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Volumen 2, Asunto 5 (2010)

Artículo de investigación

Apolipoprotein E-Deficient Lipoproteins Induce Foam Cell Formation by Activation of PERK-EIF-2α Signaling Cascade

YanFeng Zhao, ZhongMao Guo, XingHua Lin, LiChun Zhou, Emmanuel U. Okoro, GuoHuang Fan, Raju Ramaswamy and Hong Yang

Transformation of macrophages into foam cells by apolipoprotein (Apo) E-de fi cient, ApoB48-containing (E Ö¾ /B48) lipoproteins has been shown to be associated with increased phosphorylation of eukaryotic initiation factor-2 α (eIF- 2 α ). The present report examined the causal relationship between eIF-2 α phosphorylation and lipid accumulation in macrophages induced by E Ö¾ /B48 lipoproteins. E Ö¾ /B48 lipoproteins increased eIF-2 α phosphorylation and cholesterol ester accumulation, while lipoprotein degradation decreased and lysosomal acid lipase and cathepsin B mRNA translation was inhibited in mouse peritoneal macrophages (MPMs). These responses were overcome by overexpression of a nonphosphorylatable eIF-2 α mutant in MPMs. Incubation of MPMs with E Ö¾ /B48 lipoproteins also increased the phosphorylation of RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK), but not other eIF-2 α kinases. Overexpression of a nonphosphorylatable PERK mutant inhibited PERK and eIF-2 α phosphorylation, and alleviated cholesterol ester accumulation induced by E Ö¾ /B48 lipoproteins. PERK is an eIF-2 α kinase activated by endoplasmic reticulum (ER) stress. Taken together, fi ndings from this report suggest that induction of ER stress, i.e ., activation of the PERK-eIF2 α signaling cascade, is a mechanism by which E Ö¾ /B48 lipoproteins down-regulate lysosomal hydrolase synthesis, inhibit lysosomal lipoprotein degradation, and increase intracellular lipoprotein and cholesterol ester accumulation, resulting in foam cell formation.

Artículo de investigación

Rapid and Specific Approach for Direct Measurement of Topiramate in Human Plasma by LC-MS/MS: Application for Bioequivalence Study

S. R. Kuchekar, M. L. Kundlik and B. H. Zaware

A rapid liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quanti fi cation of topiramate in heparinized human plasma. The plasma samples were prepared by solid phase extraction (SPE) method without drying and then reconstitution. Topiramate and the topiramate d12 (Internal Standard IS) were chromatographed on a Betasil C18 column at a fl ow rate of 0.5 ml/min. The total run time was 1.80 min. An electrospray ionization interface was selected for ionization of analyte and IS. The mass transition [M-H] ions used for detection were m/z 338.10 → 78.20 for topiramate, m/z 350.40 → 90.10 for IS. The method was linear in the concentration range of 10–4200 ng/ml with r ≥ 0.9982. Recovery of topiramate and IS ranged from 78.20 to 87.74%. The validated method has been successfully used to analyze human plasma samples for application in 100 mg fasted pharmacokinetic studies.

Artículo de investigación

Hepatic Endosome Protein Profiling in Apolipoprotein E Deficient Mice Expressing Apolipoprotein B48 but not B100

AnShu Chen, ZhongMao Guo, LiChun Zhou and Hong Yang

Liver cells absorb apolipoprotein (Apo) B48-carrying lipoproteins in ApoE’s absence, albeit not as ef fi ciently as the ApoE-mediated process. Our objective was to identify differentially expressed hepatic endosome proteins in mice expressing ApoB48 but lacking ApoE and ApoB100 expression ( ApoE -/- /B48/48 ). We puri fi ed early and late endosomes from ApoE -/- /B48/48 and wild-type mouse’s livers. In ApoE -/- /B48/48 mouse’s hepatic endosomes, proteomic analysis revealed elevated protein levels of major urinary protein 6 (MUP), calreticulin, protein disul fi de isomerases (PDI) A1, and A3. These proteins are capable of interacting with lipids/lipoproteins and triggering receptor- mediated endocytosis. In addition, hepatic endosomes from ApoE -/- /B48/48 mice exhibited signi fi cantly reduced protein levels of haptoglobin, hemopexin, late endosome/lysosome interacting protein, cell division control protein 2 homolog, γ -soluble Nethylmaleimide- sensitive factor attachment protein, vacuolar ATP synthase catalytic subunit A1, dipeptidyl peptidases II, cathepsin B, D, H, and Z. These proteins participate in plasma heme clearance, receptor-mediated signaling, membrane fusion, endosomal/lysosomal acidi fi cation, and protein degradation. The signi fi cance of increasing endosomal MUP, calreticulin and PDIs in ApoE -/- /B48/48 mouse liver cells is not clear; however, reducing endosomal/ lysosomal membrane proteins and hydrolases might be, at least partially, responsible for the retarded clearance of plasma ApoB-carrying lipoproteins in ApoE -/- /B48/48 mice.

Artículo de investigación

Bioencapsulation of Praziquantel in Adult Artemia

Matthew C. Allender, Mike Kastura, Robert George, Frank Bulman, Jason Yarborough and Sherry Cox

A description of bioencapsulation of praziquantel in adult Artemia for 2.5 g/L, 5 g/L, and 10 g/L treatment baths is presented. Praziquantel was detected in adult brine shrimp tissue after enrichment periods of 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr, and 24 hr. The assays were performed using high performance liquid chromatography. There was variable uptake by Artemia at all three bath treatments over time. Despite early variability, all three baths showed a terminal increase in praziquantel concentration. Highest concentration of praziquantel was seen in the initial sample (5 g/L) or the last sample (2.5 g/L and 10 g/L). The highest concentration of praziquantel at any one point was observed in the 5 g/L treatment bath at 15 minutes. Based on percentage, more praziquantel was incorporated into shrimp at the 10 g/L than either of the other treatments. Non-predictable fl uctuations were seen in the concentration of praziquantel in both the treatment water and control water. Concentration of praziquantel in the control water increased in each treatment group over each of the fi nal three time points. Neither total praziquantel in the treatment bath (shrimp and water) or the control bath were consistent among any treatment group. Survival of shrimp was not affected by concentration, but decreased over time in all treatment baths comparatively. It can be concluded that praziquantel can be successfully, but not reliably, bioencapsulated in adult Artemia .

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