Lähetti-RNA:han sitoutuvat proteiinit tyypin 1 angiotensiini II -reseptorin säätelijöinä

The renin-angiotensin system is a key regulator of blood pressure and electrolyte homeostasis. Most hemodynamic responses to angiotensin II are mediated via angiotensin II type 1 receptor (AT1R). The 3 -untranslated region (3 UTR) of AT1R mRNA is important in posttranscriptional regulation of AT1R expression. It contains multiple adenylate-uridylate-rich elements that are recognized by various RNA-binding proteins (RNBP) affecting the stability, conformation, subcellular localization, and translation of their target mRNAs. The aim of this study was to identify novel AT1R 3 UTR-binding RNBPs and understand their physiological role in AT1R function. AT1R 3 UTR-associated RNBPs were isolated from human vascular smooth muscle cells by protein affinity purification using AT1R 3 UTR as a probe. Mass spectrometric identification of the isolated proteins led to identification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hu antigen R (HuR) and TIA1 cytotoxic granule-associated RNA-binding protein (TIA-1). GAPDH was identified as a novel AT1R mRNA-binding protein that suppresses AT1R translation. GAPDH binds to a hairpin-structure rich in adenosine and uridine in the proximal region of AT1R 3 UTR. Hydrogen peroxide-induced oxidative stress increased AT1R expression by dissociating GAPDH from AT1R 3 UTR and relieving it from the GAPDH-mediated suppression. Insulin-induced increase in AT1R expression was shown to be posttranscriptionally mediated by HuR. Insulin stabilized AT1R mRNA in a 3 UTR-dependent manner and HuR was shown to bind AT1R 3 UTR. Insulin induced nucleocytoplasmic translocation of HuR to cytoplasm and increased HuR binding to AT1R 3 UTR. This increased AT1R expression via stabilization of AT1R mRNA. AT1R avoids the endoplasmic reticulum (ER) stress-mediated translational suppression. Binding of TIA-1 to AT1R 3 UTR was shown to suppress AT1R expression. TIA-1 normally colocalized with AT1R mRNA in the cytoplasm. However, during ER stress TIA-1 was dissociated and directed to translationally silenced stress granules, while AT1R mRNA remained excluded from them. Thus, AT1R mRNA avoids aggregation to SGs and TIA-1-mediated translational suppression during ER stress. In conclusion, oxidative stress, insulin, and ER stress regulates AT1R expression posttranscriptionally by affecting the binding of GAPDH, HuR, and TIA-1 to its mRNA 3 UTR.Reniini-angiotensiinijärjestelmä on keskeinen verenpaineen ja kehon elektrolyytitasapainon säätelijä. Angiotensiini II:n vaikutukset välittyvät pääasiassa tyypin 1 angiotensiini II -reseptorin (AT1R) kautta. AT1R:n lähetti-RNA:n (mRNA) 3 -pään transloitumaton alue (3 UTR) on keskeinen AT1R:n transkription jälkeisessä säätelyssä. Se sisältää useita adenylaatti-uridylaattipitoisia alueita, jotka ovat RNA:ta sitovien proteiinien kohteita. Ne vaikuttavat kohde-mRNA-molekyyliensä puoliintumisaikaan, konformaatioon, solunsisäiseen sijaintiin ja translaatioon. Tutkimuksen tavoitteena oli tunnistaa uusia AT1R:n 3 UTR:ään sitoutuvia proteiineja ja ymmärtää niiden fysiologiset vaikutukset AT1R:n toiminnassa. Käyttäen AT1R:n 3 UTR:ää koettimena siihen sitoutuvat proteiinit eristettiin ihmisen verisuonten sileälihassoluista. Sitoutuneista proteiineista tunnistettiin massaspektrometrialla glyseraldehydi-3-fosfaattidehydrogenaasi (GAPDH), HuR (Hu antigen R) ja TIA-1 (TIA1 cytotoxic granule-associated RNA binding protein). GAPDH havaittiin AT1R:n mRNA:ta sitovaksi proteiiniksi, joka estää sen translaatiota. GAPDH sitoutuu AT1R:n 3 UTR:n alussa sijaitsevaan adenosiiniä ja uridiinia sisältävään hiuspinnirakenteeseen. Vetyperoksidin aiheuttama hapetusstressi lisäsi AT1R:n ilmentymistä irrottamalla GAPDH:n 3 UTR:stä estäen näin GAPDH:n negatiivisen vaikutuksen. Insuliinin lisäämä AT1R:n ilmentyminen osoitettiin johtuvan HuR:n välittämästä transkription jälkeisestä säätelystä. Insuliini vakautti AT1R:n mRNA:ta 3 UTR:n kautta ja HuR:n osoitettiin sitoutuvan tälle alueelle. Insuliini lisäsi HuR:n siirtymistä tumasta solulimaan ja sitoutumista AT1R:n 3 UTR:ään. Tämä lisäsi AT1R:n ilmentymistä vakauttamalla sen mRNA:ta. AT1R välttää endoplasmakalvoston (ER) stressin aiheuttaman translaation eston. TIA-1 vähensi AT1R:n ilmentymistä 3 UTR:n välityksellä. Tavallisesti TIA-1 ja AT1R:n mRNA kolokalisoituvat solulimassa, mutta ER-stressin aikana TIA-1 siirtyy translationaalisesti hiljennettyihin stressigranuloihin AT1R:n mRNA:n jäädessä niiden ulkopuolelle. Näin ollen AT1R:n mRNA välttää kertymisen stressigranuloihin ja TIA-1:n välittämän translationaalisen hiljentämisen ER-stressin aikana. Yhteenvetona: hapetusstressi, insuliini ja ER-stressi säätelevät AT1R:n ilmentymistä transkription jälkeisellä tasolla vaikuttamalla GAPDH:n, HuR:n ja TIA-1:n sitoutumiseen sen mRNA:n 3 UTR-alueelle

Spatial Analysis of Northern Goshawk Territories in the Black Hills, South Dakota

The Northern Goshawk (Accipiter gentilis) is the largest of the three North American species of Accipiter and is more closely associated with older forests than are the other species. Its reliance on older forests has resulted in concerns about its status, extensive research into its habitat relationships, and litigation. Our objective was to model the spatial patterns of goshawk territories in the Black Hills, South Dakota, to make inferences about the underlying processes. We used a modification of Ripley\u27s K function that accounts for inhomogeneous intensity to determine whether territoriality or habitat determined the spacing of goshawks in the Black Hills, finding that habitat conditions rather than territoriality were the determining factor. A spatial model incorporating basal area of trees in a stand of forest, canopy cover, age of trees \u3e23 cm in diameter, number of trees per hectare, and geographic coordinates provided good fit to the spatial patterns of territories. There was no indication of repulsion at close distances that would imply spacing was determined by territoriality. These findings contrast with those for the Kaibab Plateau, Arizona, where territoriality is an important limiting factor. Forest stands where the goshawk nested historically are now younger and have trees of smaller diameter, probably having been modified by logging, fire, and insects. These results have important implications for the goshawk\u27s ecology in the Black Hills with respect to mortality, competition, forest fragmentation, and nest-territory protection

Detection of New Delhi Metallo-β-Lactamase (Encoded by \u3ci\u3ebla\u3c/i\u3e\u3csub\u3eNDM-1\u3c/sub\u3e) in \u3ci\u3eAcinetobacter schindleri\u3c/i\u3e during Routine Surveillance

A carbapenem-resistant Alcaligenes faecalis strain was isolated from a surveillance swab of a service member injured in Afghanistan. The isolate was positive for blaNDM by real-time PCR. Species identification was reevaluated on three identification systems but was inconclusive. Genome sequencing indicated that the closest relative was Acinetobacter schindleri and that blaNDM-1 was carried on a plasmid that shared \u3e99% identity with one identified in an Acinetobacter lwoffii isolate. The isolate also carried a novel chromosomally encoded class D oxacillinase

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA-1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner.Peer reviewe

Cyclopentenone Isoprostanes Inhibit the Inflammatory Response in Macrophages

Although both inflammation and oxidative stress contribute to the pathogenesis of many disease states, the interaction between the two is poorly understood. Cyclopentenone isoprostanes (IsoPs), highly reactive structural isomers of the bioactive cyclopentenone prostaglandins PGA2 and PGJ2, are formed non-enzymatically as products of oxidative stress in vivo. We have, for the first time, examined the effects of synthetic 15-A2- and 15-J2-IsoPs, two groups of endogenous cyclopentenone IsoPs, on the inflammatory response in RAW264.7 and primary murine macrophages. Cyclopentenone IsoPs potently inhibited lipopolysaccharide-stimulated IkappaB alpha degradation and subsequent NF-kappaB nuclear translocation and transcriptional activity. Expression of inducible nitric-oxide synthase and cyclooxygenase-2 were also inhibited by cyclopentenone IsoPs as was nitrite and prostaglandin production (IC50 approximately 360 and 210 nM, respectively). 15-J2-IsoPs potently activated peroxisome proliferator-activated receptor gamma (PPARgamma) nuclear receptors, whereas 15-A2-IsoP did not, although the anti-inflammatory effects of both molecules were PPARgamma-independent. Interestingly 15-A2-IsoPs induced oxidative stress in RAW cells that was blocked by the antioxidant 4-hydroxy-TEMPO (TEMPOL) or the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. TEMPOL also abrogated the inhibitory effect of 15-A2-IsoPs on lipopolysaccharide-induced NF-kappaB activation, inducible nitricoxide synthase expression, and nitrite production, suggesting that 15-A2-IsoPs inhibit the NF-kappaB pathway at least partially via a redox-dependent mechanism. 15-J2-IsoP, but not 15-A2-IsoP, also potently induced RAW cell apoptosis again via a PPAR gamma-independent mechanism. These findings suggest that cyclopentenone IsoPs may serve as negative feedback regulators of inflammation and have important implications for defining the role of oxidative stress in the inflammatory response

Regulation of AT1R expression through HuR by insulin

Peer reviewe

Microsomal prostaglandin E synthase-2 is not essential for in vivo prostaglandin E2 biosynthesis

Prostaglandin E2 (PGE2) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE2 from the cyclooxygenase metabolite PGH2 have been described. Here, we examine the contribution of one of these enzymes to PGE2 production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE2 levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE2 synthase

Department of Pathology, Thomas Jefferson University, Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors.

BACKGROUND: Although numerous mouse models of breast carcinomas have been developed, we do not know the extent to which any faithfully represent clinically significant human phenotypes. To address this need, we characterized mammary tumor gene expression profiles from 13 different murine models using DNA microarrays and compared the resulting data to those from human breast tumors. RESULTS: Unsupervised hierarchical clustering analysis showed that six models (TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3, TgWAP-Tag, and TgC3(1)-Tag) yielded tumors with distinctive and homogeneous expression patterns within each strain. However, in each of four other models (TgWAP-T121, TgMMTV-Wnt1, Brca1Co/Co;TgMMTV-Cre;p53+/- and DMBA-induced), tumors with a variety of histologies and expression profiles developed. In many models, similarities to human breast tumors were recognized, including proliferation and human breast tumor subtype signatures. Significantly, tumors of several models displayed characteristics of human basal-like breast tumors, including two models with induced Brca1 deficiencies. Tumors of other murine models shared features and trended towards significance of gene enrichment with human luminal tumors; however, these murine tumors lacked expression of estrogen receptor (ER) and ER-regulated genes. TgMMTV-Neu tumors did not have a significant gene overlap with the human HER2+/ER- subtype and were more similar to human luminal tumors. CONCLUSION: Many of the defining characteristics of human subtypes were conserved among the mouse models. Although no single mouse model recapitulated all the expression features of a given human subtype, these shared expression features provide a common framework for an improved integration of murine mammary tumor models with human breast tumors

Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors

Comparison of mammary tumor gene-expression profiles from thirteen murine models using microarrays and with that of human breast tumors showed that many of the defining characteristics of human subtypes were conserved among mouse models