Crystal structure of the human N-Myc downstream-regulated gene 2 protein provides insight into its role as a tumor suppressor

Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/β-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/β-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.     

CC and CXC chemokines induce airway smooth muscle proliferation and survival

The increase in airway smooth muscle (ASM) mass is a major structural change in asthma. This increase has been attributed to ASM cell (ASMC) hyperplasia and hypertrophy. The distance between ASMC and the epithelium is reduced, suggesting migration of smooth muscle cells toward the epithelium. Recent studies have suggested a role of chemokines in ASMC migration toward the epithelium; however, chemokines have other biological effects. The objective of the current study is to test the hypothesis that chemokines (eotaxin, RANTES, IL-8, and MIP-1α) can directly influence ASMC mass by increasing the rate of proliferation or enhancing the survival of these cells. Human ASMCs were exposed to different concentrations of eotaxin, RANTES, IL-8, or MIP-1α. To test for proliferation, matched control and stimulated ASMC were pulsed with [(3)H]thymidine, or ASMCs were stained with BrdU and then analyzed with flow cytometry. Apoptosis was measured using Annexin V staining and flow cytometry. Expression of phosphorylated p42/p44 and MAPKs was assessed by Western blot. In a concentration-dependent manner, chemokines including eotaxin, RANTES, IL-8, and MIP-1α increased ASMC's [(3)H]thymidine incorporation and DNA synthesis. IL-8, eotaxin, and MIP-1α decreased the rate of apoptosis of ASMCs compared with the matched controls. A significant increase in phosphorylated p42/p44 MAPKs was seen after treating ASMCs with RANTES and eotaxin. Moreover, inhibition of p42/p44 MAPK phosphorylation reduced the level of chemokine-induced ASM proliferation. We conclude that chemokines might contribute to airway remodeling seen in asthma by enhancing the number and survival of ASMCs.     

Effects of insulin and high glucose on mobilization of slo1 BKCa channels in podocytes

Podocytes are dynamic polarized cells that lie on the surface of glomerular capillaries and comprise an essential component of the glomerular filtration barrier. Podocytes are affected in the earliest stages of diabetic nephropathy and insulin signaling to podocytes is essential for normal glomerular function. Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) encoded by the Slo1 gene are expressed in podocytes in a complex with multiple glomerular slit diaphragm proteins including nephrin, TRPC6 channels, and several different actin-binding proteins. Here we show that insulin increases cell surface expression of podocyte BK(Ca) channels, which is accompanied by a corresponding increase in the density of current flowing through these channels. Insulin stimulation of BK(Ca) channels was detectable in 15 min and required activation of both Erk and Akt signaling cascades. Exposure to high glucose (36.1 mM) for 24 h caused a marked reduction in the steady-state surface expression of BK(Ca) channels as well as of the slit diaphragm signaling molecule nephrin. High glucose treatment also abolished the stimulatory effects of insulin on BK(Ca) current density, although insulin continued to increase phosphorylation of Erk and Akt under those conditions. Therefore, in contrast to most other cell types, high glucose abrogates the effects of insulin in podocytes at relatively distal steps in its signaling pathway. Insulin stimulation of BK(Ca) channels in podocytes may prepare podocytes to adapt to changes in pressure gradients that occur during postprandial hyperfiltration.     

Temperature-dependent fumigant activity of essential oils against twospotted spider mite (Acari: Tetranychidae)

Fumigant activity of 34 commercial essential oils was assessed on female adults and eggs of twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) at three temperatures (5, 15, and 25 degrees C). Common thyme, cinnamon, and lemongrass oils were equally effective on twospotted spider mite adults showing 85.8-100% mortality at 5 and 10 microl/liter air at 25 degrees C. At a lower temperature of 15 degrees C, lemongrass and peppermint resulted in > or =90% mortality of adults at 10 microl/liter air. Only lemongrass was relatively active at 5 microl/liter air, at 15 degrees C. At 5 degrees C, lemongrass and peppermint caused significantly higher adult mortality than controls but only at 10 microl/liter air. Common thyme oil showed the highest ovicidal activity at 5 microl/liter air at 25 degrees C. Among the main components of common thyme and lemongrass oils, citral was lethal to twospotted spider mite adults at all tested temperatures. Carvacrol, thymol, and citral caused the same inhibitory effects on the hatch of twospotted spider mite eggs at 25 degrees C. However, citral was more active than other compounds to twospotted spider mite eggs at 15 degrees C. Therefore, we conclude that citral has the best potential for development as a fumigant against twospotted spider mite on agricultural products harvested late in the growing season.     

IDENTIFICATION OF THE HIGH‐TEMPERATURE RESPONSE GENES FROM PORPHYRA SERIATA (RHODOPHYTA) EXPRESSION SEQUENCE TAGS AND ENHANCEMENT OF HEAT TOLERANCE OF CHLAMYDOMONAS (CHLOROPHYTA) BY EXPRESSION OF THE PORPHYRA HTR2 GENE1

Temperature is one of the major environmental factors that affect the distribution, growth rate, and life cycle of intertidal organisms, including red algae. In an effort to identify the genes involved in the high‐temperature tolerance of Porphyra, we generated 3,979 expression sequence tags (ESTs) from gametophyte thalli of P. seriata Kjellm. under normal growth conditions and high‐temperature conditions. A comparison of the ESTs from two cDNA libraries allowed us to identify the high temperature response (HTR) genes, which are induced or up‐regulated as the result of high‐temperature treatment. Among the HTRs, HTR2 encodes for a small polypeptide consisting of 144 amino acids, which is a noble nuclear protein. Chlamydomonas expressing the Porphyra HTR2 gene shows higher survival and growth rates than the wild‐type strain after high‐temperature treatment. These results suggest that HTR2 may be relevant to the tolerance of high‐temperature stress conditions, and this Porphyra EST data set will provide important genetic information for studies of the molecular basis of high‐temperature tolerance in marine algae, as well as in Porphyra.     

Mechanism of inhibition of retrovirus release from cells by interferon-induced gene ISG15

Budding of retroviruses from cell membranes requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including endosome sorting complex required for transport III (ESCRT-III) protein complex and vacuolar protein sorting 4 (VPS4) and its ATPase. In response to infection, a cellular mechanism has evolved that blocks virus replication early and late in the budding process through expression of interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin. Interferon treatment of DF-1 cells blocks avian sarcoma/leukosis virus release, demonstrating that this mechanism is functional under physiological conditions. The late block to release is caused in part by a loss in interaction between VPS4 and its coactivator protein LIP5, which is required to promote the formation of the ESCRT III-VPS4 double-hexamer complex to activate its ATPase. ISG15 is conjugated to two different LIP5-ESCRT-III-binding charged multivesicular body proteins, CHMP2A and CHMP5. Upon ISGylation of each, interaction with LIP5 is no longer detected. Two other ESCRT-III proteins, CHMP4B and CHMP6, are also conjugated to ISG15. ISGylation of CHMP2A, CHMP4B, and CHMP6 weakens their binding directly to VPS4, thereby facilitating the release of this protein from the membrane into the cytosol. The remaining budding complex fails to release particles from the cell membrane. Introducing a mutant of ISG15 into cells that cannot be conjugated to proteins prevents the ISG15-dependent mechanism from blocking virus release. CHMP5 is the primary switch to initiate the antiviral mechanism, because removal of CHMP5 from cells prevents ISGylation of CHMP2A and CHMP6.     

Identification of noncoding transcripts from within CENP-A chromatin at fission yeast centromeres

The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1).     

VEGF inhibitor (Iressa) arrests histone deacetylase expression: single-cell cotransfection imaging cytometry for multi-target-multi-drug analysis

Multi-target-multi-drug approaches are needed to accelerate the process of drug discovery screening and to design efficient therapeutic strategies against diseases that involve alterations in multiple cellular targets. Herein we report single-cell cotransfection imaging cytometry to quantitatively screen drug-induced off-target effects. Vascular endothelial growth factor (VEGF) and histone deacetylase (HDAC) genes amplified from the genomic DNA were cloned in fluorescently tagged gene constructs (RFP-HDAC/YFP-VEGF). These gene constructs were cotransfected in HEK-293 cells to explore the possibility of off-target effects of 4-phenylbutyrate and Iressa on the expression of VEGF and HDAC through single-cell imaging cytometry. Iressa (10 μM) treatment at the time of cotransfection or 48 h after cotransfection of RFP-HDAC/YFP-VEGF plasmids in HEK-293 cells resulted in off-target effects on HDAC expression. These results suggest possible applications of Iressa in the treatment of diseases in which expression of both HDAC and VEGF should be inhibited. 4-Phenylbutyrate (2.0 mM) did not show any off-target effects on VEGF expression. The developed quantitative multicolor live single-cell cotransfection imaging can be employed to select better drug combinations for faster screening and greater accuracy in multi-target-multi-drug analysis by increasing the on-target/desired off-target effects and eliminating the undesirable off-target effects.