Protein Expression and Purification of Human Hormone Sensitive Lipase

The purpose of this project was to develop a new product, purified human hormone sensitive lipase (HSL), for Blue Sky Biotech\u27s product line. This was accomplished by cloning the HSL gene by gene synthesis, amplifying the annealed templates by PCR, cloning the amplicons into a plasmid in E. coli, packaging the gene into baculovirus, expressing the gene in cultured insect cells, and purifying the protein by affinity chromatography using histidine and biotin tags contained in the engineered vector. The results indicate that HSL has been purified and remains biologically active. Blue Sky Biotech will be able to sell this product to laboratories currently researching lipolysis pathways involved in diseases such as obesity, diabetes, and hyperlipidemia

Conservation of Helical Bundle Structure between the Exocyst Subunits

Background: The exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies. Methodology/Principal Findings: Using advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60 % a-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners. Conclusions/Significance: Although all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folde

Biological, clinical and population relevance of 95 loci for blood lipids.

Plasma concentrations of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides are among the most important risk factors for coronary artery disease (CAD) and are targets for therapeutic intervention. We screened the genome for common variants associated with plasma lipids in >100,000 individuals of European ancestry. Here we report 95 significantly associated loci (P < 5 x 10(-8)), with 59 showing genome-wide significant association with lipid traits for the first time. The newly reported associations include single nucleotide polymorphisms (SNPs) near known lipid regulators (for example, CYP7A1, NPC1L1 and SCARB1) as well as in scores of loci not previously implicated in lipoprotein metabolism. The 95 loci contribute not only to normal variation in lipid traits but also to extreme lipid phenotypes and have an impact on lipid traits in three non-European populations (East Asians, South Asians and African Americans). Our results identify several novel loci associated with plasma lipids that are also associated with CAD. Finally, we validated three of the novel genes-GALNT2, PPP1R3B and TTC39B-with experiments in mouse models. Taken together, our findings provide the foundation to develop a broader biological understanding of lipoprotein metabolism and to identify new therapeutic opportunities for the prevention of CAD

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Pulmonary Metastasectomy in Pediatric Solid Tumors

Metastatic disease and the complications of treating metastatic disease are the primary causes of mortality in children with solid malignancies. Nearly 25% of children with solid tumors have metastatic disease at initial diagnosis and another 20% develop metastases during or after treatment. The most common location of these metastases is the lung. The role of surgery in metastatic disease depends greatly on the histology of the primary. In general, tumors that are refractory to adjuvant therapies are most appropriate for pulmonary metastasectomy. This article will summarize the indications for metastasectomy in pediatric solid tumors and discuss the ongoing debate over the technique of metastasectomy in osteosarcoma

Similarity between full-length exocyst subunits.

<p>HMM P-values of the comparisons are indicated for the full length proteins. SGD identifiers are indicated in the first column of the table.</p

Recombinant Sec10(145–827) is soluble.

<p>Several Sec10p truncation constructs designed using secondary structure predictions are not generally soluble. (<i>A</i>) Secondary structure prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Jones1" target="_blank">[41]</a> and schematic of several representative N- and C-terminal truncations tested. The secondary structure prediction is schematically depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone-0004443-g001" target="_blank">Figure 1</a>. Truncations 1–589 and 590–871 were derived from dominant negative constructs described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Roth1" target="_blank">[27]</a>. (<i>B</i>) <i>E. coli</i> cells were transformed with Sec10p truncation variants cloned with an N-terminal His₆-tag in the vector pET15b (Novagen). Expression was induced by addition of IPTG to 0.1 mM, and growth was continued at 15°C for 14–18 h. Cells were pelleted, lysed and the insoluble (P) material was separated from the soluble material (S) by centrifugation; these were run on a 10% SDS-PAGE gel and stained with Coomassie blue dye. Asterisks indicate the migration of each construct. For each construct except Sec10(145–827), very little of the His₆-tagged protein was in the soluble fraction. Although the Sec10(75–859) construct initially appeared promising, it was sticky and aggregated after partial purification on Ni-NTA resin. The right hand lane contains Sec10(145–827) after purification by Ni-NTA resin and gel filtration chromatography.</p

The exocyst subunits have similar helical bundle structures.

<p>(<i>A</i>) The known structures of the exocyst subunits are shown: Exo70p (PDB ID 2B1E), Exo84CT (PDB ID 2D2S), Sec15CT (PDB ID 2A2F), Sec6CT (PDB ID 2FJI). Molecular graphics were generated with PyMOL (<a href="http://pymol.sourceforge.net/" target="_blank">http://pymol.sourceforge.net/</a>). Exo84CT is aligned with the N-terminal helical bundles of Exo70p, while Sec15CT and Sec6CT are aligned with the C-terminal bundles of Exo70p. (<i>B</i>) Secondary structure predictions for all of the exocyst subunits. The black horizontal lines represent the sequence of each yeast exocyst subunit. The predicted α-helices (magenta) and β-strands (cyan) are indicated by vertical bars above each line. The height of the bars is proportional to the confidence of the secondary structure prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Jones1" target="_blank">[41]</a>. Red blocks underline regions of the known structures. Green blocks underline the best hits to exocyst structures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone-0004443-t001" target="_blank">Table 1</a>).</p