Midkine Controls Arteriogenesis by Regulating the Bioavailability of Vascular Endothelial Growth Factor A and the Expression of Nitric Oxide Synthase 1 and 3

Midkine is a pleiotropic factor, which is involved in angiogenesis. However, its mode of action in this process is still ill defined. The function of midkine in arteriogenesis, the growth of natural bypasses from pre-existing collateral arteries, compensating for the loss of an occluded artery has never been investigated. Arteriogenesis is an inflammatory process, which relies on the proliferation of endothelial cells and smooth muscle cells. We show that midkine deficiency strikingly interferes with the proliferation of endothelial cells in arteriogenesis, thereby interfering with the process of collateral artery growth. We identified midkine to be responsible for increased plasma levels of vascular endothelial growth factor A (VEGFA), necessary and sufficient to promote endothelial cell proliferation in growing collaterals. Mechanistically, we demonstrate that leukocyte domiciled midkine mediates increased plasma levels of VEGFA relevant for upregulation of endothelial nitric oxide synthase 1 and 3, necessary for proper endothelial cell proliferation, and that non-leukocyte domiciled midkine additionally improves vasodilation. The data provided on the role of midkine in endothelial proliferation are likely to be relevant for both, the process of arteriogenesis and angiogenesis. Moreover, our data might help to estimate the therapeutic effect of clinically applied VEGFA in patients with vascular occlusive diseases

Separation of river network–scale nitrogen removal among the main channel and two transient storage compartments

Author Posting. © American Geophysical Union, 2011. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Water Resources Research 47 (2011): W00J10, doi:10.1029/2010WR009896.Transient storage (TS) zones are important areas of dissolved inorganic nitrogen (DIN) processing in rivers. We assessed sensitivities regarding the relative impact that the main channel (MC), surface TS (STS), and hyporheic TS (HTS) have on network denitrification using a model applied to the Ipswich River in Massachusetts, United States. STS and HTS connectivity and size were parameterized using the results of in situ solute tracer studies in first- through fifth-order reaches. DIN removal was simulated in all compartments for every river grid cell using reactivity derived from Lotic Intersite Nitrogen Experiment (LINX2) studies, hydraulic characteristics, and simulated discharge. Model results suggest that although MC-to-STS connectivity is greater than MC-to-HTS connectivity at the reach scale, at basin scales, there is a high probability of water entering the HTS at some point along its flow path through the river network. Assuming our best empirical estimates of hydraulic parameters and reactivity, the MC, HTS, and STS removed approximately 38%, 21%, and 14% of total DIN inputs during a typical base flow period, respectively. There is considerable uncertainty in many of the parameters, particularly the estimates of reaction rates in the different compartments. Using sensitivity analyses, we found that the size of TS is more important for DIN removal processes than its connectivity with the MC when reactivity is low to moderate, whereas TS connectivity is more important when reaction rates are rapid. Our work suggests a network perspective is needed to understand how connectivity, residence times, and reactivity interact to influence DIN processing in hierarchical river systems.This work was supported by the National Science Foundation through DEB- 0614282, BCS-0709685 and the Plum Island Long Term Ecological Research site (NSF OCE-0423565)

Analyse von AU-reichen Elementen in der Hefe Pichia pastoris

Die strenge Regulation von mRNA Stabilität und Translation durch spezifische “cis-acting“-Sequenzen und „trans-acting“-Faktoren ist für die Kontrolle der Genexpression ein essentielles Mittel. Diese Prozesse erlauben es der Zelle, das Expressionsmuster von regulatorischen Faktoren als transiente Antwort auf interne oder externe Signale wie Zellproliferation, Signaltransduktion, inflammatorische Stimuli oder (UV)-Strahlung einzustellen. Eine heute gut untersuchte „cis-acting“-Sequenz, die die mRNA-Stabilität kontrolliert, ist das „AU reiche Element“ oder „AU rich element“ (ARE), das in der 3’ untranslatierten Region (3’ UTR) von vielen, aber nicht allen, instabilen mRNAs von verschiedenen Wachstumsfaktoren, Cytokinen, Protooncogenen und Transkriptionsfakotren zu finden ist. Es wurde ein heterologes System für die Analyse von AREs in der Hefe P. pastoris etabiliert. Die zwei AREs des humanen Protoonkogen c-fos und des Cytokins TNF wurden in die 3’ UTR von P. pastoris Expressionsvektoren kloniert. Es wurde gezeigt, dass beide AREs in dieser Hefe Funktionalität besitzen und verschiedenartig reguliert werden. Dieses Ergebnis betont zusätzlich die Konservierung des ARE-regulierten Systems unter Eukaryonten und unterstreicht seine Wichtigkeit im zellulären Zusammenhang. Eine Auswahl von verschiedenen Reporterkonstrukten sowie Mutationsanalysen zeigten die Wichtigkeit zweier spezieller mRNA Hairpins, die einen signifikanten Einfluss auf Transkriptstabilität und Translation haben können. Damit konnte die Involvierung von ARE-flankierenden Sequenzen und Strukturen bei dem ARE-regulierten mRNA-Umsatz und sehr wahrscheinlich bei der Selektion von ARE-Bindungsproteinen (ARE-BP). Schließlich konnte ein Modell aufgestellt werden, das die beobachteten Effekte abhängig von der mRNA Sekundärstruktur erklärt. Um weitere für dieses Modell unterstützende Daten zu sammeln und um endogene ARE-BPs zu identifizieren, wurden Gelretardationsassays durchgeführt. Es konnte ein ca. 14 kDa großes Protein als Bindungspartner für die c-fos als auch TNF ARE Sequenzen identifizert werden.The tight regulation of mRNA stability and translation by specific cis-acting sequences and trans-acting factors is an essential means for the control of gene expression. These processes allow cells to rapidly adjust the expression pattern of regulatory factors and response transiently to internal and external signals including cell proliferation, signal transduction, inflammatory stimuli and radiation. A well known important cis-acting sequence element that controls mRNA stability is the AU-rich element (ARE) found in the 3’ untranslated region (3’UTR) of many, but not all, unstable mRNAs of various growth factors, cytokines, proto-oncogenes and transcription factors. A heterologous system for the analysis of AREs in the yeast Pichia pastoris was established. The AREs from the human proto-oncogene c-fos and the cytokine TNF were separately cloned into the 3’ UTR of P. pastoris expression vectors. It was demonstrated that both AREs are functional in this yeast and that they are regulated in different ways. This finding further stresses that the ARE-regulated system is conserved among eukaryotes and it underlines the importance of the system in the cellular context. A set of reporter constructs and mutational analysis were used to, analyse the importance of two special hairpins, which we hypothesize to have significant influence on transcript stability and translation. Thus, evidence is provided for the involvement of ARE-flanking sequences in ARE-mediated mRNA turnover and most likely in the selectivity of RNA-binding proteins. Finally, a model is proposed that explains the observed effects on protein and mRNA level in a RNA-structure-dependent manner. In order to find further support for the findings and to identify ARE-BPs gel retardation assays were performed. As a result an approximately 14 kDa protein could be identified as a binding partner of the core c-fos and TNF AREs in Pichia

NLRP10 Affects the Stability of Abin-1 To Control Inflammatory Responses

NOD-like receptors (NLR) are critical regulators of innate immune signaling. The NLR family consists of 22 human proteins with a conserved structure containing a central oligomerization NACHT domain, an N-terminal interaction domain, and a variable number of C-terminal leucine-rich repeats. Most NLR proteins function as cytosolic pattern recognition receptors with activation of downstream inflammasome signaling, NF-kappa B, or MAPK activation. Although NLRP10 is the only NLR protein lacking the leucine rich repeats, it has been implicated in multiple immune pathways, including the regulation of inflammatory responses toward Leishmania major and Shigella flexneri infection. In this study, we identify Abin-1, a negative regulator of NF-kappa B, as an interaction partner of NLRP10 that binds to the NACHT domain of NLRP10. Using S. flexneri as an infection model in human epithelial cells, our work reveals a novel function of NLRP10 in destabilizing Abin-1, resulting in enhanced proinflammatory signaling. Our data give insight into the molecular mechanism underlying the function of NLRP10 in innate immune responses

Development of the Commercial Manufacturing Process for Ipatasertib

Ipatasertib is a potent small molecule Akt kinase inhibitor currently tested in Phase III clinical trials for the treatment of metastatic castration-resistant prostate cancer and triple negative metastatic breast cancer. In this paper an overview of the development achievements towards the commercial manufacturing process is given. The convergent synthesis consists of ten steps with eight isolated intermediates and utilizes a wide range of chemical techniques and technologies to build-up this complex drug. All three stereocenters are introduced using enzyme or metal catalysis

Handling Hydrogen Peroxide Oxidations on a Large Scale: Synthesis of 5‑Bromo-2-nitropyridine

5-Bromo-2-nitropyridine was prepared from the corresponding amine via hydrogen peroxide oxidation in large scale production. This transformation initially showed low conversion, high impurity content and lack of reproducibility in lab trials. Parallel to process development, safety studies were conducted to investigate the stability of oxidant mixture, its composition and the oxidation reaction itself by reaction and adiabatic calorimetry. The resulting robust reaction conditions and appropriate safety boundaries allowed to develop a reproducible, safe protocol for the implementation of this chemistry on large scale, obtaining consistent results throughout the campaign