Anti-hyperlipidemic and insulin sensitizing activities of fenofibrate reduces aortic lipid deposition in hyperlipidemic Golden Syrian hamster

Cholesterol ester transfer protein (CETP) and apolipoprotein (apo) E are important in peroxisome proliferation activated receptor-α (PPAR-α)-mediated regulation of lipoprotein metabolism. Therefore, popularly used apolipoprotein E knockout mice are not suitable to evaluate PPAR-α agonists. In this study, we aimed to: a) evaluate hamster as a model for insulin resistance, hyperlipidemia and atherosclerosis; and b) investigate the effect of a PPAR-α activator, fenofibrate, in this model. A high fat high cholesterol (HFHC) diet increased serum cholesterol and triglycerides, but inclusion of fenofibrate in the diet decreased cholesterol and proatherogenic lipoproteins, VLDL and LDL, in a time-dependent manner. Concomitantly, serum levels of triglycerides also decreased. These reductions were attributed, in part, to the down-regulation of lipogenic genes and upregulation of lipoprotein lipase. The HFHC diet caused body weight gain and mild insulin resistance, both of which were prevented following the treatments with fenofibrate. Insulin resistance was further investigated in high fructose-fed hamsters. Fenofibrate prevented both hyperinsulinemia and hypertriglyceridemia. The insulin sensitizing activity of fenofibrate appeared to occur via reductions in protein tyrosine phophatase-1B. To determine whether lowering of lipids by fenofibrate treatment contributed to the reduced risks of developing atherosclerosis in hyperlipidemic hamsters, we measured lipid deposition in the aorta. Our results showed that fenofibrate treatment reduced aortic lipid deposition by 70%. These findings suggest that hamster may be an adequate animal model to evaluate the efficacy of lipid lowering, insulin sensitizing and antiatherosclerotic agents. We also show that fenofibrate is an effective antiatherosclerotic agent in hyperlipidemic hamster model.     

A rapid,sensitive method for simultaneous measurements of tissue levels of oxygenated derivatives of cholesterol

A mass spectrometric procedure which utilizes multiple selected ion monitoring (SIM) for measuring the tissue levels of cholest-5-en-3β,7α-diol, cholest-5-en-3β,7β-diol, cholest-5-en-3β,25-diol, and cholest-5-en-3β-ol-7-one is described. Trimethylsilyl ethers (TMS) of sterols in a lipid extract are analyzed directly by focusing the ions at $\text{m}\text{e}$ 546, 472, and 443. Endogenous cholesterol serves as an internal standard and its concentration is determined by gas chromatography. The sensitivity of this method has allowed measurement of 2 ng of oxygenated sterol which corresponded to the amount present in 1 mg of rat liver.     

Human embryonic stem cells express an immunogenic nonhuman sialic acid

Human embryonic stem cells (HESC) can potentially generate every body cell type, making them excellent candidates for cell- and tissue-replacement therapies. HESC are typically cultured with animal-derived 'serum replacements' on mouse feeder layers. Both of these are sources of the nonhuman sialic acid Neu5Gc, against which many humans have circulating antibodies. Both HESC and derived embryoid bodies metabolically incorporate substantial amounts of Neu5Gc under standard conditions. Exposure to human sera with antibodies specific for Neu5Gc resulted in binding of immunoglobulin and deposition of complement, which would lead to cell killing in vivo. Levels of Neu5Gc on HESC and embryoid bodies dropped after culture in heat-inactivated anti-Neu5Gc antibody-negative human serum, reducing binding of antibodies and complement from high-titer sera, while allowing maintenance of the undifferentiated state. Complete elimination of Neu5Gc would be likely to require using human serum with human feeder layers, ideally starting with fresh HESC that have never been exposed to animal products.     

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.     

Identification ofClostridium histolyticum collagenase hyperreactive sites in type I,II, and III collagens: Lack of correlation with local triple helical stability

The class I and IIClostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the α1(I), α2(I), α1(II), and α1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.     

Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.