Drug and Alcohol Rehabilitation: On the Specificity of Heparin/Heparan Sulfate Binding to Proteins. Anion-Binding Sites on Antithrombin and Thrombin Are Fundamentally Different.

On the Specificity of Heparin/Heparan Sulfate Binding to Proteins. Anion-Binding Sites on Antithrombin and Thrombin Are Fundamentally Different.

Filed under: Drug and Alcohol Rehabilitation

PLoS One. 2012; 7(11): e48632
Mosier PD, Krishnasamy C, Kellogg GE, Desai UR

BACKGROUND: The antithrombin-heparin/heparan sulfate (H/HS) and thrombin-H/HS interactions are recognized as prototypic specific and non-specific glycosaminoglycan (GAG)-protein interactions, respectively. The fundamental structural basis for the origin of specificity, or lack thereof, in these interactions remains unclear. The availability of multiple co-crystal structures facilitates a structural analysis that challenges the long-held belief that the GAG binding sites in antithrombin and thrombin are essentially similar with high solvent exposure and shallow surface characteristics. METHODOLOGY: Analyses of solvent accessibility and exposed surface areas, gyrational mobility, symmetry, cavity shape/size, conserved water molecules and crystallographic parameters were performed for 12 X-ray structures, which include 12 thrombin and 16 antithrombin chains. Novel calculations are described for gyrational mobility and prediction of water loci and conservation. RESULTS: The solvent accessibilities and gyrational mobilities of arginines and lysines in the binding sites of the two proteins reveal sharp contrasts. The distribution of positive charges shows considerable asymmetry in antithrombin, but substantial symmetry for thrombin. Cavity analyses suggest the presence of a reasonably sized bifurcated cavity in antithrombin that facilitates a firm ‘hand-shake’ with H/HS, but with thrombin, a weaker ‘high-five’. Tightly bound water molecules were predicted to be localized in the pentasaccharide binding pocket of antithrombin, but absent in thrombin. Together, these differences in the binding sites explain the major H/HS recognition characteristics of the two prototypic proteins, thus affording an explanation of the specificity of binding. This provides a foundation for understanding specificity of interaction at an atomic level, which will greatly aid the design of natural or synthetic H/HS sequences that target proteins in a specific manner.
HubMed – drug


Establishment and Characterization of a Highly Tumourigenic and Cancer Stem Cell Enriched Pancreatic Cancer Cell Line as a Well Defined Model System.

Filed under: Drug and Alcohol Rehabilitation

PLoS One. 2012; 7(11): e48503
Fredebohm J, Boettcher M, Eisen C, Gaida MM, Heller A, Keleg S, Tost J, Greulich-Bode KM, Hotz-Wagenblatt A, Lathrop M, Giese NA, Hoheisel JD

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.
HubMed – drug


Micro-Arrayed Human Embryonic Stem Cells-Derived Cardiomyocytes for In Vitro Functional Assay.

Filed under: Drug and Alcohol Rehabilitation

PLoS One. 2012; 7(11): e48483
Serena E, Cimetta E, Zatti S, Zaglia T, Zagallo M, Keller G, Elvassore N

INTRODUCTION: The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs) and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. METHODS: hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. RESULTS: Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, ?-actinin) and functional properties. The spontaneous contraction frequency was (0.83±0.2) Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H(2)O(2). Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H(2)O(2), but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. CONCLUSIONS: Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.
HubMed – drug



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