Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2

Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2). Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-protein-pseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.     

Expression patterns of Hox10 paralogous genes during lumbar spinal cord development

We have examined the expression of three paralogous Hox genes from E11.5 through E15.5 in the mouse spinal cord. These ages coincide with major phases of spinal cord neurogenesis, neuronal differentiation, cell migration, gliogenesis, and motor neuron cell death. The three genes, Hoxa10, Hoxc10, and Hoxd10, are all expressed in the lumbar spinal cord and have distinct expression patterns. Mutations in these three genes are known to affect motor neuron patterning. All three genes show lower levels of expression at the rostral limits of their domains, with selective regions of higher expression more caudally. Hoxa10 and Hoxd10 expression appears confined to postmitotic cell populations in the intermediate and ventral gray, while Hoxc10 is also expressed in proliferating cells in the dorsal ventricular zone. Hoxc10 and Hoxd10 expression is clearly excluded from the lateral motor columns at rostral lumbar levels but is present in this region more caudally. Double labeling demonstrates that Hoxc10 expression is correlated with ventrolateral LIM gene expression in the caudal part of the lumbar spinal cord.     

Comparative proteomic analysis of Helicobacter pylori strains associated with iron deficiency anemia

Helicobacter pylori is known to cause chronic gastritis, peptic ulcer, and gastric cancer, and has also been linked to iron deficiency anemia (IDA). To determine whether H. pylori clinical isolates correlate with the prevalence of H. pylori-associated IDA, we compared the proteomic profiles of H. pylori strains isolated from antral biopsy specimens of H. pylori-positive patients with or without IDA. Fifteen strains, including eight non-IDA and seven IDA strains, were cultured under iron-rich and iron-depleted conditions and then analyzed for protein expression profiles by 2-DE. The distances between two H. pylori strains were determined on the basis of similarities between their expression patterns of 189 protein spots, and a phylogenetic tree was constructed. The results revealed that the IDA strains formed a cluster separate from that of six non-IDA strains, with two non-IDA strains between the clusters. H. pylori strain 26695 was located in the non-IDA cluster. Protein spots displaying similar expression patterns were clustered, and 18 spots predominantly expressed in IDA strains were identified by MALDI-TOF analysis. These data indicate that the non-IDA and IDA strains can be distinguished by their protein expression profiles, suggesting that the polymorphism of H. pylori strains may be one of the factors determining the occurrence of H. pylori-associated IDA.     

Physiological 3D tissue model of the airway wall and mucosa

This protocol describes the setup, maintenance and characteristics of a tissue-engineered model of the human bronchial mucosa that can be used for basic physiology and pathophysiology studies. The model includes a well-differentiated epithelium with functional cilia, mucus secretion and subepithelial fibroblasts within type I collagen. The tissue is created within porous polymeric wells to prevent gel contraction and allow culture at the air-liquid interface. It requires at least 2 wk to be established and can be maintained thereafter for over 4 wk, with tissue differentiation moving towards a more physiologically relevant phenotype with increasing time in culture. Over time, the extracellular matrix also remodels, depositing proteins such as types III and IV collagen and fibronectin. Because it recapitulates many key anatomical and functional features of the airway wall, this model is well suited for a wide range of studies, including those on airway remodeling, transepithelial transport and inflammatory cell interactions with the mucosa. The entire protocol takes 4-6 wk, including cell expansion, depending on the extent of ciliogenesis desired.     

Cancer/Testis Antigen CAGE Exerts Negative Regulation on p53 Expression through HDAC2 and Confers Resistance to Anti-cancer Drugs

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M^R) and the human hepatic cancer cell line SNU387 (SNU387^R) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387^R cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M^R cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.     

One-step enzymatic synthesis of blue pigments from geniposide for fabric dyeing

In this study, we describe a one-step chemoenzymatic reaction for the production of natural blue pigments, in which the geniposide from Gardenia extracts is transformed by glycosidases to genipin. Genipin is then allowed to react with amino acids, thereby generating a natural blue pigment. The β-glycosidases, most notably isolase (a variant of β-glucanase), recombinant β-glucosidase, Cellulase T, and amylases, were shown to hydrolyze geniposide to produce the desired pigments, whereas the α-glycosidases did not. Among the 20 tested amino acids, glycine and tyrosine were associated with the highest dye production yields. The optimal molar ratio of geniposide to glycine, two reactants relevant to pigment production, was unity. The natural blue pigments produced in this study were used to dye cotton, silk, and wool. The color yields of the pigments were determined to be significantly higher than those of other natural dyes. Furthermore, the color fastness properties of these dyes were fairly good, even in the absence of mordant.     

TC1(C8orf4) is upregulated by IL-1beta/TNF-alpha and enhances proliferation of human follicular dendritic cells

Follicular dendritic cells (FDC) play crucial roles in immune regulation. TNF-alpha has been shown to be essential to the FDC network. However, the molecular regulation of FDC proliferation has not been characterized. Here, we show that TC1(C8orf4), a novel positive regulator of the Wnt/beta-catenin pathway in vertebrates, is upregulated by IL-1beta and TNF-alpha in the human FDC-like line HK. TC1 enhances HK cell proliferation, while TC1-knockdown inhibits the proliferation induced by IL-1beta, suggesting a role of TC1 as a regulator of FDC proliferation. The regulation by pro-inflammatory cytokines suggests that TC1 might be implicated in linking local inflammation to immune response by stimulating FDC.     

Targeted molecular dynamics simulation studies of calcium binding and conformational change in the C-terminal half of gelsolin

Gelsolin consists of six related domains (G1-G6) and the C-terminal half (G4-G6) acts as a calcium sensor during the activation of the whole molecule, a process that involves large domain movements. In this study, we used targeted molecular dynamics simulations to elucidate the conformational transitions of G4-G6 at an atomic level. Domains G4 and G6 are initially ruptured, followed by a rotation of G6 by approximately 90 degrees , which is the dominant conformational change. During this period, local conformational changes occur at the G4 and G5 calcium-binding sites, facilitating large changes in interdomain distances. Alterations in the binding affinities of the calcium ions in these three domains appear to be related to local conformational changes at their binding sites. Analysis of the relative stabilities of the G4-G6-bound calcium ions suggests that they bind first to G6, then to G4, and finally to G5.