The Puccinia species of Berkheya (Asteraceae) with description of four new species from South Africa

Puccinia species (rust fungi, Pucciniales, formerly Uredinales) occurring on Berkheya (syn. Stobaea) of Asteraceae are critically evaluated. Puccinia berkheyicola, P. stobaeae var. stobaeae and P. stobaeae var. woodii are recognized as valid species and varieties, while P. berkheyae is relegated to a synonym of P. stobaeae var. stobaeae. Lectotypes are selected for P. stobaeae var. stobaeae and P. stobaeae var. woodii. P. clanwilliamensis, P. garstfonteinii and P. monsfontium are proposed as new species for three rust fungi previously assigned to P. stobaeae; P. berkheyaephila is described as new on B. bipinnatifida. A key to the accepted species is presented.     

The bradykinin BK2 receptor mediates angiotensin II receptor type 2 stimulated rat duodenal mucosal alkaline secretion

Background  

This study investigates bradykinin and nitric oxide as potential mediators of AT2-receptor-stimulated duodenal mucosal alkaline secretion. Duodenal mucosal alkaline secretion was measured in methohexital- and α-chloralose-anaesthetised rats by means of in situ pH-stat titration. Immunohistochemistry and Western blot were used to identify the BK2 receptors.     

Recruitment of human cytomegalovirus immediate-early 2 protein onto parental viral genomes in association with ND10 in live-infected cells

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise co-localizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.     

Synthesis and radiopharmacological characterization of 2beta-carbo-2'-[18F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane ([18F]MCL-322) as a PET radiotracer for imaging the dopamine transporter (DAT)

The fluoroalkyl-containing tropane derivative 2beta-carbo-2'-fluoroethoxy-3beta-(4-bromo-phenyl)tropane (MCL-322) is a highly potent and moderately selective ligand for the dopamine transporter (DAT). The compound was labeled with the short-lived positron emitter (18)F in a single step by nucleophilic displacement of the corresponding tosylate precursor MCL-323 with no-carrier-added [(18)F]fluoride. The positron emission tomography (PET) radiotracer 2beta-carbo-2'-[(18)F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane [(18)F]MCL-322 was obtained in decay-corrected radiochemical yields of 30-40% at a specific radioactivity of 1.6-2.4Ci/mumol (60-90GBq/mumol) at the end-of-synthesis (EOS). Small animal PET, ex vivo and in vivo biodistribution experiments in rats demonstrated a high uptake in the striatum (3.2% ID/g) 5min after injection, which increased to 4.2% ID/g after 60min. The uptake in the cerebellum was 1.8% ID/g and 0.6% ID/g after 5min and 60min post-injection, respectively. Specific binding to DAT of [(18)F]MCL-322 was confirmed by blocking experiments using the high affinity DAT ligand GBR 12909. The radiopharmacological characterization was completed with metabolite and autoradiographic studies confirming the selective uptake of [(18)F]MCL-322 in the striatum. It is concluded that the simple single-step radiosynthesis of [(18)F]MCL-322 and the promising radiopharmacological data make [(18)F]MCL-322 an attractive candidate for the further development of a PET radiotracer potentially suitable for clinical DAT imaging in the human brain.     

Elevated amount of Toll-like receptor 4 mRNA in bronchial epithelial cells is associated with airway inflammation in horses with recurrent airway obstruction

Recurrent airway obstruction (RAO) is characterized by neutrophilic airway inflammation and obstruction, and stabling of susceptible horses triggers acute disease exacerbations. Stable dust is rich in endotoxin, which is recognized by Toll-like receptor (TLR) 4. In human bronchial epithelium, TLR4 stimulation leads to elevation of interleukin (IL)-8 mRNA expression. The zinc finger protein A20 negatively regulates this pathway. We hypothesized that TLR4 and IL-8 mRNA and neutrophil numbers are elevated and that A20 mRNA is not increased in RAOs during stabling compared with controls and with RAOs on pasture. We measured the maximal change in pleural pressure (DeltaPpl(max)), determined inflammatory cell counts in bronchoalveolar lavage fluid (BAL), and quantified TLR4, IL-8, and A20 mRNA in bronchial epithelium by quantitative RT-PCR. We studied six horse pairs, each pair consisting of one RAO and one control horse. Each pair was studied when the RAO-affected horse had airway obstruction induced by stabling and after 7, 14, and 28 days on pasture. Stabling increased BAL neutrophils, DeltaPpl(max), and TLR4 (4.14-fold change) significantly in RAOs compared with controls and with RAOs on pasture. TLR4 correlated with IL-8 (R2 = 0.75). Whereas stabling increased IL-8 in all horses, A20 was unaffected. IL-8 was positively correlated with BAL neutrophils (R2 = 0.43) and negatively with A20 (R2 = 0.44) only in RAO-affected horses. Elevated TLR4 expression and lack of A20 upregulation in bronchial epithelial cells from RAO-affected horses may contribute to elevated IL-8 production, leading to exaggerated neutrophilic airway inflammation in response to inhalation of stable dust.     

A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and apoptosis

Protein phosphatase 1 (PP1) catalytic subunits typically combine with other proteins that modulate their activity, direct them to distinct substrates, or serve as substrates for PP1. More than 50 PP1-interacting proteins (PIPs) have been identified so far. Given there are approximately 10 000 phosphoproteins in mammals, many PIPs remain to be discovered. We have used arrays containing 100 carefully selected antibodies to identify novel PIPs that are important in cell proliferation and cell survival in murine fetal lung epithelial cells and human A549 lung cancer cells. The antibody arrays identified 31 potential novel PIPs and 11 of 17 well-known PIPs included as controls, suggesting a sensitivity of at least 65%. A majority of the interactions between PP1 and putative PIPs were isoform- or cell type-specific. We confirmed by co-immunoprecipitation that 9 of these proteins associate with PP1: APAF-1, Bax, E-cadherin, HSP-70, Id2, p19Skp1, p53, PCNA, and PTEN. We examined two of these interactions in greater detail in A549 cells. Exposure to nicotine enhanced association of PP1 with Bax (and Bad), but also induced inhibitory phosphorylation of PP1. In addition to p19Skp1, PP1alpha antibodies also coprecipitated cullin 1, suggesting that PP1alpha is associated with the SCF1 complex. This interaction was only detectable during the G1/S transition and S phase. Forced loss of PP1 function decreased the levels of p27Kip1, a well-known SCF1 substrate, suggesting that PP1 may rescue proteins from ubiquitin/proteasome-mediated destruction. Both of these novel interactions are consistent with PP1 facilitating cell cycle arrest and/or apoptosis.     

Dynamics of the phosphoinositide 3-kinase p110δ interaction with p85α and membranes reveals aspects of regulation distinct from p110α

Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.     

Genome-wide meta-analysis identifies regions on 7p21 (AHR) and 15q24 (CYP1A2) as determinants of habitual caffeine consumption

We report the first genome-wide association study of habitual caffeine intake. We included 47,341 individuals of European descent based on five population-based studies within the United States. In a meta-analysis adjusted for age, sex, smoking, and eigenvectors of population variation, two loci achieved genome-wide significance: 7p21 (P = 2.4 × 10(-19)), near AHR, and 15q24 (P = 5.2 × 10(-14)), between CYP1A1 and CYP1A2. Both the AHR and CYP1A2 genes are biologically plausible candidates as CYP1A2 metabolizes caffeine and AHR regulates CYP1A2.     

Optimization of Escherichia coli cultivation methods for high yield neuropeptide Y receptor type 2 production

The recombinant expression of human G protein-coupled receptors usually yields low production levels using commonly available cultivation protocols. Here, we describe the development of a high yield production protocol for the human neuropeptide Y receptor type 2 (Y2R), which provides the determination of expression levels in a time, media composition, and process parameter dependent manner. Protein was produced by Escherichia coli in a defined medium composition suitable for isotopic labeling required for investigations by nuclear magnetic resonance spectroscopy. The Y2 receptor was fused to a C-terminal 8x histidine tag by means of the pET vector system for easy one-step purification via affinity chromatography, yielding a purity of 95-99% for every condition tested, which was determined by SDS-PAGE and Western blot analysis. The Y2 receptor was expressed as inclusion body aggregates in complex media and minimal media, using different carbon sources. We investigated the influences of media composition, temperature, pH, and set specific growth rate on cell behavior, biomass wet weight specific and culture volume specific amounts of the target protein, which had been identified by inclusion body preparation, solubilization, followed by purification and spectrometric determination of the protein concentration. The developed process control strategy led to very high reproducibility of cell growth and protein concentrations with a maximum yield of 800 μg purified Y2 receptor per gram wet biomass when glycerol was used as carbon source in the mineral salt medium composition (at 38 °C, pH 7.0, and a set specific growth rate of 0.14 g/(gh)). The maximum biomass specific amount of purified Y2 receptor enabled the production of 35 mg Y2R per liter culture medium at an optical density (600 nm) of 25.     

Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.