Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.     

The translation initiation factor, PeIF5B, from Pisum sativum displays chaperone activity

We earlier documented the structural and functional characterization of PeIF5B factor from Pisum sativum that shows strong homology to the universal translation initiation factor eIF5B (Rasheedi et al., 2007, 2010 [12] and [13]). We now show that PeIF5B is an unusually thermo-stable protein resisting temperatures up to 95 °C. PeIF5B prevents thermal aggregation of heat labile proteins, such as citrate synthase (CS) and NdeI, under heat stress or chemical denaturation conditions and promotes their functional folding. It also prevents the aggregation of DTT induced insulin reduction. GTP appears to stimulate PeIF5B-mediated chaperone activity. In-vivo, PeIF5B over expression significantly enhances, the viability of Escherichia coli cells after heat stress (50 °C). These observations lead us to conclude that PeIF5B, in addition to its role in protein translation, has chaperone like activity and could be likely involved in protein folding and protection from stress.     

Induction of polyclonal CD8+ T cell activation and effector function by Pertussis toxin

Pertussis toxin (PTX) has pronounced adjuvant activity and strongly enhances innate and adaptive immune responses, including increased antibody production and Th1/Th2 cytokine production. Adjuvant effects of PTX on Th1 and Th2 cells are primarily mediated via CD80/86 costimulation via enhanced expression of these molecules by APCs. However, it has remained unresolved whether PTX modulates the expression of costimulatory and inhibitory molecules on CD4+ and CD8+ T cells. To address this question, we determined the expression kinetics of CD28, CTLA-4, and CD40L on spleen CD4+ and CD8+ T cells after incubation with PTX. The results show that PTX upregulated the expression of CD28 by CD8+ T cells, but not by CD4+ T cells. In contrast, the expression of CTLA-4 and CD40L was not substantially altered on CD4+ or CD8+ T cells. CD28 upregulation by CD8+ T cells was paralleled by upregulation of CD69 and the induction of IFN-γ, Granzyme B (GrB), and IL-17. CD8+ T cell activation and cytokine production could be substantially blocked with anti-CD80 and CD86 antibodies, consistent with CD28 mediated signaling. Treatment of highly purified CD8+ T cells with PTX resulted in upregulation of CD28 and CD69, and production of IFN-γ. Incubation with CD28 mAb further enhanced this effect, suggesting that PTX has direct effects on CD8+ T cells which are enhanced by CD80/86-mediated costimulation provided by APCs.     

Physical mapping of the mouse tilted locus identifies an association between human deafness loci DFNA6/14 and vestibular system development

The tilted (tlt) mouse carries a recessive mutation causing vestibular dysfunction. The defect in tlt homozygous mice is limited to the utricle and saccule of the inner ear, which completely lack otoconia. Genetic mapping of tlt placed it in a region orthologous with human 4p16.3-p15 that contains two loci, DFNA6 and DFNA14, responsible for autosomal dominant, nonsyndromic hereditary hearing impairment. To identify a possible relationship between tlt in mice and DFNA6 and DFNA14 in humans, we have refined the mouse genetic map, assembled a BAC contig spanning the tlt locus, and developed a comprehensive comparative map between mouse and human. We have determined the position of tlt relative to 17 mouse chromosome 5 genes with orthologous loci in the human 4p16.3-p15 region. This analysis identified an inversion between the mouse and human genomes that places tlt and DFNA6/14 in close proximity.     

Hsp90 is involved in the formation of P-bodies and stress granules

Previously, we found that treatment of cells with the Hsp90 inhibitor geldanamycin (GA) leads to a substantial reduction in the number of processing bodies (P-bodies), and also alters the size and subcellular localization of stress granules. These findings imply that the chaperone activity of Hsp90 is involved in the formation of P-bodies and stress granules. To verify these observations, we examined whether another Hsp90 inhibitor radicicol (RA) affected P-bodies and stress granules. Treatment with RA reduced the level of the Hsp90 client protein Argonaute 2 and the number of P-bodies. Although stress granules still assembled in RA-treated cells upon heat shock, they were smaller and more dispersed in the cytoplasm than those in untreated cells. Furthermore eIF4E and eIF4E-transporter were dissociated selectively from stress granules in RA-treated cells. These observations were comparable to those obtained upon treatment with GA in our previous work. Thus, we conclude that abrogation of the chaperone activity of Hsp90 affects P-body formation and the integrity of stress granules.     

Genomic changes following host restriction in bacteria

Many genomic sequences have been recently published for bacteria that can replicate only within eukaryotic hosts. Comparisons of genomic features with those of closely related bacteria retaining free-living stages indicate that rapid evolutionary change often occurs immediately after host restriction. Typical changes include a large increase in the frequency of mobile elements in the genome, chromosomal rearrangements mediated by recombination among these elements, pseudogene formation, and deletions of varying size. In anciently host-restricted lineages, the frequency of insertion sequence elements decreases as genomes become extremely small and strictly clonal. These changes represent a general syndrome of genome evolution, which is observed repeatedly in host-restricted lineages from numerous phylogenetic groups. Considerable variation also exists, however, in part reflecting unstudied aspects of the population structure and ecology of host-restricted bacterial lineages.     

Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice

Bioactive components from bitter melon (BM) have been reported to improve glucose metabolism in vivo, but definitive studies on efficacy and mechanism of action are lacking. We sought to investigate the effects of BM bioactives on body weight, muscle lipid content and insulin signaling in mice fed a high-fat diet and on insulin signaling in L6 myotubes. Male C57BL/6J mice were randomly divided into low-fat diet control (LFD), high-fat diet (HFD) and HFD plus BM (BM) groups. Body weight, body composition, plasma glucose, leptin, insulin and muscle lipid profile were determined over 12 weeks. Insulin signaling was determined in the mouse muscle taken at end of study and in L6 myotubes exposed to the extract. Body weight, plasma glucose, insulin, leptin levels and HOMA-IR values were significantly lower in the BM-fed HFD group when compared to the HFD group. BM supplementation significantly increased IRS-2, IR β, PI 3K and GLUT4 protein abundance in skeletal muscle, as well as phosphorylation of IRS-1, Akt1 and Akt2 when compared with HFD (P<.05 and P<.01). BM also significantly reduced muscle lipid content in the HFD mice. BM extract greatly increased glucose uptake and enhanced insulin signaling in L6 myotubes. This study shows that BM bioactives reduced body weight, improved glucose metabolism and enhanced skeletal muscle insulin signaling. A contributing mechanism to the enhanced insulin signaling may be associated with the reduction in skeletal muscle lipid content. Nutritional supplementation with this extract, if validated for human studies, may offer an adjunctive therapy for diabetes.     

Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region

UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F⁶¹⁹ in the motif stacks partly over cis-Pro⁶²⁰ in one conformation, whereas it is swung out from cis-P⁶²⁰, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCP-binding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP.     

Spatial complexity of mechanisms controlling a bacterial cell cycle

Cell cycle progression in Caulobacter is governed by a multilayered regulatory network linking chromosome replication with polar morphogenesis and cell division. Temporal and spatial regulation have emerged as the central themes, with the abundance, activity and subcellular location of key structural and regulatory proteins changing over the course of the cell cycle. An additional layer of complexity was recently uncovered, showing that each segment of the chromosome is located at a specific cellular position both during and after the completion of DNA replication, raising the possibility that this positioning contributes to temporal and spatial control of gene expression.     

Signal acquisition and analysis for cortical control of neuroprosthetics

Work in cortically controlled neuroprosthetic systems has concentrated on decoding natural behaviors from neural activity, with the idea that if the behavior could be fully decoded it could be duplicated using an artificial system. Initial estimates from this approach suggested that a high-fidelity signal comprised of many hundreds of neurons would be required to control a neuroprosthetic system successfully. However, recent studies are showing hints that these systems can be controlled effectively using only a few tens of neurons. Attempting to decode the pre-existing relationship between neural activity and natural behavior is not nearly as important as choosing a decoding scheme that can be more readily deployed and trained to generate the desired actions of the artificial system. These artificial systems need not resemble or behave similarly to any natural biological system. Effective matching of discrete and continuous neural command signals to appropriately configured device functions will enable effective control of both natural and abstract artificial systems using compatible thought processes.     

Oxygen and cytokine-dependent changes in choline phospholipid saturation in hematopoietic progenitor cells detected by MALDI-TOF mass spectrometry

The adaptation of cells to a changing environment is normally accompanied by rapid and/or chronic remodeling of membrane lipids. In order to understand the role played by membrane lipid metabolism in such responses, it is necessary to characterize in more detail the changes in membrane composition occurring in response to defined stimuli. There has been intense interest in characterizing the “stem cell niche” in recent years and an emerging consensus that stem cells are located in regions of low oxygen tension and probably well-isolated from the blood supply.We report here the use of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry to monitor changes in the composition and saturation degree of choline phospholipids of hematopoietic progenitor (FDCPmix) cells under standard nutrient-rich culture conditions and at low oxygen and low glucose concentrations. We found that the increase in proliferation rate driven by high concentrations of interleukin-3 (IL-3) is associated with a decrease in membrane phosphatidylcholine (PC) 18:0/20:4 and sphingomyelin (SM) together with an increase in PC 18:0/18:2 and dihydro SM. Furthermore, this effect is most pronounced under low oxygen and low glucose conditions, independent of cell proliferation rates.     

Migration of connective tissue-derived cells is mediated by ultra-low concentration gradient fields of EGF

The directed migration of cells towards chemical stimuli incorporates simultaneous changes in both the concentration of a chemotactic agent and its concentration gradient, each of which may influence cell migratory response. In this study, we utilized a microfluidic system to examine the interactions between epidermal growth factor (EGF) concentration and EGF gradient in stimulating the chemotaxis of connective tissue-derived fibroblast cells. Cells seeded within microfluidic devices were exposed to concentration gradients established by EGF concentrations that matched or exceeded those required for maximum chemotactic responses seen in transfilter migration assays. The migration of individual cells within the device was measured optically after steady-state gradients had been experimentally established. Results illustrate that motility was maximal at EGF concentration gradients between .01- and 0.1-ng/(mL.mm) for all concentrations used. In contrast, the number of motile cells continually increased with increasing gradient steepness for all concentrations examined. Microfluidics-based experiments exposed cells to minute changes in EGF concentration and gradient that were in line with the acute EGFR phosphorylation measured. Correlation of experimental data with established mathematical models illustrated that the fibroblasts studied exhibit an unreported chemosensitivity to minute changes in EGF concentration, similar to that reported for highly motile cells, such as macrophages. Our results demonstrate that shallow chemotactic gradients, while previously unexplored, are necessary to induce the rate of directed cellular migration and the number of motile cells in the connective tissue-derived cells examined.     

Human CD8+ T cells display a differential ability to undergo cytokine-driven bystander activation

A subset of CD44^hiCD8⁺ T cells in some, but not all mice, can be induced to rapidly secrete IFNγ during infection with Listeria monocytogenes. This response is dependent on the presence of both IL-12 and IL-18 and does not require engagement of the T cell receptor. In this study, we demonstrate that human CD8⁺ T cells also vary widely in their ability to secrete IFNγ within 15 h of either Listeria infection or cytokine stimulation. The magnitude of the rapid IFNγ response correlated more closely with the intrinsic responsiveness of the T cells to cytokine stimulation rather than the amount of IL-12 produced. CD8⁺ T cells from 2 out of 16 blood donors (12.5%) failed to generate a significant IFNγ response. These results demonstrate that bystander activation of CD8⁺ T cells varies among individuals and validate further study of the differential responses observed using BALB/c vs. C57BL/6 mice.     

Circling,deafness, and yellow coat displayed by yellow submarine (ysb) and light coat and circling (lcc) mice with mutations on chromosome 3

We describe here two mouse mutants, yellow submarine (Ysb) and light coat and circling (Lcc). Ysb arose as the result of insertions of a transgene, pAA2, into the genome. Lcc is an independent, radiation-induced mutation. Both mutants are characterized by recessive circling behavior and deafness, associated with a non-segregating, semi-dominant yellow coat color. Complementation tests showed that Ysb and Lcc are allelic. We attribute the yellow coat in Ysb and Lcc mice to the absence of black awl overhairs, increased agouti zigzag underhairs, and the presence of agouti awls with long subapical yellow pigment. Chromosomal mapping and genomic characterization showed the Ysb and Lcc mutations involve complex chromosomal rearrangements in overlapping regions of mouse chromosome 3, A2/A3-B/C and B-E1, respectively. Ysb and Lcc show for the first time, to our knowledge, the presence of genes in the B-C region of chromosome 3 important for balance and hearing and the pigmentation and specification of coat hair.     

Characterization of a Mouse Gene (Fbxw6) That Encodes a Homologue of Caenorhabditis elegans SEL-10

The SCF complex is a type of ubiquitin ligase that consists of the invariable components SKP1, CUL1, and RBX1 as well as a variable component, known as an F-box protein, that is the main determinant of substrate specificity. The Caenorhabditis elegans F-box- and WD40-repeat-containing protein SEL-10 functionally and physically associates with LIN-12 and SEL-12, orthologues of mammalian Notch and presenilin, respectively. We have now identified a gene (which we call Fbxw6) that encodes a mouse homologue (F-box–WD40 repeat protein 6, or FBW6) of SEL-10 and is expressed mainly in brain, heart, and testis. Co-immunoprecipitation analysis showed that FBW6 interacts with SKP1 and CUL1, indicating that these three proteins form an SCF complex. Comparison of the genomic organization of Fbxw6, which is located on mouse chromosome 3.3E3, with that of mouse Fbxw1, Fbxw2, and Fbxw4 showed only a low level of similarity, indicating that these genes diverged relatively early and thereafter evolved independently.