Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor alpha chain recognition.

Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor alpha chain (hIL-5R alpha). A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor alpha chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5R alpha were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues Glu-110 and Trp-111, and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important alpha chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5R alpha.

Platelet-derived growth factor stimulates the secretion of hyaluronic acid by proliferating human vascular smooth muscle cells.

Total glycans from the cell layer and the culture medium of human vascular smooth muscle cells (VSMC) that had been cultivated in the presence of platelet-derived growth factor (PDGF) were isolated and purified by gel filtration after Pronase and DNase digestion and alkaliborohydride treatment. Measurements of the content of neutral hexoses and uronic acids revealed that PDGF stimulates total glycan synthesis by proliferating VSMC in a linear fashion from 24 h to 72 h of incubation. In contrast, total glycan synthesis by human fibroblasts, epithelial cells, or endothelial cells was not affected by PDGF, indicating cell-type specificity. Chemical, biochemical, and enzymological characterization of the total glycans synthesized by VSMC showed that PDGF stimulates the secretion of a 340-kDa glycan molecule in a time-dependent manner from 24 h to 72 h. This molecule is highly acidic, shares a common structure with hyaluronic acid, and exhibits a potent antiproliferative activity on VSMC. These results suggest that VSMC in response to PDGF are capable of controlling their own growth and migration by the synthesis of a specific form of hyaluronic acid with antiproliferative potency, which may be involved in the regulation of the local inflammatory responses associated with atherosclerosis.

Three-dimensional functional magnetic resonance imaging of human brain on a clinical 1.5-T scanner.

Functional magnetic resonance imaging (fMRI) is a tool for mapping brain function that utilizes neuronal activity-induced changes in blood oxygenation. An efficient three-dimensional fMRI method is presented for imaging brain activity on conventional, widely available, 1.5-T scanners, without additional hardware. This approach uses large magnetic susceptibility weighting based on the echo-shifting principle combined with multiple gradient echoes per excitation. Motor stimulation, induced by self-paced finger tapping, reliably produced significant signal increase in the hand region of the contralateral primary motor cortex in every subject tested.

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.

The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase.

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene. The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets over a span of 744 aa, suggesting that OCRL may be a similar enzyme. We engineered a construct of the OCRL cDNA that encodes amino acids homologous to the platelet 5-phosphatase for expression in baculovirus-infected Sf9 insect cells. This cDNA encodes aa 264-968 of the OCRL protein. The recombinant protein was found to catalyze the reactions also carried out by platelet 5-phosphatase II. Thus OCRL converts inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, and it converts inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. Most important, the enzyme converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The relative ability of OCRL to catalyze the three reactions is different from that of 5-phosphatase II and from that of another 5-phosphatase isoenzyme from platelets, 5-phosphatase I. The recombinant OCRL protein hydrolyzes the phospholipid substrate 10- to 30-fold better than 5-phosphatase II, and 5-phosphatase I does not cleave the lipid at all. We also show that OCRL functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in OCRL-expressing Sf9 cells. These results suggest that OCRL is mainly a lipid phosphatase that may control cellular levels of a critical metabolite, phosphatidylinositol 4,5-bisphosphate. Deficiency of this enzyme apparently causes the protean manifestations of Lowe syndrome.

Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Platelet-activating factor downregulates the expression of liver X receptor-α and its target genes in human neutrophils

Liver X receptors (LXRs) are ligand-activated members of the nuclear receptor superfamily that regulate the expression of genes involved in lipid metabolism and inflammation, although their role in inflammation and immunity is less well known. It has been reported that oxysterols/LXRs may act as anti-inflammatory molecules, although opposite actions have also been reported. In this study, we investigated the effect of platelet-activating factor (PAF), a proinflammatory molecule, on LXRα signalling in human neutrophils. We found that PAF exerted an inhibitory effect on mRNA expression of TO901317-induced LXRα, ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and sterol response element binding protein 1c. This negative action was mediated by the PAF receptor, and was dependent on the release of reactive oxygen species elicited by PAF, as it was enhanced by pro-oxidant treatment and reversed by antioxidants. Current data also support the idea that PAF induces phosphorylation of the LXRα molecule in an extracellular signal-regulated kinase 1/2-mediated fashion. These results suggest that a possible mechanism by which PAF exerts its proinflammatory effect is through the downregulation of LXRα and its related genes, which supports the notion that LXRα ligands exert a modulatory role in the neutrophil-mediated inflammatory response.

The EU’s Human Rights Promotion in China and Myanmar: Trading Rights for Might? EU Diplomacy Paper No. 5, October 2014

This paper aims to answer two questions: generally, to what extent the human rights promotion of the European Union (EU) in third countries is consistent, and more specifically, why the EU’s approach towards human rights promotion in China and Myanmar differs despite similar breaches of human rights. It compares the EU’s approach to the two countries over two time periods in the late 1980s and 1990s in the context of the EU’s evolving human rights promotion. Based on the two case studies, this paper finds that the EU’s human rights promotion in third countries varies significantly. Whereas one would expect the EU’s approach to become increasingly assertive throughout the 1990s, this has only been the case with Myanmar. China’s economic and political importance to the EU appears to have counterweighed the general rise in European attention to third countries’ human rights records. In other words, this paper finds that commercial interests take precedence over human rights concerns in case of important trading partners.