Syntheses and structures of four antimony complexes with planar tridentate pyridine ligands

The syntheses and structures of four antimony chloride complexes with tridentate N,N,O Schiff base ligands are reported. The tridentate ligands derived from 2-acetylpyridine and various acid hydrazides all lost a proton upon coordination. The ligand was either negative or zwitterionic depending on the acid hydrazide. The complexes are water soluble although a hydrolysis reaction can occur. The appearance of the μ-dichloro-μ-oxo-tetrachlorodiantimonate(III) anion in one of the complexes was unexpected but appears to be related to the temperatures used in the synthesis. The variation in the distances in the various complexes and the anion are discussed using bond valence sum calculations.     

Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism

How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behavior of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.     

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

CYP2J2 epoxygenase is a membrane bound cytochrome P450 that converts omega‐3 and omega‐6 fatty acids into physiologically active epoxides. In this work, we present a comprehensive comparison of the effects of N‐terminal modifications on the properties of CYP2J2 with respect to the activity of the protein in model lipid bilayers using Nanodiscs. We demonstrate that the complete truncation of the N‐terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation in the lipid bilayers of Nanodiscs. Notably, the introduction of silent mutations at the N‐terminus was used to express full length CYP2J2 in E. coli while maintaining wild‐type functionality. We further show that lipid bilayers are essential for the productive use of NADPH for ebastine hydroxylation by CYP2J2. Taken together, it was determined that the presence of the N‐terminus is not as critical as the presence of a membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This suggests that adopting the native‐like conformation of CYP2J2 and cytochrome P450 reductase in lipid bilayers is essential for effective use of reducing equivalents from NADPH for ebastine hydroxylation.     

The adsorption of adrenocorticotropin-(1-24)-tetracosapeptide to lecithin bilayer membranes formed from liposomes

The interaction of the peptide hormone adrenocorticotropin (ACTH_1-24) with solvent-free planar lipid bilayers has been studied by use of the capacitance minimization method. The membranes were formed from artificial vesicles according to the method described by Schindler. In contrast to analogous studies with hexane-containing membranes, experiments with these vesicle-derived bilayers were completely reproducible and gave no indication that ACTH_1-24 spans such hexane-free bilayers.     

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.     

Interactions of angiotensin II with membranes using a combination of differential scanning calorimetry and 31P NMR spectroscopy

Summary This study of angiotensin II (ANG II) membrane interactions uses a combination of³¹P NMR spectroscopy and differential scanning calorimetry (DSC), two valuable and complementary techniques which can provide useful information about the thermotropic and dynamic properties of peptide hormones in membranes. The major conclusion from the calorimetric experiments is that ANG II affects the phase properties of hydrated dipalmitoyl-phosphatidylcholine (DPPC) bilayers by mainly broadening the pretransition area. Preliminary³¹P NMR data seem to confirm the DSC results by showing that ANG II produces a lowering of the pretransition temperature but affects only minimally the main phase transition. In combination, the results from the two methods may indicate that the hormone produces its effects on the phospholipid head groups while its effects on the bilayer alkyl chains are not significant. Such results can be interpreted to mean that ANG II closely interacts with the phospholipid head groups perhaps up to the level of the interface, but does not enter deeper into the membrane bilayer.     

Confocal microscopy of giant vesicles supports the absence of HIV-1 neutralizing 2F5 antibody reactivity to plasma membrane phospholipids

The broadly neutralizing anti-HIV-1 2F5 monoclonal antibody recognizes a gp41 epitope proximal to the viral membrane. Potential phospholipid autoreactivity at cell surfaces has raised concerns about the use of this antibody for development of vaccines or immunotherapy. In this study, confocal microscopy of giant unilamellar vesicles (GUVs) was used to assess 2F5 reactivity with phospholipids assembled into bilayers with surface charge and curvature stress approximating those of the eukaryotic plasma membranes. Antibody partitioning into lipid bilayers required the specific recognition of membrane-inserted epitope, indicating that 2F5 was unable to directly react with GUV phospholipids, even under fluid phase segregation conditions. Our results thus support the feasibility of raising 2F5-like neutralizing responses through vaccination, and the medical safety of mAb infusions.     

Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A2 and Humicola lanuginosa lipase

An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A₂ (PLA₂) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates.