45 research outputs found
Identification of Domains of the HPV11 E1 Protein Required for DNA Replication in Vitro
AbstractThe HPV E1 and E2 proteins along with cellular factors, are required for replication of the viral genome. In this study we show that in vitro synthesized HPV11 E1 can support DNA replication in a cell-free system and is able to cooperate with E2 to recruit the host polymerase α primase to the HPV origin in vitro. Deletion analysis revealed that the N-terminal 166 amino acids of E1, which encompass a nuclear localization signal and a cyclin E-binding motif, are dispensable for E1-dependent DNA replication and for recruitment of pol α primase to the origin in vitro. A shorter E1 protein lacking the N-terminal 190 amino acids supported cell-free DNA replication at less than 25% the efficiency of wild-type E1 and was active in the pol α primase recruitment assay. An even shorter E1 protein lacking a functional DNA-binding domain due to a truncation of the N-terminal 352 amino acids was inactive in both assays despite the fact that it retains the ability to associate with E2 or pol α primase in the absence of ori DNA. We provide additional functional evidence that E1 interacts with pol α primase through the p70 subunit of the complex by showing that p70 can be recruited to the HPV origin by E1 and E2 in vitro, that the domain of E1 (amino acids 353–649) that binds to pol α primase in vitro is the same as that needed for interaction with p70 in the yeast two-hybrid system, and that exogenously added p70 competes with the interaction between E1 and pol α primase and inhibits E1-dependent cell-free DNA replication. On the basis of these results and the observation that pol α primase competes with the interaction between E1 and E2 in solution, we propose that these three proteins assemble at the origin in a stepwise process during which E1, following its interaction with E2, must bind to DNA prior to interacting with pol α primase
SerpinA3N is a novel hypothalamic gene upregulated by a high-fat diet and leptin in mice
Background: Energy homeostasis is regulated by the hypothalamus but fails when animals are fed a high-fat diet (HFD), and leptin insensitivity and obesity develops. To elucidate the possible mechanisms underlying these effects, a microarray-based transcriptomics approach was used to identify novel genes regulated by HFD and leptin in the mouse hypothalamus. Results: Mouse global array data identified serpinA3N as a novel gene highly upregulated by both a HFD and leptin challenge. In situ hybridisation showed serpinA3N expression upregulation by HFD and leptin in all major hypothalamic nuclei in agreement with transcriptomic gene expression data. Immunohistochemistry and studies in the hypothalamic clonal neuronal cell line, mHypoE-N42 (N42), confirmed that alpha 1-antichymotrypsin (α1AC), the protein encoded by serpinA3, is localised to neurons and revealed that it is secreted into the media. SerpinA3N expression in N42 neurons is upregulated by palmitic acid and by leptin, together with IL-6 and TNFα, and all three genes are downregulated by the anti-inflammatory monounsaturated fat, oleic acid. Additionally, palmitate upregulation of serpinA3 in N42 neurons is blocked by the NFκB inhibitor, BAY11, and the upregulation of serpinA3N expression in the hypothalamus by HFD is blunted in IL-1 receptor 1 knockout (IL-1R1−/−) mice. Conclusions: These data demonstrate that serpinA3 expression is implicated in nutritionally mediated hypothalamic inflammation
Estrogen Receptor β-Selective Agonists Stimulate Calcium Oscillations in Human and Mouse Embryonic Stem Cell-Derived Neurons
Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERα and ERβ on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERβ, but not ERα. The non-selective ER agonist 17β-estradiol (E2) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERα agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERβ agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E2. The ERβ agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERβ activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERβ signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds
Measuring empathy in pediatrics: validation of the Visual CARE measure
Background: Empathy is a key element of “Patient and Family Centered Care”, a clinical approach recommended
by the American Academy of Pediatrics. However, there is a lack of validated tools to evaluate paediatrician
empathy. This study aimed to validate the Visual CARE Measure, a patient rated questionnaire measuring physician
empathy, in the setting of a Pediatric Emergency Department (ED).
Methods: The empathy of physicians working in the Pediatric ED of the University Hospital of Udine, Italy, was
assessed using an Italian translation of the Visual Care Measure. This test has three versions suited to different age
groups: the 5Q questionnaire was administered to children aged 7–11, the 10Q version to those older than 11, and
the 10Q–Parent questionnaire to parents of children younger than 7.
The internal reliability, homogeneity and construct validity of the 5Q and 10Q/10Q–Parent versions of the Visual
Care Measure, were separately assessed. The influence of family background on the rating of physician empathy
and satisfaction with the clinical encounter was also evaluated.
Results: Seven physicians and 416 children and their parents were included in the study. Internal consistency
measured by Cronbach’s alpha was 0.95 for the 10Q/10Q–Parent versions and 0.88 for the 5Q version. The
item-total correlation was > 0.75 for each item. An exploratory factor analysis showed that all the items load
onto the first factor.
Physicians’ empathy scores correlated with patients’ satisfaction for both the 10Q and 10Q–Parent questionnaires
(Spearman’s rho = 0.7189; p < 0.001) and for the 5Q questionnaire (Spearman’s rho = 0.5968; p < 0,001). Trust in the
consulting physician was lower among immigrant parents (OR 0.43. 95% CI 0.20–0.93).
Conclusions: The Visual Care Measure is a reliable second-person test of physician empathy in the setting of a
Pediatric Emergency Room. More studies are needed to evaluate the reliability of this instrument in other pediatric
settings distinct from the Emergency Room and to further evaluate its utility in measuring the impact of
communication and empathy training programmes for healthcare professionals working in pediatrics