneally with bromodeoxyuridine (BrdU) to detect proliferation among progenitor cells and to follow their fate in the dentate gyrus. The rats were sacrificed either 1 day or 4 weeks after BrdU injections. This experimental paradigm allows for discrimination between proliferative effects and survival effects on the newborn progenitors elicited by different housing conditions. The number of newborn cells in the dentate gyrus was not altered 1 day after BrdU injections. In contrast, the number of surviving progenitors 1 month after BrdU injections was markedly increased in animals housed in an enriched environment. The relative ratio of neurogenesis and gliogenesis was not affected by environmental conditions, as estimated by double‐labeling immunofluorescence staining with antibodies against BrdU and either the neuronal marker calbindin D28k or the glial marker GFAp, resulting in a net increase in neurogenesis in animals housed in an enriched environment. Furthermore, we show that adult rats housed in an enriched environment show improved performance in a spatial learning test. The results suggest that environmental cues can enhance neurogenesis in the adult hippocampal region, which is associated with improved spatial memory. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 569–578, 1999