Reproductive Organ and Vascular Specific Promoter of the Rice Plasma Membrane Ca2+ATPase Mediates Environmental Stress Responses in Plants

Background

Plasma membrane Ca²⁺ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca²⁺) from the cell, hence regulating Ca²⁺ level within cells. Though plant Ca²⁺ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.

Results

The 1478 bp promoter sequence of rice plasma membrane Ca²⁺ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca²⁺ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The −1478 to −886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for −1210 and −886 bp flanking region. The −1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The −1210 and −886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the −886 bp and −519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.

Conclusions

The rice plasma membrane Ca²⁺ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.     

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.     

Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.     

Synthesis,conformational analysis and biological properties of a dicarba derivative of the antimicrobial peptide,brevinin-1BYa

Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the frog Rana boylii, displays broad-spectrum antimicrobial activity and potent haemolytic activity. This study investigates the effects on conformation and biological activity of replacement of the intramolecular disulphide bridge in the peptide by a non-reducible dicarba bond. Dicarba-brevinin-1BYa was prepared by microwave irradiation of [Agl¹⁸,Agl²⁴]-brevinin-1BYa (Agl = allylglycine) in the presence of a second generation Grubbs’ catalyst. Circular dichroism spectroscopy in 50% trifluoroethanol-water indicated that the degree of α-helicity of the dicarba derivative (22%) was less than that of brevinin-1BYa (27%) but comparable to that of the acyclic derivative [Ser¹⁸,Ser²⁴]-brevinin-1BYa (23%). Dicarba-brevinin-1BYa showed a two-fold increase in potency against reference strains of Escherichia coli, Staphylococcus aureus, and Candida albicans compared with the native peptide and displayed potent bactericidal activity against clinical isolates of methicillin-resistant S. aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MIC in the range 1–8 μM). Compared with brevinin-1BYa and [Ser¹⁸,Ser²⁴]-brevinin-1BYa, the dicarba derivative was associated with increased cytotoxicity against human erythrocytes (2.5-fold), MDA-MB-231 breast carcinoma cells (1.3-fold) and HepG2 hepatoma-derived cells (1.5-fold).     

New Delhi metallo-��-lactamase-1: local acquisition in Ontario, Canada, and challenges in detection

New Delhi metallo-β-lactamase-1 (NDM-1) is a recently identified metallo-β-lactamase that confers resistance to carbapenems and all other β-lactam antibiotics, with the exception of aztreonam. NDM-1 is also associated with resistance to many other classes of antibiotics. The enzyme was first identified in organisms isolated from a patient in Sweden who had previously received medical treatment in India, but it is now recognized as endemic throughout India and Pakistan and has spread worldwide. The gene encoding NDM-1 has been found predominantly in Escherichia coli and Klebsiella pneumoniae. We describe the isolation NDM-1–producing organisms from two patients in Toronto, Ontario. To the best of our knowledge, this is the first report of an organism producing NDM-1 that was locally acquired in Canada. We also discuss the evidence that NDM-1 can affect bacterial species other than E. coli and K. pneumoniae, the limited options for treatment and the difficulty laboratories face in detecting organisms that produce NDM-1.New Delhi metallo-β-lactamase-1 (NDM-1) is a metallo-β-lactamase that confers resistance to carbapenems and all other β-lactam antibiotics, with the exception of aztreonam. It is predominantly found in the Enterobacteriaeceae. It was first identified in Escherichia coli and Klebsiella pneumoniae isolated from a patient in Sweden who had previously received medical treatment in India. It is now recognized as endemic throughout India and Pakistan and has spread worldwide due to travel, “medical tourism” and the ability of the genetic element encoding the enzyme to transfer between bacteria.^1–3 Three reports of organisms producing NDM-1 in Canada have been published to date. In each instance, the organisms were isolated from the urinary tracts of patients who had recently been admitted to hospitals in India. Two of the isolates were strains of K. pneumoniae and one was a strain of E. coli.^4–6 Additional reports of isolation of organisms producing NDM-1 from patients in Canada have been presented in the lay press.Organisms that produce NDM-1 have been associated with resistance to classes of antibiotics other than the β-lactams, thus severely limiting options for treatment.² Infection control guidance regarding the management of colonization by or infection with organisms that produce carbapenemases, such as NDM-1, have recently been published by Canadian and European authorities.^7–9 An essential component of these recommendations is the rapid and accurate identification of the organisms in a clinical microbiology laboratory. The Clinical Laboratory Standards Institute (CLSI) and the United States Centers for Disease Control and Prevention (CDC) recommend screening for the production of carbapenemase using the Modified Hodge Test.^10,11 If the result of that test is positive, then the presence and type of carbapenemase can be confirmed by polymerase chain reaction.⁴Herein, we summarize two additional instances in which organisms producing NDM-1 were isolated from patients in Canada and the first where the organism appears to have been acquired in Canada.     

Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepacia in a model of cystic fibrosis

Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.     

Disuse-related decline in trabecular bone structure

Sedentary life style may degrade bone mass and microstructure resulting in osteoporosis. We characterized trabecular bone structural properties to determine if the LRP5 G171V mutation will protect against disuse-related bone loss. Forty-eight adult male mice representing three genotypes (WT = wild type, KO = LRP5-knockout +/−, HBM = High bone with the LRP5 G171V mutation) were each randomly divided between control and disuse (4 week hindlimb suspension) groups. Trabecular bone volume fraction (BV/TV) declined in all the three genotypes. Trabecular thickness was lower in the HBM and LRP5 (+/−) KO disuse groups when compared to their respective controls. While the remaining measures of bone structure (Trabecular number, connectivity density, apparent and tissue density) were lower, the trabecular separation increased in the LRP5 (+/−) with disuse. Although the absolute loss in BV/TV was similar, the relative loss due to disuse was far greater in the LRP5 (+/−) mice (67%) than in the HBM mice (14%). The disuse caused 20% decrease in trabecular number and thickness for LRP5 (+/−), while the decline was between 6 and 11% for the HBM and WT mice.     

Differential response of three cell types to dual stress of nitric oxide and radiation

The perception of toxicity to nitric oxide (NO) and irradiation (IR) by three different cell types has been studied. The three cell types are the macrophage like RAW264.7 cells, EL4 lymphoma cells, and splenocytes, which represent the different components of a tumor. These three cell types respond differently to NO donors (SNP and SNAP) and radiation treatment. The macrophages were found to be most radio-resistant and insensitive to NO donors. The innate resistance of the macrophages was not due to its antioxidant defense system since there was no significant activation of the enzymes (superoxide dismutases, catalase, and glutathione peroxidase) in RAW264.7 cells after NO donor and irradiation. But the cell cycle arrest of the three cell types was different from each other. The EL4 cells were found to arrest in the G2/M phase while the macrophages were found arrested in the G1 phase of the cell cycle. Such specific killing of the tumor cell in response to NO donor while sparing the macrophages can be of immense importance to radiotherapy.     

Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin

Human gene-2 (H2) relaxin is a member of the insulin-relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity.