Exploring oxidative modifications of tyrosine:an update on mechanisms of formation, advances in analysis and biological consequences

Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

Preparation and characterization of gas-filled liposomes:Can they improve oil recovery?

Although well known for delivering various pharmaceutical agents, liposomes can be prepared to entrap gas rather than aqueous media and have the potential to be used as pressure probes in magnetic resonance imaging (MRI). Using these gas-filled liposomes (GFL) as tracers, MRI imaging of pressure regions of a fluid flowing through a porous medium could be established. This knowledge can be exploited to enhance recovery of oil from the porous rock regions within oil fields. In the preliminary studies, we have optimized the lipid composition of GFL prepared using a simple homogenization technique and investigated key physico-chemical characteristics (size and the physical stability) and their efficacy as pressure probes. In contrast to the liposomes possessing an aqueous core which are prepared at temperatures above their phase transition temperature (Tc), homogenization of the phospholipids such as 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocoline (DSPC) in aqueous medium below their Tc was found to be crucial in formation of stable GFL. DSPC based preparations yielded a GFL volume of more than five times compared to their DPPC counter part. Although the initial vesicle sizes of both DSPC and DPPC based GFL were about 10 μm, after 7 days storage at 25°C, the vesicle sizes of both formulations significantly (p < 0.05) increased to 28.3 ± 0.3 μm and 12.3 ± 1.0 μm, respectively. When the DPPC preparation was supplemented with cholesterol at a 1:0.5 or 1:1 molar ratio, significantly (p < 0.05) larger vesicles were formed (12-13 μm), however, compared to DPPC only vesicles, both cholesterol supplemented formulations displayed enhanced stability on storage indicating a stabilizing effect of cholesterol on these gas-filled vesicles. In order to induce surface charge on the GFL, DPPC and cholesterol (1: 0.5 molar ratio) liposomes were supplemented with a cationic surfactant, stearylamine, at a molar ratio of 0.25 or 0.125. Interestingly, the ζ potential values remained around neutrality at both stearylamine ratios suggesting the cationic surfactant was not incorporated within the bilayers of the GFL. Microscopic analysis of GFL confirmed the presence of spherical structures with a size distribution between 1-8 μm. This study has identified that DSPC based GFL in aqueous medium dispersed in 2% w/v methyl cellulose although yielded higher vesicle sizes over time were most stable under high pressures exerted in MRI. Copyright © Informa Healthcare USA, Inc.

Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Synthesis of hydrazine by the fixation process

A critical review of the literature concerning organic derivatives of hydrazine, the ammonia-chlorine reaction and the electrolytic formation of hydrazine has been carried out. Apparatus was constructed to study the electrolysis of liquid ammonia, the formation of chloramine and the fixation of chloramine with a ketone to form an isohydrazone. In the latter case the reaction was carried out in a 3" diameter stirred tank and also in a 1" diameter, 2' high column reactor where the liquid phase was continuously recirculated. Two methods of analysis of azines and isohydrazones in a ketone solution have been developed. One is a colorimetric technique using p-dimethylaminobenzaldehyde and the other involves the hydrolysis of the organic derivative to hydrazine sulphate. Hydrazine was detected in low concentration in some of the electrolytic experiments carried out but it was concluded that this method did not show sufficient promise to warrant further investigation. The gas phase formation of chloramine and acetone isohydrazone has also been studied but in this system difficulties were encountered with the chlorine jet blocking with ammonium chloride. The formation of isohydrazones in a stirred tank reactor has been investigated in some detail and the effect of several parameters was determined. The yield was found to be extremely sensitive to chlorine concentration and in order to obtain yields of more than 90 per cent, the molar concentration of chlorine in the gas phase had to be of the order of 5 per cent. An optimum temperature in the region of 0°C was also detected. These results disagree with those quoted in previous studies but extensive experimental work has confirmed the information presented in this thesis. It has also been shown that at high yields the chloramine formation reaction took place in the gas phase.

High-sensitivity differential scanning calorimetry for measurement of steroid entrapment in nebulised liposomes generated from proliposomes

A novel approach to the determination of steroid entrapment in the bilayers of aerosolised liposomes has been introduced using high-sensitivity differential scanning calorimetry (DSC). Proliposomes were dispersed in water within an air-jet nebuliser and the energy produced during atomisation was used to hydrate the proliposomes and generate liposome aerosols. Proliposomes that included the steroid beclometasone dipropionate (BDP) produced lower aerosol and lipid outputs than steroid-free proliposomes. Size analysis and transmission electron microscopy showed an evidence of liposome formation within the nebuliser, which was followed by deaggregation and size reduction of multilamellar liposomes on nebulisation to a two-stage impinger. For each formulation, no difference in thermal transitions was observed between delivered liposomes and those remaining in the nebuliser. However, steroid (5 mole%) lowered the onset temperature and the enthalpy of the pretransition, and produced a similar onset temperature and larger enthalpy of the main transition, with broadened pretransition and main transitions. This indicates that BDP was entrapped and exhibited an interaction with the liposome phospholipid membranes. Since the pretransition was depressed but not completely removed and no phase separation occurred, it is suggested that the bilayers of the multilamellar liposomes can entrap more than 5 mole% BDP. Overall, liposomes were generated from proliposomes and DSC investigations indicated that the steroid was entrapped in the bilayers of aerosolised multilamellar vesicles.