The pH and ionic composition of the sub-embryonic fluid of the Japanese quail (Coturnix c. japonica)

The current theory of acid-base chemistry regards [H+] as a dependent variable: changes in pH of a fluid can only occur by alteration of strong ion concentrations ([Na+], [K+], [Cl-]). The objective of this study was to determine whether manipulation of sub-embryonic fluid strong ion composition would alter [H+] in the direction predicted by theory. Changes to fluid strong ion concentrations decreased pH in the way predicted and the changes in Cl- suggest a passive distribution. Also, changes in organic anions suggest an essential role for these in acid-base chemistry of this fluid. It was notable that both Na and HCO3- were unaffected by the treatments emphasising the importance of these two ions in fluid production by the quail blastoderm

Can A State Commit A Crime? Definitely, Yes!

As is well known, the International Law Commission (ILC) decided in 1976 to include an article in its Draft Articles on State Responsibility that makes a distinction between normal international wrongful acts, which it called delicts, on the one hand, and exceptionally grave breaches of international law which it called international crimes, on the other hand

The Charter of the United Nations: A Commentary of Bruno Simma\u27s Commentary

Review of The Charter of the United Nations: A Commentary (Bruno Simma, Hermann Mosler, Albrecht Randelzhofer, Christian Tomuschat, Rüdiger Wolfrum, Andreas Paulus, Eleni Chaitobu eds.

The Charter of the United Nations: A Commentary of Bruno Simma\u27s Commentary

Review of The Charter of the United Nations: A Commentary (Bruno Simma, Hermann Mosler, Albrecht Randelzhofer, Christian Tomuschat, Rüdiger Wolfrum, Andreas Paulus, Eleni Chaitobu eds.

Systematic inference of regulatory networks that drive cytokine-stimulus integration by T cells

Differenzierungsentscheidungen von Zellen werden durch die Integration mehrerer Stimuli bestimmt. Die Differenzierung von Helfer-T-Zellen (Th-Zellen) ist hierfür ein gut untersuchtes Beispiel: reife Th-Zellen entwickeln sich beim Kontakt mit einem für sie spezifischen Antigen zu einem spezialisierten Subtyp, der von den in ihrer Umgebung vorhandenen Zytokinen abhängt und exprimieren dann einen spezifischen Mastertranskriptionsfaktor. Die häufigsten Th-Zell-Subtypen sind T-bet-exprimierende Th1-Zellen und GATA-3-exprimierende Th2-Zellen. Neuere Entdeckungen bezüglich der Plastizität von Th-Zell-Subtypen sowie die Existenz von T-bet+GATA-3+ Hybrid-Phänotypen haben die detaillierte Untersuchung vom Differenzierungsprozessen von Th-Zellen mit komplexer Zytokinsignale motiviert. Dazu haben wir systematisch die Zytokine IFN-g, IL-12 und IL-4 während der primären Differenzierung Th-Zellen titriert und Signaltransduktion und Zielgenexpression quantifiziert. Der Umfang und die Komplexität der Daten machten eine systematische Analyse notwendig, um involvierte Mechanismen genau zu identifizieren. Lineare Regressionsanalyse wurde verwendet, um die Netzwerktopologie zu extrahieren, wobei schon bekannte und zahlreiche neue Interaktionen vorausgesagt wurden. Die prognostizierte Netzwerktopologie wurde dann verwendet, um ein mechanistisches, mathematisches Modell der Zytokinsignalintegration zu entwickeln. Diese Methode hat ein hochgradig vernetztes regulatorisches Netzwerk inferiert. Bisher nicht beschriebene Funktionen von STAT-Proteine, die die Neuverkabelung des Netzwerkes während der Differenzierung vermitteln, wurden vorhergesagt. Ausgewählte neue Interaktionen wurden in gezielten genetischen Experimenten bestätigt. Während gegenseitige Inhibitionsmotive oft als kanonische digitale Schalter interpretiert werden, funktioniert das Th-Zell-Netwerk als ein Rheostat, der Variationen der Zytokinsignale in graduelle Expressionsänderungen der Mastertranskriptionsfaktoren übersetzt. Unsere Arbeit erklärt mechanistisch das beobachtete Kontinuum von Th-Zelldifferenzierungszuständen entlang der Th1-Th2-Achse und beschreibt eine quantitative Methode für die datenbasierte Inferenz zellulärer Netzwerke der Signalintegration.Cell-fate decisions are governed by the integration of multiple stimuli. Th cell differentiation is a well-studied example thereof: mature Th cells differentiate into a specialised subtype upon encounter with their cognate antigen depending on the polarising cytokines present in their environment and start expressing specific master transcription factors. The most common Th cell subtypes are T-bet-expressing Th1 cells and GATA-3-expressing Th2 cells. Recent discoveries concerning the plasticity of Th cell subtypes as well as the existence of stable T-bet+GATA-3+ hybrid Th1/2 phenotypes have stimulated the detailed study of the differentiation process under different assumptions than the hitherto valid paradigm of single master transcription factor expression by using complex cytokine signals as inputs. Here, we developed a data-based approach for inferring the molecular network underlying the differentiation of T-bet- and/or GATA-3 expressing lymphocytes. We performed systematic titrations of the polarising cytokines IFN-g, IL-12 and IL-4 during primary differentiation of Th cells and quantified signal transduction as well as target-gene expression. The size and complexity of the dataset made a systematic analysis necessary to identify the mechanisms involved. To extract the network topology, we used linear regression analysis, retrieving known regulatory mechanisms and predicting numerous novel ones. This network topology was used to develop a mechanistic mathematical model of cytokine signal integration. This approach inferred a highly connected regulatory network. Previously undescribed functions of STAT proteins mediating network rewiring during differentiation were predicted. Selected new interactions were confirmed by experiments using gene-deficient cells. Importantly, while mutual-inhibition motifs are often considered canonical digital switches, the inferred Th-cell network acts as a rheostat, generating a continuum of differentiated states along the Th1-Th2 axis. This work explains the observed Th1-Th2 cell fate continuum mechanistically and provides a quantitative framework for the data-based inference of cellular signal integration networks

The role of the co-receptor neuropilin-1 in human vascular smooth muscle cells.

Neuropilin-1 (NRP1) is a co-receptor required for neuronal and vascular development, which binds to class 3 semaphorins and VEGFs. NRP1 has been strongly implicated in VEGF-induced endothelial cell migration. VEGF has been shown to regulate vascular smooth muscle cell (VSMC) function in vitro. Evidence from mutant mice also suggests that NRP1 disruption in vivo can affect VSMC as well as endothelial function. I therefore investigated the role of NRPs in VSMC biological functions and more particularly in their migration. Western blotting showed that NRP1 and the related molecule, NRP2, were strongly expressed in human coronary artery SMC (HCASMC), whereas the major VEGF signalling receptor VEGFR2/KDR was not detectable. A high molecular weight NRP1-immunoreactive band (>250 kDa) was also strongly expressed in HCASMC, but was not detected either in cognate Human coronary artery endothelial cells (HCAEC) or in Human umbilical vein EC. The high molecular weight species was decreased significantly by treating the SMCs with chondroitinase, an enzyme that specifically chondroitin sulphate (CS) residues found in CS proteoglycan. Treatment with heparitinase, an enzymethat specifically heparan sulphate (HS) residues also resulted in a decrease of the highmolecular weight band but to a lesser extent than chondroitinase. Finally, treatment of SMC with both enzymes caused the complete disappearance of the high molecular weight species. Hence, in SMCs, in addition to the known NRP1 species at 130 kDa, NRP1 exists as a glycosaminoglycan containing either chondroitin sulphate or heparan sulphate polysaccharide chains. Mutational analysis of candidate O-linked glycosylation sites in the NRP1 extracellular domain showed that glycosylation occurred at serine 612. The importance of this gly-cosaminoglycan (GAG) modification was assessed by generating a construct of NRP1 lacking this GAG modification, called S612A. This was done by generation of an adenovirus NRP1 mutant with an alanine residue instead of the serine found in the wild-type species, however, the over-expression of the S612A NRP1 mutant in VSMC caused no signicant difference in PDGF-induced HCASMC migration. VEGF was able to bind significantly to ECs and SMCs but did not induce a significant migratory response of SMCs in contrast to PDGF-AA and PDGF-BB. PDGF-BB-induced HCASMC migration in transwell assays was inhibited by EG3287, a NRP1-specific antagonist, which blocks the ability of VEGF-A^1^6^5 to bind to NRP1. Furthermore, the migratory response to PDGF-BB was significantly decreased by siRNA-mediated knockdown of NRP1, NRP2 or a neuropilin interacting protein (NIP1 or synectin), and by pre-treatment with soluble NRP1 or NRP1 b1 domain (NRP1 VEGF binding domain). NRP1 knockdown also inhibited the migratory response to PDGF-AA. NRP1 was found to physically interact with PDGFR\alpha, but not with PDGFR\beta, as determined by co-immunoprecipitation. PDGFR\alpha, but not PDGFR\beta, phosphorylation was decreased in response to PDGF-AA and PDGF-BB when NRP1 was knocked down in HCASMC. Intracellular signalling in response to PDGF-BB stimulation was investigated in HCASMCs with NRP1 knockdown. PDGF-BB stimulated tyrosine phosphorylation of the adapter protein p130Cas, which has been stronlgy implicated in cellular and molecular processes involved in cell migration. NRP1 knockdown reduced p130Cas phosphorylation, but had little effect on signalling pathways, such as ERK1/2, Akt, cofilin, Hsp27 and FAK. To investigate the contribution of the NRP1 intracellular domain in PDGF-induced migration and signalling, I generated a NRP1 construct lacking the intracellular domain by introducing a stop codon after the transmembrane domain. Overexpression of NRP1 lacking its C-terminus in HCASMC resulted in a decrease of PDGF-induced migration and activation of phospho-p130Cas. Furthermore, p130Cas knockdown also inhibited PDGF- induced HCASMC migration, thus reinforcing the importance of p130Cas phosphorylation in NRP1-dependent cell migration. The findings that NRP1 is strongly expressed in HCASMC in a CS-GAG and a HS-GAG modified form and plays a role in the chemotactic response to PDGF-BB, highlight the possible involvement of NRPs in neotintima formation in vasculoproliferative diseases