Regulation of Smooth Muscle Cell Proliferation by β-Catenin/T-Cell Factor Signaling Involves Modulation of Cyclin D1 and p21 Expression

We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of β-catenin. In this study we demonstrate directly that growth factors stimulate β-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether β-catenin/TCF signaling regulates VSMC proliferation via modulation of the β-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited β-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of β-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by β-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting β-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and β-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 β-catenin/TCF responsive genes

Regulation of smooth muscle cell proliferation by β-catenin/T-cell factor signaling involves modulation of cyclin D1 and p21 expression

We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of β-catenin. In this study we demonstrate directly that growth factors stimulate β-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether β-catenin/TCF signaling regulates VSMC proliferation via modulation of the β-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited β-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of β-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by β-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting β-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and β-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 β-catenin/TCF responsive genes

Body color pattern and aggressiveness related to behavioral context and light intensity in an Amazonian cichlid, Laetacara fulvipinnis

The aim of this study was to describe agonistic interaction and color patterns relative to social status and to evaluate the effects of light on aggressiveness in Laetacara fulvipinnis. Eight agonistic events were observed: frontal display, mouth fight, nipping, chase, parallel display, threat, undulation, and flight. Four body color patterns were related to the social context. Two light levels were also tested: groups of juveniles were maintained for 15 days under low (228 ± 38.60 lx) or high (1435.92 ± 481.40 lx) light. Color pattern a was mainly observed during escalating agonistic interactions. Other patterns were exhibited during flight, rest, and attack. High light decreased the latency to fighting; increased the frequency of threats, total attacks, and flight; and destabilized the social hierarchy. These findings indicate that light intensity increases aggressiveness, interferes with the stability of hierarchies, and may result in a stressful situation with negative effects on animal welfare. © 2017 Informa UK Limited, trading as Taylor & Francis Group

Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.

peer reviewedAgrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop

Exploring the Fuzzy Boundaries of Discourse Markers Through Manual and Automatic Annotation

Discourse markers are non-propositional linguistic items that are notoriously difficult to identify as well as to categorize. We can observe several borderline phenomena and overlaps with other formal and functional categories, for example inserts, adverbials, contextualization cues, pragmatic force modifiers and so on. By way of addressing such overlaps as well as the disambiguation between discourse marker uses and their source categories, the chapter presents a comparison of automated and manual annotation of oral discourse markers (discourse markers). Firstly, an overview of the criterial features of discourse markers that are relevant to disambiguation is presented. Secondly, the UCREL Semantic Analysis System (USAS) and its disambiguation methods are briefly discussed. In the third part of the chapter manual and automatic decisions about categorization are compared with a view to addressing the margin of error reported to apply in general semantic annotation as well as the question of what formal-functional properties of the relevant discourse markers might explain possible differences between manual and automatic annotation