Discovery of antibacterial cyclic peptides that inhibit the ClpXP protease

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA-tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.     

Lack of S-Adenosylmethionine Results in a Cell Division Defect in Escherichia coli

The enzyme S-adenosylmethionine (SAM) synthetase, the Escherichia coli metK gene product, produces SAM, the cell’s major methyl donor. We show here that SAM synthetase activity is induced by leucine and repressed by Lrp, the leucine-responsive regulatory protein. When SAM synthetase activity falls below a certain critical threshold, the cells produce long filaments with regularly distributed nucleoids. Expression of a plasmid-carried metK gene prevents filamentation and restores normal growth to the metK mutant. This indicates that lack of SAM results in a division defect.     

Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.     

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.     

Role of IKK/NF-κB signaling in extinction of conditioned place aversion memory in rats

The inhibitor κB protein kinase/nuclear factor κB (IKK/NF-κB) signaling pathway is critical for synaptic plasticity. However, the role of IKK/NF-κB in drug withdrawal-associated conditioned place aversion (CPA) memory is unknown. Here, we showed that inhibition of IKK/NF-κB by sulphasalazine (SSZ; 10 mM, i.c.v.) selectively blocked the extinction but not acquisition or expression of morphine-induced CPA in rats. The blockade of CPA extinction induced by SSZ was abolished by sodium butyrate, an inhibitor of histone deacetylase. Thus, the IKK/NF-κB signaling pathway might play a critical role in the extinction of morphine-induced CPA in rats and might be a potential pharmacotherapy target for opiate addiction.     

Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus

MicroRNAs (miRNAs) regulate the abundance of target mRNAs by guiding cleavage at sequence complementary regions. In this study, artificial miRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV) were constructed using Arabidopsis pre-miRNA159a as the backbone. The constructs included six single amiRNAs targeting motifs A, B1, B2, C, D of E, and two triple amiRNAs targeting motifs AB1E or B2DC. Processing of pre-amiRNAs was confirmed by agro-infiltration, and transgenic Nicotiana benthamiana plants expressing each amiRNA were generated. Single amiRNA transgenic lines expressing amiR-LB2 or amiR-LD showed resistance to WSMoV by delaying symptom development. Triple amiRNA lines expressing amiR-LB2, amiR-LD and amiR-LC provided complete resistance against WSMoV, with no indication of infection 28 days after inoculation. Resistance levels were positively correlated with amiRNA expression levels in these single and triple amiRNA lines. The triple amiR-LAB1E line did not provide resistance to WSMoV. Similarly, the poorly expressed amiR-LC and amiR-LE lines did not provide resistance to WSMoV. The amiR-LA- and amiR-LB1-expressing lines were susceptible to WSMoV, and their additional susceptibility to the heterologous Turnip mosaic virus harbouring individual target sequences indicated that these two amiRNAs have no effect in vivo. Transgenic lines expressing amiR-LB2 exhibited delayed symptoms after challenge with Peanut bud necrosis virus having a single mismatch in the target site. Overall, our results indicate that two amiRNAs, amiR-LB2 and amiR-LD, of the six designed amiRNAs confer moderate resistance against WSMoV, and the triple construct including the two amiRNAs provides complete resistance.     

Time-dependent cross ratio estimation for bivariate failure times

In the analysis of bivariate correlated failure time data, it is important to measure the strength of association among the correlated failure times. One commonly used measure is the cross ratio. Motivated by Cox's partial likelihood idea, we propose a novel parametric cross ratio estimator that is a flexible continuous function of both components of the bivariate survival times. We show that the proposed estimator is consistent and asymptotically normal. Its finite sample performance is examined using simulation studies, and it is applied to the Australian twin data.     

Wolbachia in field populations of Forcipomyia taiwana (Diptera: Ceratopogonidae) in Taiwan

Wolbachia pipientis is an endosymbiotic alpha-proteobacterium that is found in numerous insects and arthropods. Only a few studies have been made on Wolbachia in blood-sucking midges. In this study, we identified and determined the molecular characteristics of Wolbachia strains in Forcipomyia taiwana (Shiraki), a blood-sucking midge found in Taiwan and southern China. Our results indicate that all F. taiwana individuals captured in Nantou County, Taiwan were infected with Wolbachia strains closely related to the A-supergroup wAlbA. Moreover, 2 individuals captured at low-abundance locations were also infected with a B-supergroup Wolbachia strain. We observed that 63% F. taiwana individuals captured at the low-abundance locations harbored the wAlbA-like strain which has a particular substitution pattern (D51G, T84A, and A85V) in the Wolbachia surface protein. Taken together, our results indicate that distinct Wolbachia strains exist in F. taiwana populations in the field.     

Invasion of the African sharp-tooth catfish <Emphasis Type=Italic>Clarias gariepinus</Emphasis> (Burchell, 1822) in South China

We record here the invasion of the African sharp-tooth catfish Clarias gariepinus in the South China biodiversity hotspot, an area rich in endemic and endangered fish fauna. C. gariepinus was introduced into the natural systems as escapees from aquaculture ponds. These catfishes are very large, top predators, and thus have the potential to cause serious threats to the native fish fauna. The impact of C. gariepinus needs more investigation with emphasis on developing techniques for controlling its dispersal.