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ApoA-II is the second most abundant protein on HDL making up ∼ 20% of the total protein but its functions have still only been partially characterized. Recent methodological improvements have allowed for the recombinant expression and characterization of human apoA-II which shares only 55% sequence homology with murine apoA-II. Here we describe the purification of the two most common polymorphic variants of apoA-II found in inbred mouse strains, differing at 3 amino acid sites. C57BL/6 mice having variant apoA-II(a) have lower plasma HDL levels than FVB/N mice that have variant apoA-II(b). Characterization of the helical structure of these two variants reveals a more alpha-helical structure for the FVB/N apoA-II. These changes do not alter the lipid or HDL binding of the two apoA-II variants, but significantly increase the ability of the FVB/N variant to promote both ABCA1 and ABCG1 mediated cellular cholesterol efflux. These differences may be differentially altering plasma HDL apoA-II levels. In vivo, neither C57 nor FVB apoA-II protein levels are affected by the absence of apoE, while an apoE/apoA-I double deficiency results in a 50% decrease of plasma FVB apoA-II but results in undetectable levels of C57 apoA-II in the plasma. FVB apoA-II is able to form an HDL particle in the absence of apoE or apoA-I.

LIGHT/TNFSF14 is a costimulatory molecule expressed on activated T cells for activation and maintenance of T cell homeostasis. LIGHT over expressed in T cells also down regulates hepatic lipase levels in mice through lymphotoxin beta receptor (LTβR) signaling. It is unclear whether LIGHT regulates hepatic lipase directly by interacting with LTβR expressing cells in the liver or indirectly by activation of T cells, and whether Kupffer cells, a major cell populations in the liver that expresses the LTβR, are required. Here we report that LIGHT expression via an adenoviral vector (Ad-LIGHT) is sufficient to down regulate hepatic lipase expression in mice. Depletion of Kupffer cells using clodronate liposomes had no effect on LIGHT-mediated down regulation of hepatic lipase. LIGHT-mediated regulation of hepatic lipase is also independent of LIGHT expression by T cells or activation of T cells. This is demonstrated by the decreased hepatic lipase expression in the liver of Ad-LIGHT infected recombination activating gene deficient mice that lack mature T cells and by the Ad-LIGHT infection of primary hepatocytes. Hepatic lipase expression was not responsive to LIGHT when mice lacking LTβR globally or only on hepatocytes were infected with Ad-LIGHT. Therefore, our data argues that interaction of LIGHT with LTβR on hepatocytes, but not Kupffer cells, is sufficient to down regulate hepatic lipase expression and that this effect can be independent of LIGHT's costimulatory function.

BackgroundCytokines and growth factors are critical reagents used in cell therapy and regenerative medicine, yet their biological activity and consistency can vary significantly based on the expression system used. HEK293-expressed growth factors offer advantages in terms of authentic post-translational modifications and improved bioactivity.MethodsIn this study, we performed various cell-based assays to compare the bioactivity, cell expansion and cell differentiation potential of cytokines derived from HEK293 and E. coli.ResultsWe highlight the significant improvement in bioactivity and performance of HEK293-derived growth factors further leading to enhancing cell expansion and differentiation. Human Betacellulin (BTC) protein expressed in HEK293 shows 100 times more bioactivity than E. coli derived BTC. Brain-derived neurotrophic factor (BDNF) expressed in HEK293 cells demonstrated enhanced efficacy in promoting neuronal differentiation of SHSY5Y cells compared to E. coli derived. Similarly, HEK293-expressed interleukin-34 (IL-34) significantly improved the generation and maturation of macrophages. Furthermore, the combination of HEK293-derived IL-2, IL-7, and IL-15 resulted in robust expansion of T cells with a favorable phenotype for immunotherapeutic applications.ConclusionsThese findings not only support the use of HEK293-expressed growth factors as biologically superior reagents but also a way to reduce cost of cell expansion and differentiation workflows.

Tocopherols and tocotrienols all possess to varying levels the vitamin E activity of α-tocopherol, the most bioactive form of the vitamin in vivo. α-Tocopherol is the most abundant vitamer in vivo, potentially explaining its higher bioactivity. This is despite higher dietary levels of other vitamers. The preferential retention of α-tocopherol appears to be at the level of elimination. Urinary metabolites of tocopherols, with the phytyl tail truncated to a three-carbon carboxylated moiety, were reported previously. The goal of this work was to elucidate the pathway(s) by which these metabolites are formed. Incubations of tocopherols in hepatocyte culture produced all expected intermediates in the predicted pathway of catabolism of vitamin E. This pathway involves ω-hydroxylation of a terminal methyl group of the phytyl tail, followed by step-wise removal of two or three carbon units by a β-oxidation mechanism. Analysis of microsomal enzyme activity led to the elucidation of CYP4F2 as the major P450 involved in the initial ω-hydroxylation reaction. Substrate-specificity of this enzyme was high, with activity toward γ-tocopherol being 10-fold greater than toward α-tocopherol. Sesamin, known to raise plasma γ-tocopherol to levels near those of α-tocopherol in vivo, was a potent inhibitor of this enzyme. Together, these data supported the hypothesis that the ω-oxidation pathway is an important regulator of vitamin E status. Kinetic analyses revealed key features of the tocopherol molecule which govern the affinity and activity of the enzyme toward its substrates. These studies also revealed an allosteric nature of CYP4F2. The finding that α-tocopherol is a positive effector of γ-tocopherol metabolism may explain why supplementation with α-tocopherol decreases plasma concentrations of non-α-tocopherols. Tocotrienols differed from tocopherols in their effects on lateral and rotational mobility of the microsomal membrane components, and inhibited the activity of membrane-bound P450 enzymes, possibly through inhibition of interaction of components of the cytochrome P450 multi-enzyme complex. These effects may play a role in the cytotoxic nature of the tocotrienols and highlight the importance of the tocopherol-ω-oxidation pathway in minimizing their concentration in biomembranes.

Pandeia is the exposure time calculator (ETC) system developed for the James Webb Space Telescope (JWST) that will be used for creating JWST proposals. It includes a simulation-hybrid Python engine that calculates the two-dimensional pixel-by-pixel signal and noise properties of the JWST instruments. This allows for appropriate handling of realistic point spread functions, MULTIACCUM detector readouts, correlated detector readnoise, and multiple photometric and spectral extraction strategies. Pandeia includes support for all the JWST observing modes, including imaging, slitted/slitless spectroscopy, integral field spectroscopy, and coronagraphy. Its highly modular, data-driven design makes it easily adaptable to other observatories. An implementation for use with WFIRST is also available.