Solid Phase Synthesis of a Functionalized Bis-Peptide Using …
Published 5/15/2012
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In JoVE (1)
Other Publications (4)
Articles by Wissam A. AbouAlaiwi in JoVE
Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease
1Department of Pharmacology, University of Toledo, College of Pharmacy and Pharmaceutical Sciences, 2Department of Emergency and Intensive Care, ProMedica Sponsored Research
Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.
Other articles by Wissam A. AbouAlaiwi on PubMed
Ciliary Polycystin-2 is a Mechanosensitive Calcium Channel Involved in Nitric Oxide Signaling Cascades
Cardiovascular complications such as hypertension are a continuous concern in patients with autosomal dominant polycystic kidney disease (ADPKD). The PKD2 encoding for polycystin-2 is mutated in approximately 15% of ADPKD patients. Here, we show that polycystin-2 is localized to the cilia of mouse and human vascular endothelial cells. We demonstrate that the normal expression level and localization of polycystin-2 to cilia is required for the endothelial cilia to sense fluid shear stress through a complex biochemical cascade, involving calcium, calmodulin, Akt/PKB, and protein kinase C. In response to fluid shear stress, mouse endothelial cells with knockdown or knockout of Pkd2 lose the ability to generate nitric oxide (NO). Consistent with mouse data, endothelial cells generated from ADPKD patients do not show polycystin-2 in the cilia and are unable to sense fluid flow. In the isolated artery, we further show that ciliary polycystin-2 responds specifically to shear stress and not to mechanical stretch, a pressurized biomechanical force that involves purinergic receptor activation. We propose a new role for polycystin-2 in transmitting extracellular shear stress to intracellular NO biosynthesis. Thus, aberrant expression or localization of polycystin-2 to cilia could promote high blood pressure because of inability to synthesize NO in response to an increase in shear stress (blood flow).
A Comparative Study of Embedded and Anesthetized Zebrafish in Vivo on Myocardiac Calcium Oscillation and Heart Muscle Contraction
The zebrafish (Danio rerio) has been used as a model for studying vertebrate development in the cardiovascular system. In order to monitor heart contraction and cytosolic calcium oscillations, fish were either embedded in methylcellulose or anesthetized with tricaine. Using high-resolution differential interference contrast and calcium imaging microscopy, we here show that dopamine and verapamil alter calcium signaling and muscle contraction in anesthetized zebrafish, but not in embedded zebrafish. In anesthetized fish, dopamine increases the amplitude of cytosolic calcium oscillation with a subsequent increase in heart contraction, whereas verapamil decreases the frequency of calcium oscillation and heart rate. Interestingly, verapamil also increases myocardial contraction. Our data further indicate that verapamil can increase myocardial calcium sensitivity in anesthetized fish. Taken together, our data reinforce in vivo cardiac responses to dopamine and verapamil. Furthermore, effects of dopamine and verapamil on myocardial calcium and contraction are greater in anesthetized than embedded fish. We suggest that while the zebrafish is an excellent model for a cardiovascular imaging study, the cardio-pharmacological profiles are very different between anesthetized and embedded fish.
Endothelial Cells from Humans and Mice with Polycystic Kidney Disease Are Characterized by Polyploidy and Chromosome Segregation Defects Through Survivin Down-regulation
Autosomal-dominant polycystic kidney disease (ADPKD) is the most common hereditary and systemic disorder associated with various cardiovascular complications. It has been implicated with dysfunction in primary cilia. We and others have shown that the immediate function of endothelial cilia is to sense extracellular signal. The long-term function of cilia is hypothesized to regulate cell cycle. Here, we show that ciliary function (polycystins) and structure (polaris) are required for proper cellular division. Cilia mutant cells undergo abnormal cell division with apparent defects in mitotic spindle formation, cellular spindle assembly checkpoint and centrosome amplification. Down-regulation of the chromosomal passenger survivin contributes to these abnormalities, which further result in cell polyploidy. Re-expression of survivin restores a competent spindle assembly checkpoint and reduces polyploidy. Aged animals show a more severe phenotype in cellular division, consistent with progression of cardiovascular complications seen in older ADPKD patients. For the first time, we show that structure and function of mechanosensory cilia are crucial in maintaining proper cellular proliferation. Furthermore, developmental aging plays a crucial role in the progression of these abnormal cellular phenotypes. We propose that abnormal function or structure of primary cilia not only causes failure to transmit extracellular signals, but also is associated with cytokinesis defects in both mice and humans with polycystic kidney disease.
Primary Cilia Regulates the Directional Migration and Barrier Integrity of Endothelial Cells Through the Modulation of Hsp27 Dependent Actin Cytoskeletal Organization
Cilia are mechanosensing organelles that communicate extracellular signals into intracellular responses. Altered functions of primary cilia play a key role in the development of various diseases including polycystic kidney disease. Here, we show that endothelial cells from the oak ridge polycystic kidney (Tg737(orpk/orpk) ) mouse, with impaired cilia assembly, exhibit a reduction in the actin stress fibers and focal adhesions compared to wild-type (WT). In contrast, endothelial cells from polycystin-1 deficient mice (pkd1(null/null) ), with impaired cilia function, display robust stress fibers, and focal adhesion assembly. We found that the Tg737(orpk/orpk) cells exhibit impaired directional migration and endothelial cell monolayer permeability compared to the WT and pkd1(null/null) cells. Finally, we found that the expression of heat shock protein 27 (hsp27) and the phosphorylation of focal adhesion kinase (FAK) are downregulated in the Tg737(orpk/orpk) cells and overexpression of hsp27 restored both FAK phosphorylation and cell migration. Taken together, these results demonstrate that disruption of the primary cilia structure or function compromises the endothelium through the suppression of hsp27 dependent actin organization and focal adhesion formation, which may contribute to the vascular dysfunction in ciliopathies.