Evidence for coordinated induction and repression of ecto-5'-nucleotidase (CD73) and the A2a adenosine receptor in a human B cell line

In the human B cell line P493-6 two mitogenic signals, the EpsteinBarr virus nuclear antigen 2 (EBNA2) and myc, can be independently regulated by means of an estrogen receptor fusion construct or an inducible expression vector, respectively. Shut off of EBNA2, either in the presence or absence of myc, leads to a significant increase in enzymatic activity and surface expression of ecto-5nucleotidase (CD73) as well as an increased adenosine receptor response in cyclic AMP formation. Shut off of myc expression has a small additional positive effect on CD73 activity. Among the four different subtypes of adenosine receptors, the A2a receptor exclusively is subject to regulation in this system, which is substantiated by pharmacologic data (specific agonists and inhibitors), as well as on the mRNA level. With upregulated CD73 and A2a, cells also respond to 5AMP with increased cyclic AMP formation. Turn on of EBNA2 has the reverse effect of repression of CD73 and A2a expression. The time course of both induction and repression of CD73 and A2a is rather slow

Modeling nonlinear optical interactions of focused beams in bulk crystals and thin films: A phenomenological approach

Coherent nonlinear optical micro-spectroscopy is a frequently used tool in modern material science, as it is sensitive to many different local observables, which comprise, among others, crystal symmetry and vibrational properties. The richness in information, however, may come with challenges in data interpretation, as one has to disentangle the many different effects like multiple reflections, phase jumps at interfaces, or the influence of the Guoy-phase. In order to facilitate interpretation, the work presented here proposes an easy-to-use semi-analytical modeling ansatz, that bases upon known analytical solutions using Gaussian beams. Specifically, we apply this ansatz to compute nonlinear optical responses of (thin film) optical materials. We try to conserve the meaning of intuitive parameters like the Gouy-phase and the nonlinear coherent interaction length. In particular, the concept of coherence length is extended, which is a must when using focal beams. The model is subsequently applied to exemplary cases of second-harmonic and third-harmonic generation. We observe a very good agreement with experimental data and furthermore, despite the constraints and limits of the analytical ansatz, our model performs similarly well as when using more rigorous simulations. However, it outperforms the latter in terms of computational power, requiring more than three orders less computational time and less performant computer systems

Ecto 5′-Nucleotidase and Nonspecific Alkaline Phosphatase: TWO AMP-HYDROLYZING ECTOENZYMES WITH DISTINCT ROLES IN HUMAN AIRWAYS

In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 microM [(3)H]adenosine was not transported to the mucosal compartment. Messenger RNA for the cytosolic AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial epithelial cells, suggesting that mucosal adenosine did not originate from intracellular pools. In contrast, extracellular 0.1 mm ATP was rapidly dephosphorylated into adenosine on the mucosal epithelial surface. We identified two ectonucleotidases that mediated the conversion of AMP to adenosine: ecto 5'-nucleotidase (ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and serosal epithelial surfaces displayed ecto 5'-NT activity (K(m) = 14 microM, V(max) = 0.5 nmol x min(-1) x cm(-2)), whereas AP activity was restricted to the mucosal surface (K(m,)(high) = 36 microM, V(max) = 1.2 nmol x min(-1) x cm(-2); K(m,)(low) = 717 microM, V(max) = 2.8 nmol x min(-1) x cm(-2)). In bronchial cultures and tissues, ecto 5'-NT accounted for >80% of total activity toward 0.01 mm AMP, compared with <15% for 5 mm AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5'-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5'-NT and NS AP in the regulation of adenosine concentrations on airway surfaces

Development and testing of a novel sulfur dioxide sonde

A novel technique has been developed to measure sulfur dioxide (SO2) using a modification of the existing electrochemical concentration cell (ECC) ozonesonde technology. The current sonde-based method to measure SO2 (i.e., the dual-sonde approach) involves launching two ozonesondes together, with one of the sondes having a filter to remove SO2 at the inlet. The SO2 profile is determined by taking the difference between the measurements from the two instruments. The dual-sonde method works well in typical tropospheric conditions when [O3]&gt;[SO2] but saturates when [SO2]&gt;[O3] and has large uncertainties in the upper troposphere and lower stratosphere that would limit its effectiveness in measuring SO2 from an explosive volcanic eruption. Due to these limitations, several modifications were made to create a single-sonde system that would directly measure SO2 (i.e., the SO2 sonde). These modifications included (1) a positively biased ECC current, (2) the addition of an O3 removal filter, and (3) the addition of a sample dryer. The SO2 sonde measures SO2 as a reduction in the cell current. There was a strong correlation (r2&gt;0.94) between the SO2 sonde and a Thermo 43c analyzer during controlled laboratory tests and pre-flight tests. Varying humidity levels affected the SO2 sonde's sensitivity (avg =84.6±31.7 ppbv µA−1, 1σ RSD =37 %) during initial field tests, which was resolved by adding a sample dryer upstream of the O3 removal filter and pump inlet. This modification significantly reduced the variability and increased the sensitivity of the SO2 measurements (avg =47±5.8 ppbv µA−1, 1σ RSD =12 %). Field tests included measurements near Kīlauea volcano (before and during the 2018 eruption of the Lower East Rift Zone), Costa Rica's Turrialba volcano, and anthropogenic plumes from the Athabasca oil sands region of Alberta, Canada. This single-SO2-sonde system is an effective, inexpensive instrument for measuring both ground-based and vertical profiles of SO2 from anthropogenic and natural sources (i.e., volcanic eruptions) over a wide range of concentrations.</p

Genomic structure and chromosomal localization of the human deoxycytidine kinase gene.

Deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 1.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have violated genomic clones encompassing its entire coding and 5' flanking regions and delineated all the exon/intron boundaries.Deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 1.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have violated genomic clones encompassing its entire coding and 5' flanking regions and delineated all the exon/intron boundaries

Synthesis of dicationic diaryltriazines nucleic acid binding agents

— The synthesis of 2,4-bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1,3,5-triazine 6a and 2,4-bis[4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenyl]-1,3,5-triazine 6b in 3 steps from either 4-bromobenzamidine or 4-(carbamoyl)benzamidine is reported. The synthesis of 4,6-bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-2-dimethylamino-1,3,5-triazine 9a and 4,6-bis[4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenyl]-2-dimethylamino-1,3,5-triazine 9b in 2 steps from 1,4-dicyanobenzene is also described. The compounds 6b and 9b bind strongly to DNA model sequences and inhibit topoisomerase II from 2 microbial sources. Compounds 6a and 9a bind to both DNA and RNA model sequences whereas 6b and 9b essentially do not bind to the RNA model

Structural Insights into the Inhibition of Cytosolic 5′-Nucleotidase II (cN-II) by Ribonucleoside 5′-Monophosphate Analogues

Cytosolic 5′-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5′-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5′-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues

Kolloquium Forschende Frauen 2016 und 2017: Beiträge Bamberger Nachwuchswissenschaftlerinnen

Die Buchreihe „Forschende Frauen in Bamberg“ begleitet das gleichnamige Forschungskolloquium der Frauenbeauftragten der Otto-Friedrich-Universität Bamberg. Der neunte Band unserer Reihe umfasst als Besonderheit sowohl das Kolloquium 2016 als auch 2017. Anlässlich des von uns für 2016 ausgerufenen „Genderjahres“ haben alle in diesem Buch veröffentlichten Beiträge eine Gemeinsamkeit: Untersucht werden Fragestellungen, die sich mit Genderthemen befassen, beziehungsweise Geschlechterunterschiede aufzeigen oder Frauen in den Fokus nehmen

Synthesis of dicationic diarylpyridines as nucleic-acid binding agents

The syntheses of 2,6-bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]pyridine 7, 2-[4-(4,5-dihydro-1H-imidazol-2-yl)-phenyl]-6-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]pyridine 8 and 2,6-bis[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]pyridine 9 in five steps from the appropriately substituted bromoacetophenone are described. 3,5-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]pyridine 13 is also reported, prepared in four steps from 4-bromophenylacetonitrile. The preparation of 2,5-bis[4-(4,5-dihydro-1H-imidazol-2-yl)-phenyl]pyridine 18 from 4-bromoacetophenone in six steps is presented. The dications bind to poly dA·dT in the order 7 > 13 > 18 > 8 > 9; the order of binding to poly A·U is 7 > 13 > 8 > 9; 18 essentially does not bind to the RNA model. Only 7 inhibits topoisomerase II at millimolar concentrations. The dicationic compounds that were tested against Pneumonocystis carinii in the immuno-suppressed rat model show only modest activity and are moderately toxic. Some of the compounds demonstrated modest anti-HIV-1 activity and selectivity in primary lymphocytes