Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2 weeks, or from normal human subjects treated either placebo, 20 mg, or 150 mg anacetrapib daily for 2 weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.     

Cloning,purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1

Thermostable amylopullulanases can catalyse the hydrolysis of both α-1,4 and α-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 °C. Apu-Tk was also active at a broad range of pH (4–7), with an optimum pH of ~5.0–5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% β-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications.     

Biosynthesis of capsinoid is controlled by the Pun1 locus in pepper

Pungency in pepper (Capsicum annuum L.) has unique characteristics due to the alkaloid compound group, capsaicinoids, which includes capsaicin. Although capsaicinoids have been proved to have pharmacological and physiological effects on human health, the application of capsaicinoids has been limited because of their pungency. Capsinoids found in non-pungent peppers share closely related structures with capsaicinoids and show similar biological effects. Previous studies demonstrated that mutations in the p-AMT gene were related to the production of capsinoids; however, the pathway of capsinoid synthesis has not yet been fully elucidated. In this study, we performed genetic analysis to determine the mechanism of capsinoid synthesis using a F6 recombinant inbred line population. In this population, the presence/absence of capsinoids co-segregated with the genotype of the Pun1 locus, without exception. In addition, we screened the patterns of capsinoid synthesis and the correlation between the Pun1 locus and capsinoid synthesis in p-AMT mutant accessions. In Capsicum germplasms, we selected amino-acid-substituted mutants in the PLP binding domain of the p-AMT gene. Capsinoids were not synthesized with the recessive pun1 gene, regardless of the p-AMT genotype, and no relationship was found between p-AMT mutant type and capsinoid content. We concluded that the Pun1 gene, which is responsible for capsaicinoid synthesis, also controls capsinoid synthesis.     

Efficient and fine mapping of RMES1 conferring resistance to sorghum aphid Melanaphis sacchari

Melanaphis sacchari causes serious damage to sorghum (Sorghum bicolor (L.) Moench) growth, development and productivity in many countries. A dominant gene (RMES1) conferring resistance to M. sacchari has been found in the grain sorghum variety Henong 16 (HN16), but fine mapping of the RMES1 locus remains to be reported. In this study, genetic populations segregating for RMES1 were prepared with HN16 and BTx623 as parental lines. The latter had been used for sorghum genome sequencing but was found to be susceptible to M. sacchari in this work. A total of 11 molecular markers were mapped to the short arm of chromosome 6 harboring RMES1. The closest markers flanking the RMES1 locus were Sb6m2650 and Sb6rj2776, which delimited a chromosomal region of about 126 kb containing five predicted genes. The utility of the newly identified DNA markers for tagging RMES1 in molecular breeding of M. sacchari resistance, and further efforts in cloning RMES1, are discussed.     

Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress–injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.     

The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.     

Spatial and temporal population genetic variation and structure of Nothotsuga longibracteata (Pinaceae), a relic conifer species endemic to subtropical China

Nothotsuga longibracteata, a relic and endangered conifer species endemic to subtropical China, was studied for examining the spatial-temporal population genetic variation and structure to understand the historical biogeographical processes underlying the present geographical distribution. Ten populations were sampled over the entire natural range of the species for spatial analysis, while three key populations with large population sizes and varied age structure were selected for temporal analyses using both nuclear microsatellites (nSSR) and chloroplast microsatellites (cpSSR). A recent bottleneck was detected in the natural populations of N. longibracteata. The spatial genetic analysis showed significant population genetic differentiation across its total geographical range. Notwithstanding, the temporal genetic analysis revealed that the level of genetic diversity between different age class subpopulations remained constant over time. Eleven refugia of the Last Glacial Maximum were identified, which deserve particular attention for conservation management.     

A Test of Current Models for the Mechanism of Milk‐Lipid Droplet Secretion

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin‐2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N‐linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN‐enhanced green fluorescent protein was highly mobile in areas of mouse milk‐lipid droplets that had not undergone post‐secretion changes, and endogenous mouse BTN comprised only 0.5–0.7% (w/w) of the total protein, i.e. over 50‐fold less than in the milk‐lipid droplets of cow and other species. These data are incompatible with models of milk‐lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk .     

Nitrogen Translocation to Fresh Litter in Northern Hardwood Forest

Nitrogen immobilization in fresh litter represents a significant N flux in forest ecosystems, and changes in this process resulting from atmospheric N deposition could have important implications for ecosystem responses. We conducted two leaf decay experiments, using ¹⁵N-labeled sugar maple leaf litter, to quantify N transport from old litter and soil to fresh litter during early stages of decomposition, and we examined the influence of litter N concentration and soil N availability on upward N transfer in a northern hardwood forest. After one year of decay, the average N transfer from soil to fresh litter (2.63 mg N g^?1 litter) was much higher than the N transfer from older litter (1- to 2-years-old) to fresh litter (0.37 mg N g^?1 litter). We calculated the ratio of annual N transfer per unit of excess ¹⁵N pool for these two N sources. The ratio was not significantly different between old litter and soil, suggesting that fungi utilize N in the old litter and mineral soil pools for transport to decaying fresh litter with similar efficiency. Initial litter N concentration had a significant effect on upward N flux into decaying leaf litter, whereas no effect of soil N fertilization was observed. Reduction in the flux from soil to fresh litter owing to anthropogenic N inputs probably contributes significantly to changing soil N dynamics. Future work is needed on fungal N acquisition and transport as well as the fungal taxa involved in this process and their responses to changing environments.