Expression of Drosophila Adenosine Deaminase in Immune Cells during Inflammatory Response

Extra-cellular adenosine is an important regulator of inflammatory responses. It is generated from released ATP by a cascade of ectoenzymes and degraded by adenosine deaminase (ADA). There are two types of enzymes with ADA activity: ADA1 and ADGF/ADA2. ADA2 activity originates from macrophages and dendritic cells and is associated with inflammatory responses in humans and rats. Drosophila possesses a family of six ADGF proteins with ADGF-A being the main regulator of extra-cellular adenosine during larval stages. Herein we present the generation of a GFP reporter for ADGF-A expression by a precise replacement of the ADGF-A coding sequence with GFP using homologous recombination. We show that the reporter is specifically expressed in aggregating hemocytes (Drosophila immune cells) forming melanotic capsules; a characteristic of inflammatory response. Our vital reporter thus confirms ADA expression in sites of inflammation in vivo and demonstrates that the requirement for ADA activity during inflammatory response is evolutionary conserved from insects to vertebrates. Our results also suggest that ADA activity is achieved specifically within sites of inflammation by an uncharacterized post-transcriptional regulation based mechanism. Utilizing various mutants that induce melanotic capsule formation and also a real immune challenge provided by parasitic wasps, we show that the acute expression of the ADGF-A protein is not driven by one specific signaling cascade but is rather associated with the behavior of immune cells during the general inflammatory response. Connecting the exclusive expression of ADGF-A within sites of inflammation, as presented here, with the release of energy stores when the ADGF-A activity is absent, suggests that extra-cellular adenosine may function as a signal for energy allocation during immune response and that ADGF-A/ADA2 expression in such sites of inflammation may regulate this role

Changes in Adenosine Deaminase Activity in Patients with Type 2 Diabetes Mellitus and Effect of DPP-4 Inhibitor Treatment on ADA Activity

BackgroundDipeptidyl peptidase 4 (DPP-4, also known as CD26) binds with adenosine deaminase (ADA) to activate T lymphocytes. Here, we investigated whether ADA activity is specifically affected by treatment with DPP-4 inhibitor (DPP4I) compared with other anti-diabetic agents.MethodsFasting ADA activity, in addition to various metabolic and biochemical parameters, were measured in 262 type 2 diabetes mellitus (T2DM) patients taking various anti-diabetic agents and in 46 non-diabetic control subjects.ResultsADA activity was increased in T2DM patients compared with that in non-diabetic control subjects (mean±standard error, 23.1±0.6 U/L vs. 18.6±0.8 U/L; P<0.05). ADA activity was correlated with fasting plasma glucose (r=0.258, P<0.05), HbA1c (r=0.208, P<0.05), aspartate aminotransferase (r=0.325, P<0.05), and alanine aminotransferase (r=0.248, P<0.05). Compared with the well-controlled T2DM patients (HbA1c<7%), the poorly controlled group (HbA1c>9%) showed significantly increased ADA activity (21.1±0.8 U/L vs. 25.4±1.6 U/L; P<0.05). The effect of DPP4I on ADA activity in T2DM patients did not differ from those of other oral anti-diabetic agents or insulin. T2DM patients on metformin monotherapy showed a lower ADA activity (20.9±1.0 U/L vs. 28.1±2.8 U/L; P<0.05) compared with that of those on sulfonylurea monotherapy.ConclusionOur results show that ADA activity is increased in T2DM patients compared to that in non-diabetic patients, is positively correlated with blood glucose level, and that DPP4I has no additional specific effect on ADA activity, except for a glycemic control- or HbA1c-dependent effect

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications

Präklusionsvorschriften des öffentlichen Rechts im Spannungsfeld zwischen Verfahrensbeschleunigung, Einzelfallgerechtigkeit und Rechtsstaatlichkeit: zur Vereinbarkeit der Präklusion mit dem Grundgesetz und mit dem Europarecht

Niedzwicki M. Präklusionsvorschriften des öffentlichen Rechts im Spannungsfeld zwischen Verfahrensbeschleunigung, Einzelfallgerechtigkeit und Rechtsstaatlichkeit: zur Vereinbarkeit der Präklusion mit dem Grundgesetz und mit dem Europarecht. Schriften zum öffentlichen Recht; 1051. Berlin: Duncker &amp; Humblot; 2007

Präklusionsvorschriften des öffentlichen Rechts im Spannungsfeld zwischen Verfahrensbeschleunigung, Einzelfallgerechtigkeit und Rechtsstaatlichkeit | Zur Vereinbarkeit der Präklusion mit dem Grundgesetz und mit dem Europarecht

Matthias Niedzwicki beschäftigt sich mit der Frage der Vereinbarkeit materieller Präklusionsvorschriften des öffentlichen Rechts mit dem Grundgesetz und mit dem Europäischen Gemeinschaftsrecht. Dabei stehen Präklusionsvorschriften des Umwelt- und Technikrechts im Vordergrund.Das Interesse an einer zügigen Genehmigung/Zulassung von Projekten steht dem Interesse an einem rechtsstaatlichen Schutz individueller Rechte und einer "Verfahrenskontrolle" der Genehmigung gegenüber. Durch den Erlass unanfechtbarer Verwaltungsakte sollen Vorhabenträger Investitionssicherheit erhalten; Verfahren sollen beschleunigt werden.Materielle Präklusionsvorschriften müssen sich insbesondere an Art. 103, 19, 14, 12, 8, 2 GG und an Art. 234, 10 EGV messen lassen. Dabei geht der Verfasser auf die Judikatur des BVerwG, des BVerfG und des EuGH ein. Er zeigt, mit empirischem Material belegt, dass die Investitionssicherheit durch materielle Präklusion nicht wesentlich gestärkt wird und dass diese Vorschriften zur Verfahrensbeschleunigung entbehrlich sind.Am Ende der Untersuchung wird deutlich, dass die derzeitigen materiellen Präklusionsvorschriften weder mit dem Grundgesetz noch mit dem Europarecht in Einklang zu bringen sind

Alat peningkatan mutu

Judul asli Continuous Improvement tools, Jilid 2.vii, 127 p. : il.; 21 c