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FIGURE 1.

IFNα-inducible expression of family members. , NIH3T3 cells were treated with IFNα for 3 or 6 h or left untreated as indicated. Total RNA was subsequently isolated, and the expression of (), (), (), 5 (), and () was analyzed by real time RT-PCR, using specific primers and as an internal control. The data are expressed as fold increase over control untreated samples and represent the means ± S.E. of several experiments for ( = 3), ( = 7), ( = 6), ( = 4), and ( = 5). , NIH3T3 cells were treated with IFNα for 48 h, and after cell lysis, the proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN2 antibody. , NIH3T3 cells were treated with mouse IFNα, as indicated. Cytosolic and nuclear fractions were obtained, and the proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN2 antibody. Immunoblotting with antibodies against Lamin A and GAPDH was also performed to control for successful separation of nuclear and cytosolic fractions.

To better understand the regulation of SLFN proteins during engagement of the Type I IFN receptor, we generated and used an anti-SLFN2 antibody to directly examine the expression of SLFN2 protein after IFNα treatment of cells. This antibody was custom-generated via a commercial vendor against a conserved region in the N terminus of the protein and detects a single band at ∼44 kDa, which is consistent with the predicted molecular mass of SLFN2. As shown in Easy Street Holly Clog r2TQ4X8
F , base-line expression of SLFN2 in NIH3T3 cells was clearly detectable, but treatment of cells with IFNα resulted in up-regulation of the expression of the protein ( Fig. 1 F ). We also examined the subcellular localization of the protein. In a previous study, it was shown that overexpressed FLAG-tagged SLFN2 in HEK-293T cells is exclusively expressed in the cytoplasm ( Volcom All Day Long Sandal brJJNek4g8
). However, FLAG tagging could theoretically interfere with the structural properties and localization of the protein, and the potential translocation of endogenous SLFN2 in response to cytokine treatment has not been known. In studies in which the localization of the endogenous protein was directly determined using the newly generated anti-SLFN2 antibody, we found that endogenous SLFN2 is exclusively expressed in the cytoplasm, and IFNα treatment does not induce its translocation to the nucleus ( Fig. 1 G ).

IFNα binding to the type I IFN receptor results in activation of STAT1, STAT2, and STAT3 transcription factors, which form various homo- and/or heterodimers that can bind specific sequences in the promoters of IFN inducible genes ( MICHAEL Michael Kors Carlson Flat b6ih9pIZ8J
7 ). In addition, IFNα-mediated gene transcription is regulated by auxiliary pathways, such as the p38 MAPK pathway ( 7 Kim Rogers® Jenna 2 Boot HleflB46Vw
). To define the roles of distinct STAT proteins and the p38 MAPK in SLFN gene expression, experiments were performed using cells with targeted disruption of STAT1 , STAT3 , or p38α genes. In initial studies, STAT1 −/− MEFs and STAT1 +/+ parental MEFs were treated with mouse IFNα, and mRNA expression for SLFN1 , SLFN2 , SLFN3 , SLFN5 , and SLFN8 was determined. IFNα-dependent expression of all SLFN genes was decreased in STAT1 knock-out MEFs compared with parental MEFs, and the effect ranged from a partial impairment ( SLFN3 ) to completely defective transcription ( SLFN1 , 2 , 5 , and 8 ) ( Fig. 2 ). Similarly, IFNα-inducible SLFN expression was examined in STAT3 knock-out MEF cells. The induction of expression of SLFN1 , SLFN2 , SLFN3 , and SLFN8 genes was decreased in STAT3 knock-out cells although not abrogated ( Fig. 3 , A–C ). The expression of SLFN5 was completely STAT3-independent, and in fact, SLFN5 expression was enhanced in STAT3 knock-out cells ( Fig. 3 D ).

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FIG 1

IFN-α-induced expression of genes in human renal cell carcinoma cells. (A) 786-0 cells were treated with IFN-α for the indicated time. Total mRNA was isolated, and expression of , , , and was measured by real-time RT-PCR, using GAPDH as a housekeeping gene. Data are expressed as the fold increase over control untreated samples and represent means ± standard errors of results from four independent experiments. Paired two-tailed test analysis showed a two-tailed value of 0.0108 for SLFN5 at 6 h compared to control. (B) 786-0 cells were treated with IFN-α as indicated, and after cell lysis, total proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN5 antibody or an anti-GAPDH antibody. (C) ACHN cells were treated with IFN-α for the indicated times. Total RNA was isolated, and expression of , , , , and was measured by real-time RT-PCR using GAPDH as a housekeeping gene. Data are expressed as the fold increase over control of untreated samples and represent means ± standard errors of results from three independent experiments. Paired two-tailed test analysis showed a two-tailed value of 0.0188 for SLFN5 at 6 h compared to control. (D) ACHN cells were treated with IFN-α as indicated, and after cell lysis, proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN5 antibody or an anti-α tubulin antibody.

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FIG 2

Expression of SLFN5 in normal human renal proximal tubule epithelial cells (RPTEC). (A) RPTEC and 786-0 cells were lysed, and proteins were resolved by SDS-PAGE and immunoblotted with antibodies against SLFN5 or GAPDH, as indicated. (B) RPTEC were treated with IFN-β for the indicated time. Total mRNA was isolated, and expression of SLFN5 was measured by real-time RT-PCR, using GAPDH as a housekeeping gene. Data are expressed as the fold increase over control untreated (UT) samples and represent means ± standard errors of results from two independent experiments.

To examine the functional relevance of SLFN5 expression in RCC cells, SLFN5 protein levels were transiently knocked down by SLFN5-specific siRNA or stably downregulated by shRNA-mediated targeting ( Fig. 3A and Sigerson Morrison Kalie hfbx4LYZq
). Transfection with SLFN5 siRNA resulted in a dramatic morphological change in 786-0 cells ( Rachel Zoe Tara vxTMoc
). As expected, control cells exhibited an epithelial cell-like phenotype ( Fig. 3C , top panels), but cells in which SLFN5 was knocked down exhibited a more spindle-shaped morphology ( Fig. 3C , bottom panels). To better define the morphological changes, cells were stained with antibodies against F-actin or against vinculin, a key component of the focal adhesion complex. There was a robust decrease in the focal adhesion assembly, with loss of stress fibers, and a reduction in cell size following transient knockdown of SLFN5 ( Fig. 3D ). Similarly, stable knockdown of SLFN5 resulted in loss of stress fibers and reduction in cell size ( Fig. 3E and F ). In contrast, knockdown of SLFN11 , SLFN12 , or SLFN13 did not result in any significant change ( Nine West Lipstick 3 OEo1u
). Importantly, there was no significant change in cell viability or proliferation observed when SLFN5 was knocked down ( MIA Jasmin Dual Buckle Sandal W1keUE
). In other studies, when SLFN5 was exogenously overexpressed, there were no significant effects on cell morphology ( Franco Sarto Nova a5ZgBkbuxs
), suggesting that endogenous baseline levels are important for regulation of cell morphology.

· Ökologie und Evolution
Rieker D1647 Doris 47 W8loU
Rohde Klaus
Klaus Rohde Website: Jilley Floral Print PointyToe Pumps Cr443yU
Professor emeritus, Zoology, University of New England, Armidale NSW, Australia. Research on parasitology especially marine parasitology, evolutionary biology, ecology, ultrastructure and phylogeny of invertebrates, taxonomy. About 360 publications, books: Ecology of Marine Parasites (two editions 1982, 1993, University of Queensland Press und CABI Wallingford, Oxon.; Malay-Indonesian translation); Nonequilibrium Ecology (Cambridge University Press 2005), Marine Parasitology (editor) (CSIRO Melbourne and CABI Wallingford Oxon. 2005); Satire, Politik und Kunst (Lulu 2005). About 6000 citations (2010), h- Index 36 (2009). Clarke Medal of the Royal Society of NSW; UNE award for excellence in science. Professor emeritus, Zoologie, University of New England, Armidale NSW, Australien. Forschungen über Parasitologie insbesondere Meeresparasitologie, evolutionäre Biologie, Ökologie, Ultrastruktur und Phylogenie von Wirbellosen, Taxonomie. Etwa 360 Veröffentlichungen, Bücher: Ecology of Marine Parasites (zwei Auflagen 1982, 1993, University of Queensland Press und CABI Wallingford, Oxon.; malaiisch-indonesische Übersetzung); Nonequilibrium Ecology (Cambridge University Press 2005), Marine Parasitology (Herausgeber) (CSIRO Melbourne und CABI Wallingford Oxon. 2005); Satire, Politik und Kunst (Lulu 2005). Etwa 6000 Zitierungen (2010), h- Index 36 (2009). Clarke Medaille der Royal Society of NSW; UNE Auszeichnung für hervorragende wissenschaftliche Leistungen.

Definition

Breitengradienten im Artenreichtum werden allgemein als eine Zunahme der Artenzahlen von hohen (kalten) zu niederen (warmen) Breiten definiert. Zunahme der Diversität in niederen Breiten sind jedoch auch für Gattungen und höhere Taxa bekannt, es ist daher besser, von Breitengradienten in Biodiversität zu sprechen.

Entdeckung

Alexander von Humboldt entdeckte die Gradienten während seiner Expedition nach Südamerika in 1799, und er nahm sogar an, dass Temperatur eine Erklärung geben könnte [1] .

Die Bedeutung von Untersuchungen an Breitengradienten

Eine zentrale, wenn nicht die zentrale Frage der evolutionären Ökologie ist die nach den Ursachen für Unterschiede in der Abundanz und Diversität von Organismen in verschiedenen Habitaten (Biotopen) und Regionen. Derartige Unterschiede sind häufig zwischen Biotopen, selbst benachbarten, und im grösseren Rahmen sind sie auch zu erkennen zwischen geographischen Breiten und Längen, Höhen, und Tiefen (besonders in den Ozeanen). Die weitaus am besten bekannten Gradienten sind jedoch Breitengradienten. Dies bietet uns die Möglichkeit, die die Biodiversität verursachenden Faktoren zu finden.

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