Role of Reactive Oxygen Species in the Neural and Hormonal Regulation of the PNMT Gene in PC12 Cells

The stress hormone, epinephrine, is produced predominantly by adrenal chromaffin cells and its biosynthesis is regulated by the enzyme phenylethanolamine N-methyltransferase (PNMT). Studies have demonstrated that PNMT may be regulated hormonally via the hypothalamic-pituitary-adrenal axis and neurally via the stimulation of the splanchnic nerve. Additionally, hypoxia has been shown to play a key role in the regulation of PNMT. The purpose of this study was to examine the impact of reactive oxygen species (ROS) produced by the hypoxia mimetic agent CoCl2, on the hormonal and neural stimulation of PNMT in an in vitro cell culture model, utilizing the rat pheochromocytoma (PC12) cell line. RT-PCR analyses show inductions of the PNMT intron-retaining and intronless mRNA splice variants by CoCl2 (3.0- and 1.76-fold, respectively). Transient transfection assays of cells treated simultaneously with CoCl2 and the synthetic glucocorticoid, dexamethasone, show increased promoter activity (18.5-fold), while mRNA levels of both splice variants do not demonstrate synergistic effects. Similar results were observed when investigating the effects of CoCl2-induced ROS on the neural stimulation of PNMT via forskolin. Our findings demonstrate that CoCl2-induced ROS have synergistic effects on hormonal and neural activation of the PNMT promoter

Interventions to reduce wait times for primary care appointments: a systematic review

Abstract Background Accessibility and availability are important characteristics of efficient and effective primary healthcare systems. Currently, timely access to a family physician is a concern in Canada. Adverse outcomes are associated with longer wait times for primary care appointments and often leave individuals to rely on urgent care. When wait times for appointments are too long patients may experience worse health outcomes and are often left to use emergency department resources. The primary objective of our study was to systematically review the literature to identify interventions designed to reduce wait times for primary care appointments. Secondary objectives were to assess patient satisfaction and reduction of no-show rates. Methods We searched multiple databases, including: Medline via Ovid SP (1947 to present), Embase (from 1980 to present), PsychINFO (from 1806 to present), Cochrane Central Register of Controlled Trials (CENTRAL; all dates), Cumulative Index to Nursing and Allied Health (CINAHL; 1937 to present), and Pubmed (all dates) to identify studies that reported outcomes associated with interventions designed to reduce wait times for primary care appointments. Two independent reviewers assessed all identified studies for inclusion using pre-defined inclusion/exclusion criteria and a multi-level screening approach. Our study methods were guided by the Cochrane Handbook for Systematic Reviews of Interventions. Results Our search identified 3,960 articles that were eligible for inclusion, eleven of which satisfied all inclusion/exclusion criteria. Data abstraction of included studies revealed that open access scheduling is the most commonly used intervention to reduce wait times for primary care appointments. Additionally, included studies demonstrated that dedicated telephone calls for follow-up consultation, presence of nurse practitioners on staff, nurse and general practitioner triage, and email consultations were effective at reducing wait times. Conclusions To our knowledge, this is the first study to systematically review and identify interventions designed to reduce wait times for primary care appointments. Our findings suggest that open access scheduling and other patient-centred interventions may reduce wait times for primary care appointments. Our review may inform policy makers and family healthcare providers about interventions that are effective in offering timely access to primary healthcare

Plasmodium falciparum Adhesins Play an Essential Role in Signalling and Activation of Invasion into Human Erythrocytes

The most severe form of malaria in humans is caused by the protozoan parasite Plasmodium falciparum. The invasive form of malaria parasites is termed a merozoite and it employs an array of parasite proteins that bind to the host cell to mediate invasion. In Plasmodium falciparum, the erythrocyte binding-like (EBL) and reticulocyte binding-like (Rh) protein families are responsible for binding to specific erythrocyte receptors for invasion and mediating signalling events that initiate active entry of the malaria parasite. Here we have addressed the role of the cytoplasmic tails of these proteins in activating merozoite invasion after receptor engagement. We show that the cytoplasmic domains of these type 1 membrane proteins are phosphorylated in vitro. Depletion of PfCK2, a kinase implicated to phosphorylate these cytoplasmic tails, blocks P. falciparum invasion of red blood cells. We identify the crucial residues within the PfRh4 cytoplasmic domain that are required for successful parasite invasion. Live cell imaging of merozoites from these transgenic mutants show they attach but do not penetrate erythrocytes implying the PfRh4 cytoplasmic tail conveys signals important for the successful completion of the invasion process

Plasmodium falciparum adhesins play an essential role in signalling and activation of invasion into human erythrocytes

The most severe form of malaria in humans is caused by the protozoan parasite Plasmodium falciparum. The invasive form of malaria parasites is termed a merozoite and it employs an array of parasite proteins that bind to the host cell to mediate invasion. In Plasmodium falciparum, the erythrocyte binding-like (EBL) and reticulocyte binding-like (Rh) protein families are responsible for binding to specific erythrocyte receptors for invasion and mediating signalling events that initiate active entry of the malaria parasite. Here we have addressed the role of the cytoplasmic tails of these proteins in activating merozoite invasion after receptor engagement. We show that the cytoplasmic domains of these type 1 membrane proteins are phosphorylated in vitro. Depletion of PfCK2, a kinase implicated to phosphorylate these cytoplasmic tails, blocks P. falciparum invasion of red blood cells. We identify the crucial residues within the PfRh4 cytoplasmic domain that are required for successful parasite invasion. Live cell imaging of merozoites from these transgenic mutants show they attach but do not penetrate erythrocytes implying the PfRh4 cytoplasmic tail conveys signals important for the successful completion of the invasion process

Specific amino acid residues in the cytoplasmic domain are essential for PfRh4 function.

<p>(A) Localization of PfRh4 and PfRh2 in Rh4-WT tail, RH4-AMA1tail, Rh4-mut4tail and RH4-mut5tail lines as detected using anti-PfRh4 monoclonal and anti-PfRh2 polyclonal antibodies. Parasite nuclei were stained with DAPI. (B) Expression of PfRh4 and PfRh2 in W2mefΔ175 and all transgenic lines as detected by anti-PfRh4 and anti-PfRh2 antibody. All transgenic lines migrated as a slightly larger doublet compared to PfRh4 in W2mefΔ175, consistent with the addition of the hexa-histidine tag at the C-terminus of the protein. (C) Growth assays of transgenic lines with four and five mutations at serine and tyrosine amino acid residues in the PfRh4 cytoplasmic tail. (D) Growth assays of single mutations in the PfRh4 cytoplasmic tail. (E) Growth assays of double mutations in the PfRh4 cytoplasmic tail. In all three panels, parasitaemia was measured in neuraminidase-treated, and untreated erythrocytes after every 48 hours incubation (labelled as cycles). The parasite lines used in this experiment are displayed on the X-axis. The y-axis represents parasitaemia of neuraminidase-treated erythrocytes as a percentage of parasitaemia of the same line grown on untreated erythrocytes. Error bars represent +1 standard error of the mean. Assay performed three times on separate days, each in triplicate.</p

Identification of putative phosphosites and parasite kinase involved in modification of PfRh4 cytoplasmic tail.

<p>(A) <i>In vitro</i> kinase assays of wildtype and putative phosphosite mutations in PfRh4 cytoplasmic domains. The amino acid sequence of the PfRh4 cytoplasmic tail is shown with serines and tyrosines highlighted with the residue number. Each potential phosphosite on the PfRh4 tail was individually mutated to alanine. The all 4 lane has mutations in S1667A, S1674A, Y1680A and Y1684A and the all 5 lane has S1652A, S1667A, S1674A, Y1680A and Y1684A putative kinase sites mutated. The phosphorylation signal was quantitated and adjusted for protein loading. The loading-adjusted mutant phosphorylation signals were divided by the wildtype and plotted as a percentage of the wildtype signal (Y-axis). Autoradiograph of proteins after incubation in the <i>in vitro</i> phosphorylation assay and Coomassie gel from which protein loading was quantitated are shown in lower panels. Lane labels (X-axis) denote residues mutated to alanine. Mean percentage of wildtype phosphorylation +1 standard error of the mean are displayed. Data was averaged from four experiments performed on separate days. (B) Dosage-response curve for PfRh4 tail phosphorylation by merozoite lysate in the presence of increasing concentrations of the CK2 inhibitor TBB. PfRh4 tail phosphorylation was quantitated after incubation in <i>in vitro</i> phosphorylation assay with TBB. The phosphorylation signal for each condition was adjusted to reflect the average amount of protein loaded across each condition, determined by densitometry of the Coomassie brilliant blue stained gel. Y-axis represents loading-adjusted phosphorylation signal as a percentage of phosphorylation in the presence of DMSO (control). Autoradiograph of wildtype GST-fused PfRh4 proteins after incubation in the <i>in vitro</i> phosphorylation assay. X-axis indicates the TBB concentration with which the phosphorylation assay was incubated or DMSO. (C) <i>In vitro</i> kinase assays of PfRh and EBL cytoplasmic tails. The phosphorylation signal was quantitated and adjusted for protein loading. Autoradiograph of proteins after incubation in the <i>in vitro</i> phosphorylation assay and Coomassie brilliant blue stained gel from which protein loading was quantitated are shown. Data was averaged from four experiments performed on separate days (right panel) and standard error of the mean is shown. The following sites were mutated: EBA140 (S1159A, S1168A, T1173A), EBA175 (T1466A, mut A) and (S1489A, mut B) and in combination (mut A and B), EBA181 (S1528A, S1553A, S1557A, T1564A), PfRh2a (S3128A) and PfRh2b (S3233).</p