Development of a multiplex PCR assay based on the pilA gene sequences to detect different types of Acidovorax citrulli.

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax citrulli, is a major threat to commercial watermelon and melon production worldwide. At present, there are at least two genetically distinct sub-populations (group I and II) of A. citrulli that differ in host preference among cucurbit species and copper sensitivity. In this study, we analyzed the pilA gene sequences of 103 A. citrulli strains from China and other countries. Based on these data, we classified all tested A. citrulli strains into three types. The pilA-based type 1 strains in this study coincided with the previously established group I strains; while the type 2 strains coincided with group II strains. Ten strains that did not cluster with group I or II strains were classified into a new type, designated type 3. Based on differences in pilA sequences, we designed a multiplex PCR assay to distinguish the three A. citrulli pilus types. This multiplex PCR assay has proven to be viable for strain typing of 139 A. citrulli strains and for the detection of this pathogen in artificially inoculated seeds and leaves and naturally infected leaves and fruits. This assay proved to be rapid, accurate, reliable and applicable for early distinction of A. citrulli types associated with BFB epidemics. It may also inform the judicious and environmentally sound use of bactericides, especially copper-based compounds.

Targeting enhancer of zeste homolog 2 protects against acute kidney injury.

Despite the established oncogenic and profibrotic functions of enhancer of zeste homolog 2 (EZH2), a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3), its role in acute kidney injury (AKI) remains unclear. In this study, we demonstrated that EZH2 and H3K27me3 were upregulated in the murine kidney with AKI induced by either ischemia-reperfusion (I/R) or folic acid (FA). Pharmacologic inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) prevented tubular injury in both models as demonstrated by reduced renal dysfunction, diminished neutrophil gelatinase-associated lipocalin expression and decreased renal tubular cell death. Injury to the kidney resulted in reduced expression of E-cadherin and ZO-1, whereas EZH2 inhibition largely preserved their expression. Moreover, 3-DZNep was effective in counteracting the increased expression of matrix metalloproteinase (MMP)-2 and MMP-9, as well as the phosphorylation of Raf-1 and ERK1/2 in the injured kidney. Conversely, blocking EZH2 reversed the decrease of tissue inhibitor of metalloproteinase (TIMP)-2 and metalloproteinase (TIMP)-3, and Raf kinase inhibitor protein (RKIP) in the kidney after acute injury. Similarly, oxidant injury to cultured kidney proximal tubular epithelial cells caused a decrease in the expression of E-cadherin, ZO-1, TIMP-2/-3, and RKIP, as well as an increase in the expression of MMP-2/9 and phosphorylation of Raf-1 ERK1/2. Blocking EZH2 with 3-DZNep or SiRNA hindered these responses. Thus, these results suggest that targeting EZH2 protects against AKI through a mechanism associated with the preservation of adhesion/junctions, reduction of matrix metalloproteinases and attenuation of the Raf-1/ERK1/2 pathway.

HE4 suppresses the expression of osteopontin in mononuclear cells and compromises their cytotoxicity against ovarian cancer cells.

Ovarian cancers are known to evade immunosurveillance and to orchestrate a suppressive immune microenvironment. Here we examine the role of human epididymis protein 4 (HE4), an ovarian cancer biomarker, in immune evasion. Through modified subtractive hybridization analyses we have characterized the gene targets of HE4 in human peripheral blood mononuclear cells (PBMCs), and established a preliminary mechanism for HE4-mediated immune failure in ovarian tumours. Upon exposure of purified PMBCs to HE4, osteopontin (OPN) and dual-specificity phosphatase 6 (DUSP6) emerged as the most suppressed and up-regulated genes, respectively. SKOV3 and OVCAR8, human ovarian carcinoma cell lines, exhibited enhanced proliferation in conditioned media from HE4-exposed PBMCs, an effect that was attenuated by the addition of recombinant OPN or OPN-inducible cytokines [interleukin (IL)-12 and interferon (IFN)-Ɣ]. Additionally, upon co-culture with PBMCs, HE4-silenced SKOV3 cells were found to be more susceptible to cytotoxic cell death. The relationship between HE4 and OPN was reinforced further through the analysis of serous ovarian cancer patient samples. In these biopsy specimens, the number of OPN+ T cells correlated positively with progression free survival (PFS) and inversely with serum HE4 level. Taken together, these findings show that HE4 enhances ovarian cancer tumorigenesis by compromising OPN-mediated T cell activation.

Low expression of Abelson interactor-1 is linked to acquired drug resistance in Bcr-Abl-induced leukemia.

The basis for persistence of leukemic stem cells in the bone marrow microenvironment remains poorly understood. We present evidence that signaling cross-talk between α4 integrin and Abelson interactor-1 (Abi-1) is involved in the acquisition of an anchorage-dependent phenotype and drug resistance in Bcr-Abl-positive leukemia cells. Comparison of Abi-1 (ABI-1) and α4 integrin (ITGA4) gene expression in relapsing Bcr-Abl-positive CD34+progenitor cells demonstrated a reduction in Abi-1 and an increase in α4 integrin mRNA in the absence of Bcr-Abl mutations. This inverse correlation between Abi-1 and α4 integrin expression, as well as linkage to elevated phospho-Akt and phospho-Erk signaling, was confirmed in imatinib mesylate -resistant leukemic cells. These results indicate that the α4-Abi-1 signaling pathway may mediate acquisition of the drug-resistant phenotype of leukemic cells.

Mass measurements of the neutron-deficient 41Ti, 45Cr, 49Fe, and 53Ni nuclides: first test of the isobaric multiplet mass equation in f p-shell nuclei.

Isochronous mass spectrometry has been applied to neutron-deficient 58Ni projectile fragments at the HIRFL-CSR facility in Lanzhou, China. Masses of a series of short-lived T(z)=-3/2 nuclides including 41Ti, 45Cr, 49Fe, and 53Ni have been measured with a precision of 20-40 keV. The new data enable us to test for the first time the isobaric multiplet mass equation (IMME) in fp-shell nuclei. We observe that the IMME is inconsistent with the generally accepted quadratic form for the A=53, T=3/2 quartet. We perform full space shell model calculations and compare them with the new experimental results.

Direct mass measurements of short-lived A=2Z-1 nuclides (63)Ge, (65)As, (67)Se, and (71)Kr and their impact on nucleosynthesis in the rp process.

Mass excesses of short-lived A=2Z-1 nuclei (63)Ge, (65)As, (67)Se, and (71)Kr have been directly measured to be -46,921(37), -46,937(85), -46,580(67), and -46,320(141) keV, respectively. The deduced proton separation energy of -90(85) keV for (65)As shows that this nucleus is only slightly proton unbound. X-ray burst model calculations with the new mass excess of (65)As suggest that the majority of the reaction flow passes through (64)Ge via proton capture, indicating that (64)Ge is not a significant rp-process waiting point.

Targeted deletion of NF-kappaB p50 diminishes the cardioprotection of histone deacetylase inhibition.

We have recently demonstrated that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia-reperfusion (I/R) injury. The mechanism by which HDAC inhibition confers myocardial protection remains unknown. The purpose of this study is to investigate whether the disruption of NF-kappaB p50 would eliminate the protective effects of HDAC inhibition. Wild-type and NF-kappaB p50-deficient mice were treated with trichostatin A (TSA; 0.1 mg/kg ip), a potent inhibitor of HDACs. Twenty-four hours later, the hearts were perfused in Langendorff model and subjected to 30 min of ischemia and 30 min of reperfusion. Inhibition of HDACs by TSA in wild-type mice produced marked improvements in left ventricular end-diastolic pressure, left ventricular rate pressure product, and the reduction of infarct size compared with non-TSA-treated group. TSA-induced cardioprotection in wild-type animals was absent with genetic deletion of NF-kappaB p50 subunit. Notably, Western blot displayed a significant increase in nuclear NF-kappaB p50 and the immunoprecipitation demonstrated a remarkable acetylation of NF-kappaB p50 at lysine residues following HDAC inhibition. EMSA exhibited a subsequent increase in NF-kappaB DNA binding activity. Luciferase assay demonstrated an activation of NF-kappaB by HDAC inhibition. The pretreatment of H9c2 cardiomyoblasts with TSA (50 nmol/l) decreased cell necrosis and increased in cell viability in simulated ischemia. The resistance of H9c2 cardiomyoblasts to simulated ischemia by HDAC inhibition was eliminated by genetic knockdown of NF-kappaB p50 with transfection of NF-kappaB p50 short interfering RNA but not scrambled short interfering RNA. These results suggest that NF-kappaB p50 acetylation and activation play a pivotal role in HDAC inhibition-induced cardioprotection.

p38 Triggers late preconditioning elicited by anisomycin in heart: involvement of NF-kappaB and iNOS.

We investigated the role of stress-activated p38 MAP kinase (p38/SAPK-2) signaling in delayed preconditioning of the heart. Adult male out-bred ICR mice were treated with p38 activator, anisomycin (0.1 mg/kg IP), or vehicle (5% DMSO). Twenty-four hours later, hearts were perfused in Langendorff mode and subjected to 30 minutes of ischemia and 30 minutes of reperfusion. Improvement in postischemic recovery of end-diastolic pressure and reduction in infarct size was observed, which was abolished by SB203580, a specific p38 inhibitor, and pyrrolidinediethyldithiocarbamate (PDTC), the NF-kappaB inhibitor, but not by PD 98059, a specific inhibitor for MEK1 or 2. Transient increase in p38 phosphorylation was observed 15 minutes after anisomycin treatment which subsided by 30 minutes. Electrophoretic mobility shift assay demonstrated rapid activation of NF-kappaB DNA binding with anisomycin, peaking at 30 minutes. Western blot confirmed the accumulation of p50 and p65 in nuclear extracts after anisomycin treatment. Anisomycin-induced NF-kappaB DNA binding activity was inhibited by SB203580 and PDTC. Expression of inducible nitric oxide synthase (iNOS) mRNA, protein, and nitric oxide (NO) synthesis were enhanced in anisomycin-treated mice. SB203580 and PDTC blocked the increased expression of iNOS and increase in synthesis of NO. Selective iNOS inhibitor S-methylisothiourea abolished the protective effect of anisomycin. Furthermore, postischemic cardioprotective effect of anisomycin was absent in mice with targeted ablation of iNOS gene but not in the wild-type B6.129 mice. For the first time, these results suggest that direct pharmacological activation of p38 triggers delayed preconditioning by signaling mechanism involving NF-kappaB activation and synthesis of NO from iNOS.

Adenosine-induced late preconditioning in mouse hearts: role of p38 MAP kinase and mitochondrial K(ATP) channels.

We investigated the role of p38 mitogen-activated protein kinase (MAPK) phosphorylation and opening of the mitochondrial ATP-sensitive K(+) [(K(ATP))(mito)] channel in the adenosine A(1) receptor (A(1)AR)-induced delayed cardioprotective effect in the mouse heart. Adult male mice were treated with vehicle (5% DMSO) or the A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.1 mg/kg ip). Twenty-four hours later, hearts were subjected to 30 min of global ischemia and 30 min of reperfusion in the Langendorff mode. Genistein or SB-203580 (1 mg/kg i.p.) given 30 min before CCPA treatment was used to block receptor tyrosine kinase or p38 MAPK phosphorylation, respectively. 5-Hydroxydecanoate (5-HD; 200 microM) was used to block (K(ATP))(mito) channels. CCPA produced marked improvement in left ventricular function, which was partially blocked by SB-203580 and 5-HD and completely abolished with genistein. CCPA caused a reduction in infarct size (12.0 +/- 2.0 vs. 30.3 +/- 3.0% in vehicle), which was blocked by genistein (29.4 +/- 2.3%), SB-203580 (28.3 +/- 2.6%), and 5-HD (33.9 +/- 2.4%). CCPA treatment also caused increased phosphorylation of p38 MAPK during ischemia, which was blocked by genistein, SB-203580, and 5-HD. The results suggest that A(1)AR-triggered delayed cardioprotection is mediated by p38 MAPK phosphorylation. Blockade of cardioprotection with 5-HD concomitant with decrease in p38 MAPK phosphorylation suggests a potential role of (K(ATP))(mito) channel opening in phosphorylation and ensuing the late preconditioning effect of A(1)AR.

[Effects of amiloride and glycogen depletion on post-ischemic reperfusion injury of isovolumic perfused rat hearts].

The effects of amiloride (0.5 nmol/L) and glycogen depletion were studied to find out the possible roles of Na(+)-H+ exchange of ischemia reperfusion injury in the isovolumic perfused isolated rat hearts. The data indicated that, compared to control group, both amiloride and glycogen depletion accelerated recovery of hemodynamics and reduced leakage of LDH and the formation of myocardial MDA which was accompared by a higher activity of mitochondrial GSH-Px and lesser accumulation of intracellular Na+, Ca2+. That Na(+)-H+ and Na(+)-Ca2+ exchanges might play a critical role in post-ischemic reperfusion injury is discussed.

[The relationship between Na overload and Na/H exchange--a study in the isolated isovolumic rat hearts].

Thirty min after stabilization perfusion with oxygenated buffer, hearts were divided in four groups: (1) CONTROL GROUP: 75 min. of aerobic perfusion; (2) Low flow anoxia group: 45 min. of low flow anoxic perfusion (95% N2:5%CO2, 0.15 ml/min.) followed by 30 min. of aerobic perfusion; (3) Ouabain group: protocol same as (2), except that ouabain (200 mumol/L) was added to anoxic perfusate during low flow anoxia; (4) Ouabain+Amiloride group: protocol same as (3) except that amiloride (0.5 mmol/I) was added to perfusate during low flow anoxia. Compared with the low flow anoxia group, ouabain resulted in an additional increase in Na during reperfusion accompanied with a depressed ventricular function. The deleterious effects of ouabain could be significantly combatted by amiloride. It is concluded that a decrease in Na/K ATPase activity may contribute to Na gain in reperfused myocardium, the mechanism of which might result from stimulation of Na/H exchange.