Requirement of GM2 ganglioside activator for phospholipase D activation

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by β-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792–815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.

Crystal structures of eukaryotic translation initiation factor 5A from Methanococcus jannaschii at 1.8 Å resolution

Eukaryotic translation initiation factor 5A (eIF-5A) is a ubiquitous protein found in all eukaryotic cells. The protein is closely associated with cell proliferation in the G1–S stage of the cell cycle. Recent findings show that the eIF-5A proteins are highly expressed in tumor cells and act as a cofactor of the Rev protein in HIV-1-infected cells. The mature eIF is the only protein known to have the unusual amino acid hypusine, a post-translationally modified lysine. The crystal structure of eIF-5A from Methanococcus jannaschii (MJ eIF-5A) has been determined at 1.9 Å and 1.8 Å resolution in two crystal forms by using the multiple isomorphous replacement method and the multiwavelength anomalous diffraction method for the first crystal form and the molecular replacement method for the second crystal form. The structure consists of two folding domains, one of which is similar to the oligonucleotide-binding domain found in the prokaryotic cold shock protein and the translation initiation factor IF1 despite the absence of any significant sequence similarities. The 12 highly conserved amino acid residues found among eIF-5As include the hypusine site and form a long protruding loop at one end of the elongated molecule.

A genomic approach to gene fusion technology

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

Rates of nucleotide substitution in sexual and anciently asexual rotifers

The class Bdelloidea of the phylum Rotifera is the largest well studied eukaryotic taxon in which males and meiosis are unknown, and the only one for which these indications of ancient asexuality are supported by cytological and molecular genetic evidence. We estimated the rates of synonymous and nonsynonymous substitutions in the hsp82 heat shock gene in bdelloids and in facultatively sexual rotifers of the class Monogononta, employing distance based and maximum likelihood methods. Relative-rate tests, using acanthocephalan rotifers as an outgroup, showed slightly higher rates of nonsynonymous substitution and slightly lower rates of synonymous substitution in bdelloids as compared with monogononts. The opposite trend, however, was seen in intraclass pairwise comparisons. If, as it seems, bdelloids have evolved asexually, an equality of bdelloid and monogonont substitution rates would suggest that the maintenance of sexual reproduction in monogononts is not attributable to an effect of sexual reproduction in limiting the load of deleterious nucleotide substitutions.

The extreme C terminus of progesterone receptor contains a transcriptional repressor domain that functions through a putative corepressor.

Binding of a hormone agonist to a steroid receptor leads to the dissociation of heat shock proteins, dimerization, specific DNA binding, and target gene activation. Although the progesterone antagonist RU486 can induce most of these events, it fails to activate human progesterone receptor (hPR)-dependent transcription. We have previously demonstrated that a conformational change is a key event leading to receptor activation. The major conformational distinction between hormone- and antihormone-bound receptors occurs within the C-terminal portion of the molecule. Furthermore, hPR mutants lacking the C terminus become transcriptionally active in the presence of RU486. These results suggest that the C terminus contains a repressor domain that inhibits the transcriptional activity of the RU486-bound hPR. In this study, we have defined a 12 amino acid (12AA) region in the C terminus of hPR that is necessary and sufficient for the repressor function when fused to the C-terminal truncated hPR or to the GAL4 DNA-binding domain. Mutations in the 12AA domain (aa 917-928) generate an hPR that is active in the presence of RU486. Furthermore, overexpression of the 12AA peptide activates the RU486-bound wild-type hPR without affecting progesterone-dependent activation. These results suggest that association of the 12AA repressor region with a corepressor might inactivate hPR activity when it is bound to RU486. We propose that binding of a hormone agonist to the receptor changes its conformation in the ligand-binding domain so that association with coactivator is promoted and activation of target gene occurs.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery.

Translocation of PKN from the cytosol to the nucleus induced by stresses.

Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.

Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.

The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.

Endophytic colonization of barley (Hordeum vulgare) roots by the nematophagous fungus Pochonia chlamydosporia reveals plant growth promotion and a general defense and stress transcriptomic response

Plant crop yields are negatively conditioned by a large set of biotic and abiotic factors. An alternative to mitigate these adverse effects is the use of fungal biological control agents and endophytes. The egg-parasitic fungus Pochonia chlamydosporia has been traditionally studied because of its potential as a biological control agent of plant-parasitic nematodes. This fungus can also act as an endophyte in monocot and dicot plants, and has been shown to promote plant growth in different agronomic crops. An Affymetrix 22K Barley GeneChip was used in this work to analyze the barley root transcriptomic response to P. chlamydosporia root colonization. Functional gene ontology (GO) and gene set enrichment analyses showed that genes involved in stress response were enriched in the barley transcriptome under endophytism. An 87.5 % of the probesets identified within the abiotic stress response group encoded heat shock proteins. Additionally, we found in our transcriptomic analysis an up-regulation of genes implicated in the biosynthesis of plant hormones, such as auxin, ethylene and jasmonic acid. Along with these, we detected induction of brassinosteroid insensitive 1-associated receptor kinase 1 (BR1) and other genes related to effector-triggered immunity (ETI) and pattern-triggered immunity (PTI). Our study supports at the molecular level the growth-promoting effect observed in plants endophytically colonized by P. chlamydosporia, which opens the door to further studies addressing the capacity of this fungus to mitigate the negative effects of biotic and abiotic factors on plant crops.

Effects of ocean warming and acidification on the early stages of marine fishes

Tese de doutoramento, Ciências do Mar, Universidade de Lisboa, Faculdade de Ciências, 2016

Surdez induzida pelo trauma acústico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Compression Loading Induced Cellular Stress Response of Intervertebral Disc Cells in Organ Culture

Introduction: Mechanical stress is often associated to interverterbal disc (IVD) degeneration and the effect of mechanical loading on IVD has been studied and reviewed.1,2 Previously, expression of heat shock proteins, HSP70 and HSP27 has been found in pathological discs.3 However, there is no direct evidence on whether IVD cells respond to the mechanical loading by expression of HSPs. The objective of this study is to investigate the stress response of IVD cells during compressive loading in an organ culture. Materials and Methods: Fresh adult bovine caudal discs were cultured with compressive loading applied at physiological range. Effect of loading type (static and dynamic) and repeated loading (2 hours per day for 2 days) were studied. Nucleus pulposus (NP) and annulus fibrosus (AF) of the IVD were retrieved at different time points: right after loading and right after resting. Positive control discs were heat shocked (43°C). Cell activity was assessed and expression of stress response genes (HSP70 and HSF1) and matrix remodeling genes (ACAN, COL2, COL1, ADAMTS4, MMP3 and MMP13) were studied. Results: Cell activity was maintained in all groups. Both NP and AF expressed high level of HSP70 in heat shock groups, confirming their expression in response to stress. In NP, expression of HSP70 was up-regulated after static loading and dynamic loading with higher fold change was observed after static loading. During repeated loading, HSP70 appeared to be upregulated right after loading and decreased after resting. Such trend was not observed in AF and HSF1 levels. Expressions of matrix remodeling genes did not change significantly with loading except ADAMTS4 decreased in AF during static loading. Conclusion: This study demonstrated that NP cells upregulate expression of HSP70 in response to loading induced stress without changing cell activity and matrix remodeling significantly. Acknowledgments: This project was funded by AO Spine (AOSPN) (grant number: SRN_2011_14) and a fellowship exchange award by AO Spine Scientific Research Network (SRN).

Improved germination of the Australian natives: Hibbertia commutata, Hibbertia amplexicaulis (Dilleniaceae), Chameascilla corymbosa (Liliaceae) and Leucopogon nutans (Epacridaceae)

Hibbertia commutata (Steudel), H. amplexicaulis (Steudel), Chameascilla corymbosa [(R.Br.) F.Muell. Ex Benth.] and Leucopogon nutans (E. Pritzel) are four Australian species that are difficult to germinate during mine-site rehabilitation. Laboratory germination trails were conducted to identify dormancy mechanisms and to improve germination response. Treatments applied to all species included scarification and scarification followed by soaking seeds in smoke water (1, 5 or 10%) or gibberellic-acid solution (50, 200 or 1000 muM). Additional treatments with kinetin solution (50, 200 or 1000 muM) and smoke water (50 or 100%) were applied to scarified or unscarified seeds of C. corymbosa. Thermal-shock treatment was applied to L. nutans fruit, some of which were subsequently scarified and subjected to both smoke water (10%) and gibberellic-acid solution (1000 muM). Significant germination increases were obtained by using dormancy-breaking treatments on H. commutata ( from 12.8 to 76.0%), H. amplexicaulis (from 6.8 to 55.1%) and C. corymbosa (from 48.5 to 86.4%). Scarification alone increased germination of both Hibbertia species, suggesting that these species display a physical seed coat-imposed dormancy mechanism. Germination of H. amplexicaulis was further increased by the application of gibberellic-acid solution, indicating a possible embryo-imposed dormancy mechanism. Scarification followed by the application of smoke water produced the highest germination response for C. corymbosa seeds. Scarification alone did not significantly increase germination, inferring the existence of a smoke-responsive embryo dormancy mechanism. Seeds of L. nutans, although viable, failed to germinate and are thought to display complex seed coat- and embryo-imposed dormancy mechanisms.

Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.