Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets

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Highlights? Triacylglyceride (TG) synthesis is coupled with lipid droplet (LD) growth ? Two LD populations exist: growing LDs, containing TG enzymes, and small LDs ? Specific TG synthesis enzymes move from the ER to LDs through membrane bridges ? LD localization of TG enzymes mediates expansion of a subset of LDs     

Intracellular storage and regulated secretion of von Willebrand factor in quantitative von Willebrand disease

Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF.     

Mechanisms of the vasorelaxant effect of 1-hydroxy-2, 3, 5-trimethoxy-xanthone, isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery

1-Hydroxy-2, 3, 5-trimethoxyxanthone (HM-1) is a xanthone isolated from Halenia elliptica, a Tibetan medicinal herb. HM-1 (0.33-42.1 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 1.67+/-0.27 microM. Removal of the endothelium significantly affected the vasodilator potency of HM-1, resulting in 46% decrease in E(max) value. The endothelium-dependent effects of HM-1 was confirmed when its vasorelaxant effect was inhibited after addition of nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (100 microM) or the soluble guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM). Atropine (100 nM), flurbiprofen (10 microM), propranolol (100 microM), pyrilamine (10 microM), cimetidine (10 microM) and SQ22536 (100 microM) had no effect on the vasorelaxant activity of HM-1 indicated the non-involvement of other receptor/enzyme systems. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-1 was unaffected by potassium channel blockers such as tetraethylammonium (10 mM), iberiotoxin (100 nM), barium chloride (100 microM) and 4-aminopyridine (1 mM). The involvement of Ca(2+) channel in 5-HT-primed artery ring preparations incubated with Ca(2+)-free buffer was confirmed when HM-1 (9.93 microM) partially abolished the CaCl(2)-induced vasoconstriction (87% inhibition in intact-endothelium artery rings; 50% inhibition in endothelium-denuded rings). In the KCl-primed preparations incubated with Ca(2+)-free buffer, HM-1 (9.93 microM) produced a 27.3% inhibition in endothelium-denuded rings. HM-1 (3.31-33.1 microM) had minimal relaxant effects (14.4%-20.3%) on the contractile response generated by 10 microM phorbol 12,13-diacetate (PDA) in Ca(2+)-free solutions, suggesting minimal effects on intracellular Ca(2+) mechanisms. These findings suggest the vasodilator action of HM-1 involved both an endothelium-dependent mechanism involving NO and an endothelium-independent mechanism by inhibiting Ca(2+) influx through L-type voltage-operated Ca(2+) channels; a minor contribution to the effects of HM-1 may be related to inhibition of the protein kinase C-mediated release of intracellular Ca(2+) stores.     

Plastome phylogeny and lineage diversification of Salicaceae with focus on poplars and willows

Phylogenetic relationships and lineage diversification of the family Salicaceae sensu lato (s.l.) remain poorly understood. In this study, we examined phylogenetic relationships between 42 species from six genera based on the complete plastomes. Phylogenetic analyses of 77 protein coding genes of the plastomes produced good resolution of the interrelationships among most sampled species and the recovered clades. Of the sampled genera from the family, Flacourtia was identified as the most basal and the successive clades comprised both Itoa and Poliothyrsis, Idesia, two genera of the Salicaceae sensu stricto (s.s.) (Populus and Salix). Five major subclades were recovered within the Populus clade. These subclades and their interrelationships are largely inconsistent with morphological classifications and molecular phylogeny based on nuclear internal transcribed spacer sequence variations. Two major subclades were identified for the Salix clade. Molecular dating suggested that species diversification of the major subclades in the Populus and Salix clades occurred mainly within the recent Pliocene. In addition, we found that the rpl32 gene was lost and the rps7 gene evolved into a pseudogene multiple times in the sampled genera of the Salicaceae s.l. Compared with previous studies, our results provide a well‐resolved phylogeny from the perspective of the plastomes.     

Factors Influencing Formation of Sclerotia in Grifola umbellate （Pers.） Pilaet Under Artificial Conditions

The effects of substrate composition and temperature on myceilal growth and sclerotium production in Grlfola umbellate （Pers.） Pilaet were Investigated In the present study. The Induction of sclerotla of G. umbellate was affected greatly by the type of medium, as well as the type of carbon source. Malt-extract agar was able to induce the production of sclerotia. The production of sclerotia was also observed when the carbon source in the GPC agar medium （glucose 20 g/L, peptone 6 g/L, corn steep liquor 10 g/L, and agar 15 g/L） was replaced with glycerol or mannitol. Altering the composition of the GPC medium with milk powder, thiamine hydrochlorlde, extract of Armlllarla mellea, active clay, dlatomite, kaolin, or arginlne did not induce the production of sclerotla. A temperature range of 18-25 ℃ was suitable for both mycellai growth and sclerotium formation. Glycerol significantly Induced slerotium formation on nutrient supplemented with sawdust substrates In bottle culture. 24S-Polyporusterone A and polyporusterone B were assayed In samples of natural and cultured sclerotla. Both natural and cultured sclerotla contained 24S- polyporusterone A and polyporusterone B.     

Genome Sequence of Gallaecimonas xiamenensis Type Strain 3-C-1

Gallaecimonas xiamenensis 3-C-1^T was isolated from a crude-oil-degrading consortium enriched from the surface seawater around Xiamen Island. Here, we present the draft genome of strain 3-C-1^T, which contains 4,062,282 bp with a G+C content of 60.58% and contains 3,798 protein-coding genes and 65 tRNAs.     

Effect of Methamphetamine on Spectral Binding,Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δA_max and K_D of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δA_max (0.004±0.0003 vs. 0.0068±0.0001) and K_D (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δA_max (0.0038±0.0003 vs. 0.0055±0.0003) and K_D (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced K_D in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.     

百日咳杆菌粘附素对猪源支气管败血波氏杆菌的保护性抗原的筛选

摘要：【目的】本研究通过百日咳杆菌黏附素(PRN)基因的分段克隆表达及其在BALB/c小鼠的主动和被动免疫保护试验筛选PRN中的保护性抗原肽。【方法和结果】利用大肠杆菌进行PRN的完整蛋白、N端和C端多肽及其RI和RII区域多肽（双拷贝）的表达,命名为GST-PRN、GST-PN、GST-PC、GST-2PRI和GST-2PRII。Western blot检测证实5种表达产物均具有良好的反应原性。在主动免疫保护试验中,5种表达产物均能诱导小鼠产生较高的PRN抗体水平;当使用3 LD50的支气管败血波氏杆菌