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Mingxia Liu Jeremy Allegood Xuewei Zhu Jeongmin Seo Abraham K. Gebre Elena Boudyguina Dongmei Cheng Chia-Chi Chuang Gregory S. Shelness Sarah Spiegel John S. Parks 《The Journal of biological chemistry》2015,290(12):7861-7870
Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoMQ22A) introduces a functional signal peptidase cleavage site. Expression of apoMQ22A in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoMWT). When apoMQ22A was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoMWT. Hepatocytes isolated from both apoMWT- and apoMQ22A-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoMWT, apoMQ22A hepatocytes displayed more rapid apoM and S1P secretion but minimal apoMQ22A bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoMWT and apoMQ22A hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL. 相似文献
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Mingxia Liu Jeongmin Seo Jeremy Allegood Xin Bi Xuewei Zhu Elena Boudyguina Abraham K. Gebre Dorit Avni Dharika Shah Mary G. Sorci-Thomas Michael J. Thomas Gregory S. Shelness Sarah Spiegel John S. Parks 《The Journal of biological chemistry》2014,289(5):2801-2814
Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3–5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions. 相似文献
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TJS Alves GS Cruz V Wanderley-Teixeira AAC Teixeira JV Oliveira AA Correia 《Biotechnic & histochemistry》2014,89(4):245-255
The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest. 相似文献
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Ajith Kumar GS Binil Raj Santhosh Kumar S Sanjay G Chandrasekharan Cheranellore Kartha 《Journal of visualized experiments : JoVE》2014,(88)
Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50–60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage. 相似文献
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Y Deng J Zhao D Sakurai KM Kaufman JC Edberg RP Kimberly DL Kamen GS Gilkeson CO Jacob RH Scofield CD Langefeld JA Kelly ME Alarcón-Riquelme BIOLUPUS GENLES Networks JB Harley TJ Vyse BI Freedman PM Gaffney KM Sivils JA James TB Niewold RM Cantor W Chen BH Hahn EE Brown PROFILE BP Tsao 《Arthritis research & therapy》2012,14(Z3):A5
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Dongmei Cheng Philip S. MacArthur Shunxing Rong John S. Parks Gregory S. Shelness 《Journal of lipid research》2010,51(4):849-855
The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5′ proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3′ hepatic control region, derived from a region ∼18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3′ splice acceptor sites causing deletion of cloned 5′ untranslated mRNA sequences and, in some cases, deletion of the 5′ end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3′ splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1–exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression. 相似文献