Showing papers by "UPRRP College of Natural Sciences published in 2007"

SIZ1-Mediated Sumoylation of ICE1 Controls CBF3/DREB1A Expression and Freezing Tolerance in Arabidopsis

Abstract: SIZ1 is a SUMO E3 ligase that facilitates conjugation of SUMO to protein substrates. siz1-2 and siz1-3 T-DNA insertion alleles that caused freezing and chilling sensitivities were complemented genetically by expressing SIZ1, indicating that the SIZ1 is a controller of low temperature adaptation in plants. Cold-induced expression of CBF/DREB1, particularly of CBF3/DREB1A, and of the regulon genes was repressed by siz1. siz1 did not affect expression of ICE1, which encodes a MYC transcription factor that is a controller of CBF3/DREB1A. A K393R substitution in ICE1 [ICE1(K393R)] blocked SIZ1-mediated sumoylation in vitro and in protoplasts identifying the K393 residue as the principal site of SUMO conjugation. SIZ1-dependent sumoylation of ICE1 in protoplasts was moderately induced by cold. Sumoylation of recombinant ICE1 reduced polyubiquitination of the protein in vitro. ICE1(K393R) expression in wild-type plants repressed cold-induced CBF3/DREB1A expression and increased freezing sensitivity. Furthermore, expression of ICE1(K393R) induced transcript accumulation of MYB15, which encodes a MYB transcription factor that is a negative regulator of CBF/DREB1. SIZ1-dependent sumoylation of ICE1 may activate and/or stabilize the protein, facilitating expression of CBF3/DREB1A and repression of MYB15, leading to low temperature tolerance.

Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia

Abstract: Bacteria that consume the greenhouse gas methane are potentially important players in the atmospheric budget, with the potential to sop up methane from the Earth's crust that would otherwise contribute to the atmospheric budget. Two new methane-utilizing bacteria have been isolated independently and both break new ground: unlike previous methanotrophic isolates, which are proteobacteria, they belong to the widely distributed Verrucomicrobia phylum. And both isolates display optimum growth and methane oxidation in remarkably acidic conditions, at pHs as low as 0.8 to 2.5. Acidimethylosilex fumarolicum SolV was isolated from a fuming vent on the Solfatara volcano near Naples, Italy, and Methylokorus infernorum from hot soil in the Hell's Gate (Tikitere) geothermal area of New Zealand. In spite of the fact that acidic environments support methane cycles, extreme acidophilic methanotrophs have so far resisted isolation. The isolation and initial genomic and physiological characterization of a bacterium belonging to the Verromicrobia displaying a growth and methane oxidation optimum of pH2—2.5 is described. Aerobic methanotrophic bacteria consume methane as it diffuses away from methanogenic zones of soil and sediment1. They act as a biofilter to reduce methane emissions to the atmosphere, and they are therefore targets in strategies to combat global climate change. No cultured methanotroph grows optimally below pH 5, but some environments with active methane cycles are very acidic2,3. Here we describe an extremely acidophilic methanotroph that grows optimally at pH 2.0–2.5. Unlike the known methanotrophs, it does not belong to the phylum Proteobacteria but rather to the Verrucomicrobia, a widespread and diverse bacterial phylum that primarily comprises uncultivated species with unknown genotypes. Analysis of its draft genome detected genes encoding particulate methane monooxygenase that were homologous to genes found in methanotrophic proteobacteria. However, known genetic modules for methanol and formaldehyde oxidation were incomplete or missing, suggesting that the bacterium uses some novel methylotrophic pathways. Phylogenetic analysis of its three pmoA genes (encoding a subunit of particulate methane monooxygenase) placed them into a distinct cluster from proteobacterial homologues. This indicates an ancient divergence of Verrucomicrobia and Proteobacteria methanotrophs rather than a recent horizontal gene transfer of methanotrophic ability. The findings show that methanotrophy in the Bacteria is more taxonomically, ecologically and genetically diverse than previously thought, and that previous studies have failed to assess the full diversity of methanotrophs in acidic environments.

Social network theory in the behavioural sciences: potential applications

Abstract: Social network theory has made major contributions to our understanding of human social organisation but has found relatively little application in the field of animal behaviour. In this review, we identify several broad research areas where the networks approach could greatly enhance our understanding of social patterns and processes in animals. The network theory provides a quantitative framework that can be used to characterise social structure both at the level of the individual and the population. These novel quantitative variables may provide a new tool in addressing key questions in behavioural ecology particularly in relation to the evolution of social organisation and the impact of social structure on evolutionary processes. For example, network measures could be used to compare social networks of different species or populations making full use of the comparative approach. However, the networks approach can in principle go beyond identifying structural patterns and also can help with the understanding of processes within animal populations such as disease transmission and information transfer. Finally, understanding the pattern of interactions in the network (i.e. who is connected to whom) can also shed some light on the evolution of behavioural strategies.

Nucleocytoplasmic shuttling of BZR1 mediated by phosphorylation is essential in Arabidopsis brassinosteroid signaling.

Abstract: Phytohormone brassinosteroids (BRs) play critical roles in plant growth and development. BR acts by modulating the phosphorylation status of two key transcriptional factors, BRI1 EMS SUPPRESSOR1 and BRASSINAZOLE RESISTANT1 (BZR1), through the action of BRASSINOSTEROID INSENSITIVE1/BRI1 ASSOCIATED RECEPTOR KINASE1 receptors and a GSK3 kinase, BRASSINOSTEROID INSENSITIVE2 (BIN2). It is still unknown how the perception of BR at the plasma membrane connects to the expression of BR target genes in the nucleus. We show here that BZR1 functions as a nucleocytoplasmic shuttling protein and GSK3-like kinases induce the nuclear export of BZR1 by modulating BZR1 interaction with the 14-3-3 proteins. BR-activated phosphatase mediates rapid nuclear localization of BZR1. Besides the phosphorylation domain for 14-3-3 binding, another phosphorylation domain in BZR1 is required for the BIN2-induced nuclear export of BZR1. Mutations of putative phosphorylation sites in two distinct domains enhance the nuclear retention of BZR1 and BR responses in transgenic plants. We propose that the spatial redistribution of BZR1 is critical for proper BR signaling in plant growth and development.

In Vivo Evidence of Pseudomonas aeruginosa Nutrient Acquisition and Pathogenesis in the Lungs of Cystic Fibrosis Patients

Abstract: One of the hallmarks of Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients is very-high-cell-density (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by the quorum-sensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection are largely unknown. Here, we performed microarray studies of P. aeruginosa directly isolated from the lungs of CF patients to demonstrate its metabolic capability and virulence in vivo. In vivo microarray data, confirmed by real-time reverse transcription-PCR, indicated that the P. aeruginosa population expressed several genes for virulence, drug resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The most abundant lung surfactant lipid molecule, phosphatidylcholine (PC), induces key genes of P. aeruginosa pertinent to PC degradation in vitro as well as in vivo within the lungs of CF patients. The results support recent research indicating that P. aeruginosa exists in the lungs of CF patients as a diverse population with full virulence potential. The data also indicate that there is deregulation of several pathways, suggesting that there is in vivo evolution by deregulation of a large portion of the transcriptome during chronic infection in CF patients. To our knowledge, this is the first in vivo transcriptome analysis of P. aeruginosa in a natural infection in CF patients, and the results indicate several important aspects of P. aeruginosa pathogenesis, drug resistance, nutrient utilization, and general metabolism within the lungs of CF patients.

T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses.

Abstract: T-DNA-tagged rice plants were screened under cold- or salt-stress conditions to determine the genes involved in the molecular mechanism for their abiotic-stress response. Line 0-165-65 was identified as a salt-responsive line. The gene responsible for this GUS-positive phenotype was revealed by inverse PCR as OsGSK1 (O ryza s ativa g lycogen s ynthase k inase3-like gene 1), a member of the plant GSK3/SHAGGY-like protein kinase genes and an orthologue of the Arabidopsis b rassinosteroid in sensitive 2 (BIN2), AtSK21. Northern blot analysis showed that OsGSK1 was most highly detected in the developing panicles, suggesting that its expression is developmental stage specific. Knockout (KO) mutants of OsGSK1 showed enhanced tolerance to cold, heat, salt, and drought stresses when compared with non-transgenic segregants (NT). Overexpression of the full-length OsGSK1 led to a stunted growth phenotype similar to the one observed with the gain-of-function BIN/AtSK21 mutant. This suggests that OsGSK1 might be a functional rice orthologue that serves as a negative regulator of brassinosteroid (BR)-signaling. Therefore, we propose that stress-responsive OsGSK1 may have physiological roles in stress signal-transduction pathways and floral developmental processes.

Amygdala depotentiation and fear extinction.

Abstract: Auditory fear memory is thought to be maintained by fear conditioning-induced potentiation of synaptic efficacy, which involves enhanced expression of surface AMPA receptor (AMPAR) at excitatory synapses in the lateral amygdala (LA). Depotentiation, reversal of conditioning-induced potentiation, has been proposed as a cellular mechanism for fear extinction; however, a direct link between depotentiation and extinction has not yet been tested. To address this issue, we applied both ex vivo and in vivo approaches to rats in which fear memory had been consolidated. A unique form of depotentiation reversed conditioning-induced potentiation at thalamic input synapses onto the LA (T-LA synapses) ex vivo. Extinction returned the enhanced T-LA synaptic efficacy observed in conditioned rats to baseline and occluded the depotentiation. Consistently, extinction reversed conditioning-induced enhancement of surface expression of AMPAR subunits in LA synaptosomal preparations. A GluR2-derived peptide that blocks regulated AMPAR endocytosis inhibited depotentiation, and microinjection of a cell-permeable form of the peptide into the LA attenuated extinction. Our results are consistent with the use of depotentiation to weaken potentiated synaptic inputs onto the LA during extinction and provide strong evidence that AMPAR removal at excitatory synapses in the LA underlies extinction.

Expression of DNA Methyltransferase 1 Is Activated by Hepatitis B Virus X Protein via a Regulatory Circuit Involving the p16INK4a-Cyclin D1-CDK 4/6-pRb-E2F1 Pathway

Abstract: DNA methyltransferase 1 (DNMT1) is responsible for copying DNA methylation patterns to the daughter strands during DNA replication Its expression is frequently up-regulated in human tumors, including hepatocellular carcinoma, but the mechanism of overexpression and its biological significance remain unclear Here, we show that hepatitis B virus X protein (HBx) activates DNMT1 expression via a regulatory circuit involving the p16(INK4a)-cyclin D1-cyclin-dependent kinase (CDK) 4/6-retinoblastoma protein (pRb)-E2F1 pathway HBx induced DNA hypermethylation of p16(INK4a) promoter to repress its expression, which subsequently led to activation of G1-CDKs, phosphorylation of pRb, activation of E2F1, and finally transcriptional activation of DNMT1 Inhibition of DNMT1 activity by either treatment with 5'-Aza-2'dC or introduction of DNMT1 small interfering RNA not only abolished the DNA methylation-mediated p16(INK4a) repression but also impaired DNMT1 expression itself, suggesting a cross-talk between DNMT1 and p16(INK4a) The up-regulation of cyclin D1 by HBx is likely to serve as an initiative impulse for the circuit because it was absolutely required for the activation of DNMT1 expression We also observed that accumulated DNMT1 via this pathway inactivates E-cadherin expression through promoter hypermethylation Considering that the pRb-E2F1 pathway is commonly activated in human tumors, activation of this circuit might be widespread and a potential therapeutic target

Prognostic Significance of Metastatic Lymph Node Ratio in Node-Positive Colon Carcinoma

Abstract: The aim of this study was to evaluate the prognostic significance of the lymph node ratio between metastatic and examined lymph nodes (LNR) in patients with stage III colon cancer. A review was made of 201 patients (106 men) with stage III colon cancer of R0 resection. Lymph node (LN) disease was stratified both by the American Joint Committee on Cancer and the International Union Against Cancer nodal staging system (pN) and by quartiles of the LNR. Survival curves were made by Kaplan-Meier analysis and assessed by the log rank test. Multivariate analysis was performed by the Cox proportional hazard model. Patients ranged in age from 22 to 82 (median, 59) years with median follow-up of 52 (range, 13–96) months. The LNR increased as a function of the number metastatic LNs (P < .0001; 95% confidence interval [95% CI], .7155–.8265). Cutoff points of LNR quartiles to be the best separating patients with regard to 5-year disease-free survival (DFS) were between quartile 1 and 2, and between 3 and 4 (pNr1, 2, and 3); the 5-year DFS according to such stratification was 83.6%, 61.1%, and 20% in pNr1, pNr2, and pNr3, respectively (P < .0001). The Cox model identified the pNr as the most statistically significant covariate: pNr2 was three times (95% CI, 1.407–6.280) and pNr3 eight times more risky than pNr1 (95% CI, 3.739–18.704). Ratio-based LN staging, which reflects the number of LNs examined and the quality of LN dissection, is a potent modality for prognostic stratification in patients with LN-positive colon cancer.

Extracellular gelatinase of Enterococcus faecalis destroys a defense system in insect hemolymph and human serum.

Abstract: We isolated Enterococcus faecalis from the body fluids of dead larvae of the greater wax moth, Galleria mellonella. Extracellular gelatinase (GelE) and serine protease (SprE), both of which are considered putative virulence factors of E. faecalis, were purified from the culture supernatant of E. faecalis. In an attempt to elucidate their virulence mechanisms, purified GelE and SprE were injected into hemolymph of G. mellonella and evaluated with regard to their effects on the immune system of insect hemolymph. As a result, it was determined that E. faecalis GelE degraded an inducible antimicrobial peptide (Gm cecropin) which is known to perform a critical role in host defense during the early phase of microbial infection. The results obtained from the G. mellonella-E. faecalis infection model compelled us to assess the virulence activity of GelE against the complement system in human serum. E. faecalis GelE hydrolyzed C3a and also mediated the degradation of the alpha chain of C3b, thereby inhibiting opsonization and the formation of the membrane attack complex resultant from the activation of the complement cascade triggered by C3 activation. In contrast, E. faecalis SprE exhibited no virulence effect against the immune system of insect hemolymph or human serum tested in this study.

Anoxia tolerance in turtles: metabolic regulation and gene expression.

Abstract: Freshwater turtles of the Trachemys and Chrysemys genera are champion facultative anaerobes able to survive for several months without oxygen during winter hibernation in cold water They have been widely used as models to identify and understand the molecular mechanisms of natural anoxia tolerance and the molecular basis of the hypoxic/ischemic injuries that occur in oxygen-sensitive systems and underlie medical problems such as heart attack and stroke Peter L Lutz spent much of his career investigating turtle anaerobiosis with a particular focus on the mechanisms of brain ion homeostasis and neurotransmitter responses to anoxia exposure and the mechanisms that suppress brain ion channel function and neuronal excitability during anaerobiosis Our interests intersected over the mechanisms of metabolic rate depression which is key to long term anoxia survival Studies in my lab have shown that a key mechanism of metabolic arrest is reversible protein phosphorylation which provides coordinated suppression of the rates of multiple ATP-producing, ATP-utilizing and related cellular processes to allow organisms to enter a stable hypometabolic state Anoxia tolerance is also supported by selective gene expression as revealed by recent studies using cDNA library and DNA array screening New studies with both adult T scripta elegans and hatchling C picta marginata have identified prominent groups of genes that are up-regulated under anoxia in turtle organs, in several cases suggesting aspects of cell function and metabolic regulation that have not previously been associated with anaerobiosis These groups of anoxia-responsive genes include mitochondrially-encoded subunits of electron transport chain proteins, iron storage proteins, antioxidant enzymes, serine protease inhibitors, transmembrane solute carriers, neurotransmitter receptors and transporters, and shock proteins

Curcumin attenuates the release of pro-inflammatory cytokines in lipopolysaccharide-stimulated BV2 microglia.

Abstract: Curcumin attenuates the release of pro-inflammatory cytokines in lipopolysaccharide-stimulated BV2 microglia

Susceptibility of various oral bacteria to antimicrobial peptides and to phagocytosis by neutrophils.

Abstract: Background and Objective: The aim of this study was to compare the susceptibility of nonperiodontopathic and periodontopathic bacteria to major defense mechanisms for bacterial clearance in gingival sulcus. Material and Methods: Twenty strains of 13 oral bacterial species were studied for their susceptibility to phagocytosis by human neutrophils and to the antimicrobial peptides LL-37 and human beta defensin-3. The minimum inhibitory concentrations of LL-37 and human beta defensin-3 were determined by a liquid dilution assay, and susceptibility to phagocytosis was examined by a flow cytometric phagocytosis assay. Results: The minimum inhibitory concentrations of LL-37 and human beta defensin-3 varied greatly, depending on the strain and species. Although a significant difference between the non- and periodontopathic groups was not observed, the red-complex bacteria were more resistant to LL-37 than the others (p = 0.004). The susceptibility of oral bacteria to phagocytosis was quite variable, depending on the species but not on the strains. The periodontopathic bacteria, especially Actinobacillus actinomycetemcomitans and the red-complex triad, were more resistant to phagocytosis than were the nonperiodontopathic bacteria (p = 0.0003). In addition, bacteria resistant both to antimicrobial peptides and to phagocytosis were more common in the periodontopathic group. Conclusion: Our results indicate that immune evasion may contribute to the pathogenicity of some periodontopathic bacteria.

Distinctive Signatures of Histone Methylation in Transcribed Coding and Noncoding Human β-Globin Sequences

Abstract: The establishment of epigenetic marks, such as methylation on histone tails, is mechanistically linked to RNA polymerase II within active genes. To explore the interplay between these modifications in transcribed noncoding as well as coding sequences, we analyzed epigenetic modification and chromatin structure at high resolution across 300 kb of human chromosome 11, including the β-globin locus which is extensively transcribed in intergenic regions. Monomethylated H3K4, K9, and K36 were broadly distributed, while hypermethylated forms appeared to different extents across the region in a manner reflecting transcriptional activity. The trimethylation of H3K4 and H3K9 correlated within the most highly transcribed sequences. The H3K36me3 mark was more broadly detected in transcribed coding and noncoding sequences, suggesting that K36me3 is a stable mark on sequences transcribed at any level. Most epigenetic and chromatin structural features did not undergo transitions at the presumed borders of the globin domain where the insulator factor CTCF interacts, raising questions about the function of the borders.