Thyroid control over biomembranes: VI. Lipids in liver mitochondria and microsomes of hypothyroid rats

The lipids of liver mitochondria prepared from normal rats and from rats made hypothyroid by thyroidectomy and injection with131INa contained similar amounts, per mg protein, of total lipids, phospholipids, neutral lipids and lipid phosphorus. Hypothyroidism caused a doubling of the relative amounts of mitochondrial cardiolipins (CL; to 20.5% of the phospholipid P) and an accompanying trend (although statistically not significant) toward decreased amounts of both phosphatidylcholines (PC) and phosphatidylserines (PS), with phosphatidylethanolamines (PE) remaining unchanged. The pattern of elevated 18∶2 fatty acyl content and depleted 20∶4 acyl groups of the mitochondrial phospholipids of hypothyroid preparations was reflected to varying degrees in the resolved phospholipids, with PC showing greater degrees of abnormality than PE, and CL showing none. Hypothyroidism produced the same abnormal pattern of fatty acyl distributions in liver microsomal total lipids as was found in the mitochondria. Hypothyroid rats, when killed 6 hr after injection of [1‐14C] labeled linoleate, showed the following abnormalities: the liver incorporated less label into lipids, and converted 18∶2 not exclusively to 20∶4 (as normals do) but instead incorporated the label mainly into saturated fatty acids. These data, together with the known decrease in β‐oxidation, suggest that hypothyroidism involves possible defective step(s) in the conversion of 18∶2 to 20∶4.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142296/1/lipd0328.pd

GPCR expression using baculovirus-infected Sf9 cells

Expression of proteins in insect cells using recombinant baculoviruses has gained wide use in the G protein-coupled receptor (GPCR) community. This expression system produces high yields of functional receptor, is able to perform post-translational modifications, and is readily adaptable to large-scale culture. Here, we describe the generic methods for expressing a GPCR using baculovirus-infected insect cells, including the maintenance of insect cell culture. Data are presented for polyhedrin promoter-driven expression of a C-terminal 6 x histidine-tagged mammalian M(2) muscarinic receptor in Sf9 cells. Results demonstrate that expressed receptor could be detected and quantified using radiolabeled ligand binding, that expression was maximal at approximately 72 h post-infection, and that expression levels could be altered by addition of various ligands to cultures of infected insect cells.Amanda L. Aloia, Richard V. Glatz, Edward J. McMurchie, and Wayne R. Leifer

Scale-up and economic analysis of biodiesel production from recycled grease trap waste

Grease trap waste has been considered as a cost-effective feedstock for biodiesel production due to its high lipid content and relatively low cost for collection. However, the costly pre-treatment of this contaminated resource is currently the barrier to the commercialization of biodiesel. This study analyses the economic feasibility of biodiesel production from grease trap waste collected in Adelaide (South Australia), focussing on the environmental service providers as the potential biodiesel producers. Based on the experimental results, two different production routes with the same capacity of around 4400 t/year were simulated using Aspen Plus® V8.8, these being; esterification without using acetone as a co-solvent (1); and esterification using a co-solvent of acetone-ethanol (2). The best production price of biodiesel obtained was US$1337.5/t which would indicate that grease trap waste may be a promising feedstock for biodiesel production

Cell-free receptor-based biosensors

The ability to express and purify modified recombinant signalling proteins such that they retain their biological function in a cell-free context has provided a basis for production of molecular biosensors. Here the authors utilise G-protein coupled receptors (GPCRs) and their G-proteins to detect various binding partners in a cell-free environment. Molecular biology approaches were employed to express these proteins using baculovirus and bacteria, and to alter their characteristics to improve surface-attachment and fluorescent labelling capabilities. Ligand-mediated signalling of a GPCR could be measured (using [35S]GTPgammaS-binding assays) in a reconstituted system with recombinant proteins either free in solution or attached to Ni2+-coated beads. Affinity of histidine-tagged proteins for a Ni2+-coated surface was significantly enhanced by addition of extra histidine residues to the tag, as determined by surface plasmon resonance. This was due to the longer tag occupying, on average, a greater number of available histidine-binding sites. Further, a novel homogeneous fluorescence resonance energy transfer (FRET)-based assay has been developed to detect rearrangements in the G-protein heterotrimer. Investigation of small peptides that can be fused to G-protein subunits, allowing for site-specific fluorescent labelling, was undertaken in order to improve the resolution of the "first generation" FRET assay. By utilizing this improved G-protein heterotrimer "molecular switch", we are developing a generic technology such that a range of GPCRs could be assayed for ligand-mediated activation while attached to surfaces (e.g. on beads or as microarrays) or in solution (e.g. multi-well plates), with increased throughput.Richard V. Glatz, Wayne R. Leifert, Kelly Bailey, Tamara H. Cooper, Chris S. Barton, A. Scott Martin, Amanda Aloia, Olgatina Bucco, Lakshmi Waniganayake, Gang Wei, Burkhard Raguse, Lech Wieczorek, and Edward J. McMurchi