PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 15502188

Cell kinetic analysis in artificial skin using immunochemical methods.

Andrea Casasco·Antonia Icaro Cornaglia·Federica Riva·Marco Casasco·Alberto Calligaro
View original source ↗Export metadata ↓

Abstract

Cell kinetic studies provide important information on histogenesis in vivo and in vitro. In this regard, specific antibodies to cell cycle-related antigens have been raised and characterized, thus permitting the study of cell kinetics using immunochemical methods. Recent advances in culture technology permitted the generation of human skin equivalents in vitro. We here provide detailed practical procedures for the study of epidermal cell kinetics in a model of artificial skin using immunohistochemistry and flow cytometry. The combined application of both techniques allows a precise detection of tissue growth sites and a quantitative assessment of cell growth. Moreover, simultaneous analysis of differentiation markers and proliferation antigens may be useful to understand molecular mechanisms that regulate tissue growth and development.

Explore related subjects

Cell CycleEpidermal CellsEpidermisFlow CytometryHumansImmunohistochemistryKeratinocytesKi-67 AntigenProliferating Cell Nuclear AntigenSkin, ArtificialJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Andrea Casasco, Antonia Icaro Cornaglia, Federica Riva, Marco Casasco, Alberto Calligaro. 2005. Cell kinetic analysis in artificial skin using immunochemical methods.. https://doi.org/10.1385/1-59259-830-7%3A229

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2017

Rapid DNA replication origin licensing protects stem cell pluripotency.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

Cell Cycle↗
PubMed2007

Modelling the checkpoint response to telomere uncapping in budding yeast.

One of the DNA damage-response mechanisms in budding yeast is temporary cell-cycle arrest while DNA repair takes place. The DNA damage response requires the coordinated interaction between DNA repair and checkpoint pathways. Telomeres of budding yeast are capped by the Cdc13 complex. In the temperature-sensitive cdc13-1 strain, telomeres are unprotected over a specific temperature range leading to activation of the DNA damage response and subsequently cell-cycle arrest. Inactivation of cdc13-1 results in the generation of long regions of single-stranded DNA (ssDNA) and is affected by the activity of various checkpoint proteins and nucleases. This paper describes a mathematical model of how uncapped telomeres in budding yeast initiate the checkpoint pathway leading to cell-cycle arrest. The model was encoded in the Systems Biology Markup Language (SBML) and simulated using the stochastic simulation system Biology of Ageing e-Science Integration and Simulation (BASIS). Each simulation follows the time course of one mother cell keeping track of the number of cell divisions, the level of activity of each of the checkpoint proteins, the activity of nucleases and the amount of ssDNA generated. The model can be used to carry out a variety of in silico experiments in which different genes are knocked out and the results of simulation are compared to experimental data. Possible extensions to the model are also discussed.

Cell Cycle↗
PubMed2007

Identification of Rab11 as a small GTPase binding protein for the Evi5 oncogene.

The Evi5 oncogene has recently been shown to regulate the stability and accumulation of critical G(1) cell cycle factors including Emi1, an inhibitor of the anaphase-promoting complex/cyclosome, and cyclin A. Sequence analysis of the amino terminus of Evi5 reveals a Tre-2, Bub2, Cdc16 domain, which has been shown to be a binding partner and GTPase-activating protein domain for the Rab family of small Ras-like GTPases. Here we describe the identification of Evi5 as a candidate binding protein for Rab11, a GTPase that regulates intracellular transport and has specific roles in endosome recycling and cytokinesis. By yeast two-hybrid analysis, immunoprecipitation, and Biacore analysis, we demonstrate that Evi5 binds Rab11a and Rab11b in a GTP-dependent manner. However, Evi5 displays no activation of Rab11 GTPase activity in vitro. Evi5 colocalizes with Rab11 in vivo, and overexpression of Rab11 perturbs the localization of Evi5, redistributing it into Rab11-positive recycling endosomes. Interestingly, in vitro binding studies show that Rab11 effector proteins including FIP3 compete with Evi5 for binding to Rab11, suggesting a partitioning between Rab11-Evi5 and Rab11 effector complexes. Indeed, ablation of Evi5 by RNA interference causes a mislocalization of FIP3 at the abscission site during cytokinesis. These data demonstrate that Evi5 is a Rab11 binding protein and that Evi5 may cooperate with Rab11 to coordinate vesicular trafficking, cytokinesis, and cell cycle control independent of GTPase-activating protein function.

Cell Cycle↗