Adjuvant properties of a simplified C32 monomycolyl glycerol analogue

A simplified C₃₂ monomycolyl glycerol (MMG) analogue demonstrated enhanced immunostimulatory activity in a dioctadecyl ammonium bromide (DDA)/Ag85B-ESAT-6 formulation. Elevated levels of IFN-γ and IL-6 were produced in spleen cells from mice immunised with a C₃₂ MMG analogue comparable activity to the potent Th1 adjuvant, trehalose 6,6′-di-behenate (TDB).     

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 (T_c), 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 T_c 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.     

Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products

The advantages of liposomes as delivery systems for peptide, protein and DNA vaccines is well-recognised, unfortunately their application has been stinted by their instability during storage and their limited shelf-life. Further, sterilisation of these systems has been problematic, with degradation of the liposomes being reported after sterilisation using the various techniques available. Work form our laboratory has investigated techniques that can be applied to particulate liposomal vaccines such that they can be prepared in a freeze-dried and sterile format. In this article, we describe techniques for the lyophilisation, cryoprotection and sterilisation of liposomal vaccines. Applying these methods allows for the retention of both the chemical integrity of the lipids and the key physico-chemical characteristics of the liposomes (e.g., particle size, zeta potential, and dynamic viscosity), thus supporting the enhanced transition of liposomal vaccines from the bench to the clinic.     

An electrophoretic study of the low-molecular-weight components of myosin

1. The low-molecular-weight components of myosin freshly prepared by the standard procedure from adult rabbit skeletal muscle migrated as four main bands Ml(1), Ml(2), Ml(3) and Ml(4) on polyacrylamide-gel electrophoresis in 8m-urea. 2. The number of bands increased on storage. This change was accelerated by increasing the temperature and pH. 3. None of the bands had electrophoretic mobilities identical with those of the well-characterized proteins of the myofibril or with the sarcoplasmic proteins. 4. By varying the ionic conditions and concentration of muscle mince used for the initial extraction it was possible to change the relative proportions of the two electrophoretic bands of intermediate mobility, Ml(2) and Ml(3). 5. The four-band picture similar to that obtained with rabbit was observed with myosin isolated from skeletal muscle of the rat, mouse, hamster, pigeon and chicken. 6. Rabbit cardiac myosin gave only two bands on electrophoresis. Myosin from rabbit red muscle gave a pattern intermediate between cardiac and white-skeletal-muscle myosin, i.e. the two fastest bands were present in decreased relative amounts. 7. It is suggested that the differences in the low-molecular-weight components of myosin from different types of muscle are a consequence of differences in the isoenzyme composition of the myosins.     

A phosphorylated light-chain component of myosin from skeletal muscle

1. The low-molecular-weight components of myosin from rabbit skeletal muscle migrated as four bands on polyacrylamide-gel electrophoresis in 8m-urea but only as three in systems containing sodium dodecyl sulphate. The two bands of intermediate mobility in 8m-urea (Ml(2) and Ml(3)) had identical mobilities in sodium dodecyl sulphate. 2. The isolation of pure samples of all four low-molecular-weight components by DEAE-Sephadex chromatography is described. 3. The amino acid compositions of components Ml(2) and Ml(3) were identical. Further analyses showed the presence of 1 mol of phosphate/18500g of component Ml(2) and less than 10% of this amount in component Ml(3). Neither light component contained ribose. 4. Alkaline phosphatase from Escherichia coli converted component Ml(2) into Ml(3). Incubation with crude preparations of phosphorylase b kinase or protein kinase in the presence of ATP converted component Ml(3) into Ml(2). 5. Phosphorylation of component Ml(3) with the kinases isolated from skeletal muscle and [gamma-(32)P]ATP gave incorporation of (32)P only into component Ml(2) whether whole myosin or separated low-molecular-weight components were used. 6. High-voltage electrophoresis at pH6.5 and pH1.8 of a chymotryptic digest of (32)P-labelled component Ml(2) yielded one major radioactive peptide containing serine phosphate. 7. The amino acid sequence of this peptide was shown to be: Arg-Ala-Ala-Ala-Glu-Gly-Gly-(Ser,Ser(P))-Asn-Val-Phe. This sequence shows no obvious similarity to the site phosphorylated in the conversion of phosphorylase b into phosphorylase a by phosphorylase b kinase. 8. Evidence suggests that in vivo all the 18500-molecular-weight light chain is in the phosphorylated form. The extent of dephosphorylation that occurred during myosin extraction depended on the conditions employed.     

Elevated renal norepinephrine in proANP gene-disrupted mice is associated with increased tyrosine hydroxylase expression in sympathetic ganglia

The sympatholytic properties of atrial natriuretic peptide (ANP) contribute to its vasodilatory and natriuretic effects. High circulating catecholamine levels, along with renal dysfunction, present in proANP gene-disrupted (-/-) mice are thought to contribute to the hypertension characteristic of this model. To further understand the mechanism by which the absence of ANP leads to stimulation of sympathetic activity we measured tyrosine hydroxylase expression in mice with and without ANP. The adrenal and prevertebral ganglionic expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine production, was significantly increased in ANP -/- mice. ANP's sympatholytic properties include the depression of ganglionic and adrenal TH expression and catecholamine production. Thus, these results suggest that the absence of ANP's sympatholytic effects is not completely compensated for in ANP -/- mice. In addition, mice devoid of ANP display an increase in renal sympathetic nerve activity from birth through to adulthood which may lead to structural and/or functional changes in the early postnatal kidney that contribute to the hypertensive phenotype of ANP -/- mice. The over-activation of the sympathetic nervous system in mice lacking ANP confirms the important role of this peptide in the modulation of sympathetic nerve activity and its contribution to blood pressure homeostasis.     

Regulation of the cardiomyocyte population in the developing heart

During fetal life the myocardium expands through replication of cardiomyocytes. In sheep, cardiomyocytes begin the process of becoming terminally differentiated at about 100 gestation days out of 145 days term. In this final step of development, cardiomyocytes become binucleated and stop dividing. The number of cells at birth is important in determining the number of cardiomyocytes for life. Therefore, the regulation of cardiomyocyte growth in the womb is critical to long term disease outcome. Growth factors that stimulate proliferation of fetal cardiomyocytes include angiotensin II, cortisol and insulin-like growth factor-1. Increased ventricular wall stress leads to short term increases in proliferation but longer-term loss of cardiomyocyte generative capacity. Two normally circulating hormones have been identified that suppress proliferation: atrial natriuretic peptide (ANP) and tri-iodo-l-thyronine (T₃). Atrial natriuretic peptide signals through the NPRA receptor that serves as a guanylate cyclase and signals through cGMP. ANP powerfully suppresses mitotic activity in cardiomyocytes in the presence of angiotensin II in culture. Addition of a cGMP analog has the same effect as ANP. ANP suppresses both the extracellular receptor kinases and the phosphoinositol-3 kinase pathways. T₃ also suppresses increased mitotic activity of stimulated cardiomyocytes but does so by increasing the cell cycle suppressant, p21, and decreasing the cell cycle activator, cyclin D1.     

Microsatellite DNA analyses of a highly disjunct New Zealand tree reveal strong differentiation and imply a formerly more continuous distribution

Although New Zealand is a biodiversity hotspot, there has been little genetic investigation of why so many of its threatened and uncommon plants have naturally disjunct distributions. We investigated the small tree Pseudopanax ferox (Araliaceae), which has a widespread but highly disjunct lowland distribution within New Zealand. Genotyping of nuclear microsatellites and a chloroplast locus revealed pronounced genetic differentiation and four principal genetic clusters. Our results indicate that the disjunct distribution is a product of vicariance rather than long‐distance dispersal. This highlights the need to preserve multiple populations when disjunct distributions are the result of vicariance, rather than focusing conservation efforts on a core area, in order to retain as much as possible of a species’ evolutionary legacy and potential. Additionally, based on our genetic findings and the ecology of P. ferox, we hypothesize that it was more continuously distributed during the drier (but not maximally colder) interstadials of glacial periods and/or on the fertile soils available immediately postglacial. We further hypothesize that P. ferox belongs to a suite of species of drought‐prone and/or fertile habitats whose distributions are actually restricted during warmer and wetter interglacial periods, despite being principally of the lowlands. Our genetic data for P. ferox are also the first consistent with the survival during the Last Glacial Maxima of a lowland tree at high latitudes in the south‐eastern South Island.