Inhibin in immature rat Sertoli cell conditioned medium: a 32 kDa αβ-B dimer

Abstract Conditioned medium of cultured Sertoli cells from 21-day-old rats was used as starting material for the isolation of inhibin. Inhibin activity was monitored by the dose dependent suppression of the folliclestimulating hormone release of cultured rat pituitary cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the highly purified inhibin preparation revealed a 32 kDa protein after silver staining, which could be separated in subunits of 18 kDa and 12 kDa after reduction. Western blot analysis with an antibody recognizing the 22 N-terminal amino acids of the α-subunit of 32 kDa bovine inhibin confirmed the presence of a 32 kDa inhibin molecule under non-reducing conditions, whereas an 18 kDa a-subunit was found after reduction. An antibody recognizing the β-A subunit of inhibin did not yield a signal after Western blotting. N-terminal amino acid sequence analysis of two highly purified preparations of inhibin obtained using different methods yielded the sequence predicted for a 32 kDa αβ-B dimer on basis of cDNA nucleotide sequence. This result is in agreement with the large excess of β-B over β-A mRNA in the rat testis

The balance between Gα_i-Cdc42/Rac and Gα_12/13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate

The bioactive sphingosine-1-phosphatephosphate (S1P) is present in plasma, bound to carrier proteins, and involved in many physiological processes, including angiogenesis, inflammatory responses, and vascular stabilization. S1P can bind to several G-protein-coupled receptors (GPCRs) activating a number of different signaling networks. At present, the dynamics and relative importance of signaling events activated immediately downstream of GPCR activation are unclear. To examine these, we used a set of fluorescence resonance energy transfer-based biosensors for different RhoGTPases (Rac1, RhoA/B/C, and Cdc42) as well as for heterotrimeric G-proteins in a series of live-cell imaging experiments in primary human endothelial cells. These experiments were accompanied by biochemical GTPase activity assays and transendothelial resistance measurements. We show that S1P promotes cell spreading and endothelial barrier function through S1PR1-Gαi-Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-Gα12/13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gαi-mediated signaling is suppressed. Our results suggest that Gαq activity is not involved in S1P-mediated regulation of barrier integrity. Moreover, we show that early activation of RhoA by S1P inactivates Rac1 but not Cdc42, and vice versa. Together, our data show that the rapid S1P-induced increase in endothelial integrity is mediated by a S1PR1-Gαi-Cdc42 pathwa

Identification of guanine nucleotide exchange factors that increase Cdc42 activity in primary human endothelial cells

The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology

‘The war is a money making show’: Working-Class Attitudes to World War II and Australian Nationalism

This paper will address the conference themes of ‘class, power and social structure’ through examining industrial and ideological conflict during World War II. The paper will also address the theme of ‘class and culture’ through an examination of working-class cultural expression as a means of resistance to the government’s wartime offensive. What is overlooked in most histories of World War II is the working-class experience of the war and their understanding of nationalism, particularly as nationalism was cynically exploited by the government to undermine working-class identity and solidarity. The paper will investigate the experience of one of the most militant sections of the Australian working class: the Miners. Primary source material such as the Miners’ journal Common Cause and union records reveal opposition to the war and a much more ambiguous attitude to the national sentiment used to justify Australia’s involvement. The Miners provide an interesting case study as the union was led by the Communist Party. Therefore the union leadership initially opposed to the war then became enthusiastic supporters when Russia entered the war on the allied side. It is clear that the Miners’ union leadership found it difficult to convince the rank and file to support the war. The paper will focus upon rank and file attitudes to the war and Australian nationalism particularly during times of industrial unrest.The symposium is organised on behalf of AAHANZBS by the Business and Labour History Group, The University of Sydney, with the financial support of the University’s Faculty of Economics and Business

The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions

Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue