Two novel steroidal alkaloid glycosides from the seeds of Lycium barbarum

Two novel steroidal alkaloid glycosides, lycioside A (1) and lycioside B (2) were isolated from the seeds of Lycium barbarum. Their structures were determined by various spectroscopic analyses. Compounds 1 and 2 showed inhibitory activities with the IC(50) values of 75.3 and 72.8 μM against rat intestinal sucrase, and 63.4 and 59.1 μM against rat intestinal maltase.     

LPS-Induced Formation of Immunoproteasomes: TNF-α and Nitric Oxide Production are Regulated by Altered Composition of Proteasome-Active Sites

Stimulation of mouse macrophages with LPS leads to tumor necrosis factor (TNF-α) secretion and nitric oxide (NO) release at different times through independent signaling pathways. While the precise regulatory mechanisms responsible for these distinct phenotypic responses have not been fully delineated, results of our recent studies strongly implicate the cellular cytoplasmic ubiquitin-proteasome pathway as a key regulator of LPS-induced macrophage inflammatory responses. Our objective in this study was to define the relative contribution of specific proteasomal active-sites in induction of TNF-α and NO after LPS treatment of RAW 264.7 macrophages using selective inhibitors of these active sites. Our data provide evidence that LPS stimulation of mouse macrophages triggers a selective increase in the levels of gene and protein expression of the immunoproteasomes, resulting in a modulation of specific functional activities of the proteasome and a corresponding increase in NO production as compared to untreated controls. These findings suggest the LPS-dependent induction of immunoproteasome. In contrast, we also demonstrate that TNF-α expression is primarily dependent on both the chymotrypsin- and the trypsin-like activities of X, Y, Z subunits of the proteasome. Proteasome-associated post-acidic activity alone also contributes to LPS-induced expression of TNF-α. Taken together; our results indicate that LPS-induced TNF-α in macrophages is differentially regulated by each of the three proteasome activities. Since addition of proteasome inhibitors to mouse macrophages profoundly affects the degradation of proteins involved in signal transduction, we conclude that proteasome-specific degradation of several signaling proteins is likely involved in differential regulation of LPS-dependent secretion of proinflammatory mediators.     

Serum amyloid A directly accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice

Although serum amyloid A (SAA) is an excellent marker for coronary artery disease, its direct effect on atherogenesis in vivo is obscure. In this study we investigated the direct effect of SAA on promoting the formation of atherosclerosis in apolipoprotein E-deficient (ApoE?/?) mice. Murine SAA lentivirus was constructed and injected into ApoE?/? mice intravenously. Then, experimental mice were fed a chow diet (5% fat and no added cholesterol) for 14 wks. The aortic atherosclerotic lesion area was larger with than without SAA treatment. With increased SAA levels, the plasma levels of interleukin-6 and tumor necrosis factor-α were significantly increased. Macrophage infiltration in atherosclerotic regions was enhanced with SAA treatment. A migration assay revealed prominent dose-dependent chemotaxis of SAA to macrophages. Furthermore, the expression of monocyte chemotactic protein-1 and vascular cell adhesion molecule-1 (VCAM-1) was upregulated significantly with SAA treatment. SAA-induced VCAM-1 production was detected in human aortic endothelial cells in vitro. Thus, an increase in plasma SAA directly accelerates the progression of atherosclerosis in ApoE?/? mice. SAA is not only a risk marker for atherosclerosis but also an active participant in atherogenesis.     

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.     

Adjustment for local ancestry in genetic association analysis of admixed populations

MOTIVATION: Admixed populations offer a unique opportunity for mapping diseases that have large disease allele frequency differences between ancestral populations. However, association analysis in such populations is challenging because population stratification may lead to association with loci unlinked to the disease locus. Methods and results: We show that local ancestry at a test single nucleotide polymorphism (SNP) may confound with the association signal and ignoring it can lead to spurious association. We demonstrate theoretically that adjustment for local ancestry at the test SNP is sufficient to remove the spurious association regardless of the mechanism of population stratification, whether due to local or global ancestry differences among study subjects; however, global ancestry adjustment procedures may not be effective. We further develop two novel association tests that adjust for local ancestry. Our first test is based on a conditional likelihood framework which models the distribution of the test SNP given disease status and flanking marker genotypes. A key advantage of this test lies in its ability to incorporate different directions of association in the ancestral populations. Our second test, which is computationally simpler, is based on logistic regression, with adjustment for local ancestry proportion. We conducted extensive simulations and found that the Type I error rates of our tests are under control; however, the global adjustment procedures yielded inflated Type I error rates when stratification is due to local ancestry difference.     

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD(+), thereby indicating ethanol as one of the substrates of BmADH.