Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

Mg2+ modulates integrin–extracellular matrix interaction in vascular smooth muscle cells studied by atomic force microscopy

Atomic force microscopy (AFM) was used to investigate the interaction between α5β1 integrin and fibronectin (FN) in the presence of divalent cations. AFM probes were labeled with FN and used to measure binding strength between α5β1 integrin and FN by quantifying the force required to break single FN–integrin bonds on a physiological range of loading rates (100–10 000 pN/s). The force necessary to rupture single α5β1–FN bond increased twofold over the regime of loading rates investigated. Changes in Mg²⁺ and Ca²⁺ concentration affected the thermodynamical parameters of the interaction and modulated the binding energy. These data indicate that the external ionic environment in which vascular smooth muscle cells reside, influences the mechanical parameters that define the interaction between the extracellular matrix and integrins. Thus, in a dynamic mechanical environment such as the vascular wall, thermodynamic binding properties between FN and α5β1 integrin vary in relation to locally applied loads and divalent cations concentrations. These changes can be recorded as direct measurements on live smooth muscle cells by using AFM. Copyright © 2009 John Wiley & Sons, Ltd.     

Does being Roma matter? Ethnic boundaries and stigma spillover in musical consumption

Manele songs, an updated version of traditional Romani music, are excluded from mainstream Romanian media due to their association with the country’s large Roma minority. The genre is at the intersection between Romania’s democratic transition and growing efforts to strengthen boundaries between the country’s marginalized Romani minority and the non-Romani majority population. But we find that media discussions around manele underscore a fluid relationship between ethnic boundaries and stigmatized cultural consumption. All those who listen to manele are portrayed in negative terms by the media, no matter whether ethnic markers are used as part of the portrayal. In the context of manele, genre-based stereotypes extend beyond ethnic boundaries to assign negative social value to a wide swathe of people who consume the genre. The stigma spillover surrounding manele audiences therefore underlines the ways in which Roma identity is fluid, and anyone associated with the Roma can be relegated to the lower rungs of social status. Manele show how the political process of ethnic boundary-making draws a wide net over those located at the bottom of power hierarchies.     

Linguistic evidence supports date for Homeric epics

The Homeric epics are among the greatest masterpieces of literature, but when they were produced is not known with certainty. Here we apply evolutionary‐linguistic phylogenetic statistical methods to differences in Homeric, Modern Greek and ancient Hittite vocabulary items to estimate a date of approximately 710–760 BCE for these great works. Our analysis compared a common set of vocabulary items among the three pairs of languages, recording for each item whether the words in the two languages were cognate – derived from a shared ancestral word – or not. We then used a likelihood‐based Markov chain Monte Carlo procedure to estimate the most probable times in years separating these languages given the percentage of words they shared, combined with knowledge of the rates at which different words change. Our date for the epics is in close agreement with historians' and classicists' beliefs derived from historical and archaeological sources.     

Computer simulations of in vitro morphogenesis

One of the most important challenges of contemporary biology is understanding how cells assemble into tissues. The complexity of morphogenesis calls for computational tools able to identify the dominant mechanisms involved in shaping tissues. This narrative review presents individual-based computational models that proved useful in simulating phenomena of interest in tissue engineering (TE), a research field that aims to create tissue replacements in the laboratory. First, we briefly describe morphogenetic mechanisms. Then, we present several computational models of cellular and subcellular resolution, along with applications that illustrate their potential to address problems of TE. Finally, we analyze experiments that may be used to validate computational models of tissue constructs made of cohesive cells. Our analysis shows that the models available in the literature are not exploited to their full potential. We argue that, upon validation, a computational model can be used to optimize cell culture conditions and to design new experiments.     

Fluidization, resolidification, and reorientation of the endothelial cell in response to slow tidal stretches

Mechanical stretch plays an important role in regulating shape and orientation of the vascular endothelial cell. This morphological response to stretch is basic to angiogenesis, neovascularization, and vascular homeostasis, but mechanism remains unclear. To elucidate mechanisms, we used cell mapping rheometry to measure traction forces in primary human umbilical vein endothelial cells subjected to periodic uniaxial stretches. Onset of periodic stretch of 10% strain amplitude caused a fluidization response typified by attenuation of traction forces almost to zero. As periodic stretch continued, the prompt fluidization response was followed by a slow resolidification response typified by recovery of the traction forces, but now aligned along the axis perpendicular to the imposed stretch. Reorientation of the cell body lagged reorientation of the traction forces, however. Together, these observations demonstrate that cellular reorientation in response to periodic stretch is preceded by traction attenuation by means of cytoskeletal fluidization and subsequent traction recovery transverse to the stretch direction by means of cytoskeletal resolidification.     

Elevated expression of cav-1 in a subset of SSc fibroblasts contributes to constitutive Alk1/Smad1 activation

Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.