Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Molecular Cell Research (v.1763, #3)
Comparison of differential gene expression of hot and cold thyroid nodules with primary epithelial cell culture models by investigation of co-regulated gene sets by Markus Eszlinger; Knut Krohn; Martin Beck; David Kipling; Sarah Forbes-Robertson; Jürgen Läuter; Anke Toenjes; David Wynford-Thomas; Ralf Paschke (pp. 263-271).
Both autonomously functioning thyroid nodules (AFTNs) and cold thyroid nodules (CTNs) are characterized by an increased proliferation, however, they have opposite functional activities. Therefore, with the aim to further understand the distinct molecular pathology of each entity and to discover common mechanisms like those leading to increased proliferation in both, AFTNs and CTNs, we now compared gene expression of AFTNs and CTNs with in vitro model systems (TSH-stimulated and ras-transfected primary cultures (PC)) whose gene expression patterns can be attributed to specific molecular alterations. Since combinations of co-regulated genes are more likely to reveal molecular mechanisms, we used a procedure which groups co-regulated genes within “gene sets�. We found a co-regulated gene set in the AFTNs that overlaps with differential expression in TSH-stimulated PCs but not in CTNs or ras-transfected PCs. In addition to thyroid peroxidase and sialyltransferase 1, this set of co-regulated genes comprises metallothioneins and the G-protein-coupled receptor 56. Although their role in the thyroid is unknown so far, their appearance in one group indicates a functional relevance in TSH–TSH receptor-stimulated mechanisms. Furthermore, we identified down-regulated gene sets with concordant expression patterns in AFTNs, CTNs and ras-transfected PCs. However, these expression patterns are not of relevance in the TSH-stimulated PCs. These findings suggest that TSH-stimulated PCs can be used as a model of increased thyroid function (AFTNs), whereas the ras-transfected PCs better reflect the increased proliferation of both AFTNs and CTNs.
Keywords: Thyroid; Signal transduction; Ras; Transfection; Gene expression profiling
Nek1 shares structural and functional similarities with NIMA kinase by Erez Feige; Ohad Shalom; Shlomo Tsuriel; Nissan Yissachar; Benny Motro (pp. 272-281).
The Aspergillus NIMA serine/threonine kinase plays a pivotal role in controlling entrance into mitosis. A major function attributed to NIMA is the induction of chromatin condensation. We show here that the founder murine NIMA-related kinase, Nek1, is larger than previously reported, and that the full-length protein conserves the structural hallmarks of NIMA. Even though Nek1 bears two classical nuclear localization signals (NLS), the endogenous protein localizes to the cytoplasm. Ectopic overexpression of various Nek1 constructs suggests that the C-terminus of Nek1 bears cytoplasmic localization signal(s). Overexpression of nuclear constructs of Nek1 resulted in abnormal chromatin condensation, with the DNA mainly confined to the periphery of the nucleus. Advanced condensation phenotype was associated with nuclear pore complex dispersal. The condensation was not accompanied by up-regulation of mitotic or apoptotic markers. A similar phenotype has been described following NIMA overexpression, strengthening the notion that the mammalian Nek1 kinase has functional similarity to NIMA.
Keywords: NIMA; Nek kinase; Chromatin condensation; Cell cycle; Sub-cellular localization
Aspirin-induced blockade of NF-κB activity restrains up-regulation of glial fibrillary acidic protein in human astroglial cells by Moon-Kyoung Bae; Su-Ryun Kim; Hak-Joong Lee; Hee-Jun Wee; Mi-Ae Yoo; Sae Ock Oh; Sun-Yong Baek; Bong-Seon Kim; Jae-Bong Kim; Sik-Yoon; Soo-Kyung Bae (pp. 282-289).
The marked induction of glial fibrillary acidic protein (GFAP) has been observed in astrocytes during neuropathological processes accompanying reactive gliosis; however, the precise molecular mechanism(s) underlying this GFAP induction remains poorly resolved. Therefore, in this study, we examined whether the change of nuclear factor-kappa B (NF-κB) activity can influence GFAP expression levels. Aspirin, widely used to prevent NF-κB activity, reduced the levels of GFAP mRNA and protein in human astroglial cells including human glioblastoma A172 cells and primary human brain astrocyte cells (HBAs). Furthermore, aspirin inhibited the effects of hypoxic injury on the up-regulation of GFAP expression in HBAs. We confirmed the repressive effect of aspirin on GFAP transcription by GFAP promoter-driven reporter assay and found that one NF-κB binding site conserved in the mouse and human GFAP gene promoters is critical for this effect. To further delineate whether NF-κB is directly involved in the regulation of GFAP gene expression, we transfected A172 cells with an expression vector encoding a super-repressor IκBα protein (IκBα-SR) to specifically inhibit NF-κB activity and found the marked reduction of GFAP protein levels in IκBα-SR-transfectant cells. Taken together, our results suggest that NF-κB may play pivotal roles in GFAP gene expression.
Keywords: GFAP; NF-κB; Aspirin; A172; HBAs; Hypoxia; Reactive gliosis
Cirp protects against tumor necrosis factor-α-induced apoptosis via activation of extracellular signal-regulated kinase by Toshiharu Sakurai; Katsuhiko Itoh; Hiroaki Higashitsuji; Kohsuke Nonoguchi; Yu Liu; Hirohiko Watanabe; Tadasu Nakano; Manabu Fukumoto; Tsutomu Chiba; Jun Fujita (pp. 290-295).
Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we analyzed the effects of low culture temperature (32 °C) and cold-inducible RNA-binding protein (Cirp) expression on apoptosis in vitro. In BALB/3T3 cells treated with tumor necrosis factor (TNF)-α and cycloheximide, the down-shift in temperature from 37 °C to 32 °C increased the expression of Cirp and suppressed the apoptosis. Activation of caspase-8 was suppressed, and the level of phosphorylated extracellular signal-regulated kinase (ERK) was increased. Transduction of Cirp into the Cirp-deficient mouse fibroblasts increased the level of phosphorylated ERK and suppressed the TNF-α-induced apoptosis both at 37 °C and 32 °C. The ERK-specific inhibitor PD98059 decreased the cytoprotective effect of Cirp as well as that of low culture temperature. These data suggest that mild hypothermia protects cells from TNF-α-induced apoptosis, at least partly, via induction of Cirp, and that Cirp protects cells by activating the ERK pathway.
Keywords: Abbreviations; Cirp; cold-inducible RNA-binding protein; ERK; extracellular signal-regulated kinase; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; MEF; mouse embryonic fibroblast; Rbm3; RNA binding motif protein3; TNF; tumor necrosis factorHypothermia; Cirp; TNF-α; ERK; Apoptosis
Tissue inhibitors of metalloproteinases-1 (TIMP-1) and -2(TIMP-2) are major serum factors that stimulate the TIMP-1 gene in human gingival fibroblasts by Xiao-Kui Guo; Wan-Qian Zhao; Chihiro Kondo; Nagahiro Shimojo; Kyoko Yamashita; Takanori Aoki; Taro Hayakawa (pp. 296-304).
We demonstrate in this study that both TIMP-1 and TIMP-2 are major serum factors that stimulate the induction of TIMP-1 mRNA in quiescent human gingival fibroblasts (Gin-1 cells) at mid-G1 (6–9 h after serum stimulation) of the cell cycle, but not that of TIMP-2. When we chased the secretion of both TIMP proteins into culture medium containing 10% FCS freed of both TIMPs, TIMP-2 secretion rose to the level in 10% FCS after 24 h, but TIMP-1 secretion remained at a fairly low level even after 3 days, thus reflecting a contrastive difference in the induction of both TIMP mRNAs. The stimulating activity of TIMP-1 on the expression of the TIMP-1 gene switched over to inhibitory activity, when the TIMP-1 concentration in the culture medium exceeded about 30 ng/ml. The depletion of TIMP-1 and TIMP-2 from FCS affected remarkably the induction of c- jun and c- fos mRNAs, but not that of c-ets-1 mRNA. TIMP-1 and TIMP-2-dependent expression of AP-1 protein was further demonstrated by using nuclear extracts of Gin-1 cells in an electrophoretic mobility shift assay.
Keywords: Tissue inhibitor of metalloproteinases-1 (TIMP-1); TIMP-2; c-; jun; , c-; fos; and; c-ets-1; mRNAs; AP-1; Human gingival fibroblasts (Gin-1 cells)
Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic and/or necrotic processes and on MT isoform gene expression by Alessandro Santon; Alessia Formigari; Vincenzo Albergoni; Paola Irato / (pp. 305-312).
It has been shown in various systems that zinc is able to antagonize the catalytic properties of the redox-active transition metals iron and copper, although the process is still unclear. Probably, the protective effect of Zn against oxidative stress is mainly due to the induction of a scavenger metal binding protein such as metallothionein (MT), rather than a direct action. To support this hypothesis, in this study, the effects of Zn, Cu, Fe, Zn+Cu and Zn+Fe treatments were investigated in a fibroblast cell line corresponding to an SV40-transformed MT-1/-2 mutant (MT−/−), and in wild type (MT+/+), by valuing metal concentrations and apoptotic and/or necrotic processes. We also investigated the synthesis of MT and the levels of both MT-1 and MT-2 mRNAs. In MT+/+ cells, co-treatment with Zn+Fe caused a decrease in Fe content compared to treatment with Fe alone. After Zn and Zn+Cu exposure the expression of MT-1 and MT-2 isoforms increased with a concomitant increase in MT synthesis. Annexin V-FITC and propidium iodide staining revealed necrotic or apoptotic cells in terminal stages, especially after Fe treatments. Immunofluorescent staining with an anti-ssDNA Mab and annexin detected a lower signal in co-treated cells compared to the single treatments in both cell lines. The intensity and quantity of fluorescence resulting from anti-ssDNA and Annexin V staining of MT null cells was higher compared to wild type cells. These results suggest that Zn alone does not completely exert an anti-oxidant effect against Cu and Fe toxicity, but that induction of MT is necessary.
Keywords: Metallothionein; Apoptosis; Heavy metals; Mouse embryo; Fibroblast
Tryptase activates PKB in inflammatory reaction in ECV304 cells by Yongjie Ma; Bin Zhang; Ruizhe Qian; Chao Lu; Fengdi Zhao; Lianhua Yin (pp. 313-321).
Tryptase is involved in proteinase-activated receptor-2 (PAR-2) mediated up-regulation of IL-8 expression. The present report showed the effects of tryptase on gene expression and activation, including up-regulation IL-8 expression. The expression of mRNA for NF-κB first increased at 1 h after tryptase-treatment (1 ng/ml) and reached the plateau after 4 h. The NF-κB mRNA increased by 3-fold ( n=3, P<0.05), AP-1 by 2-fold ( n=3, P<0.05), and PKB by 10-fold ( n=3, P<0.05). However, tryptase-treatment did not affect the expression of JNK and p38 MAPK when compared with control cells at mRNA level. Furthermore, in addition to increasing phosphorylation of p38 MAPK, tryptase-treatment also increased phosphorylation of PKB by 2-fold at 15 min following the treatment. The up-regulation and phosphorylation of PKB by tryptase could be abolished by either phosphoinositol-3-kinase (PI3K) inhibitor (LY294002) at 10 μM or antisense PKB cDNA transfection. The up-regulation of NF-κB expression could be inhibited by LY294002 and antisense PKB cDNA. These results indicate that tryptase can activate PI3K-PKB pathway and enhance IL-8 expression.
Keywords: Tryptase; PAR-2; PKB; NF-κB; Inflammation; Mast cell
Mapping the Ca2+-dependent binding of an invertebrate homolog of protein phosphatase 4 regulatory subunit 2 to the small EF-hand protein, calsensin by Deepa V. Venkitaramani; D. Bruce Fulton; Amy H. Andreotti; Kristen M. Johansen; Jørgen Johansen (pp. 322-329).
The EF-hand family of calcium-binding proteins regulates cellular signal transduction events via calcium-dependent interactions with target proteins. Here, we show that the COOH-terminal tail of the leech homolog of protein phosphatase 4 regulatory subunit 2 (PP4-R2) interacts with the small neuronal EF-hand calcium-binding protein, Calsensin, in a calcium-dependent manner. Using two-dimensional NMR spectroscopy and chemical shift perturbations we have identified and mapped the residues of Calsensin that form a binding surface for PP4-R2. We show that the binding groove is formed primarily of discontinuous hydrophobic residues located in helix 1, the hinge region, and helix 4 of the unicornate-type four helix structure of Calsensin. The findings suggest the possibility that calcium-dependent modulation of phosphatase complexes through interactions with small calcium-binding proteins may be a general mechanism for regulation of signal transduction pathways.
Keywords: Abbreviations; PP4-R2; Protein phosphatase 4 regulatory subunit 2; PP4; Protein phosphatase 4; NMR; Nuclear magnetic resonance; GST; Glutathione-S-transferase; EDTA; Ethylenediaminetetraacetic acid; EGTA; Ethyleneglycol-bis(b-aminoethyl)-; N,N,N′,N′; -tetraacetic acid; HRP; Horseradish peroxidase; HSQC; Heteronuclear single quantum coherence; ORF; Open reading frame; TBS; Tris buffered saline; DAB; Diamino benzidine; RACE; Rapid amplification of cDNA ends; SMN; Survival motor neuron; SnRNPs; Small nuclear ribonucleoproteins; PMSF; Phenylmethylsulfonyl fluoride; Immunoprecipitation; Ip; mAb; Monoclonal antibody; PAb; Polyclonal antiserumNMR; Calcium-binding protein; Calsensin; Binding surface; Leech; Protein phosphatase regulation
Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation by Fabio Greco; Annarita Ciana; Daniela Pietra; Cesare Balduini; Giampaolo Minetti; Mauro Torti (pp. 330-335).
Recent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the β2 subunit of the Na+/K+-ATPase, as verified according to the recently proposed “β-profiling test� [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+-ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+-activated RBCs, suggesting a possible role for this GTPase in membrane shedding.
Keywords: Red blood cells; Rap1; Rap2; Small GTPases; Vesiculation