The protein MetAP2 is a common molecular target for both AGM-1470 and ovalicin. This finding suggests that MetAP2 may play a critical role in the proliferation of endothelial cells and may serve as a promising target for the development of new anti-angiogenic drugs.

Abstract. To understand the mechanisms of mRNA transport in eukaryotes, we have isolated Saccharomyces cerevisiae temperature-sensitive (ts) mutants which accumulate poly(A) + RNA ha the nucleus at the restrictive temperature. A total of 21 recessive mutants were isolated and classified into 16 complementation groups. Backcrossed mRNA transportdefective strains from each complementation group have been analyzed. A strain which is ts for heat shock transcription factor was also analyzed since it also shows nuclear accumulation of poly(A) + RNA at 37°C. At 37°C the mRNA of each mutant is characterized by atypically long polyA tails. Unlike ts pre-mRNA splithag mutants, these strains do not interrupt splicing of pre-mRNA at 370C; however four strains accumulate oversized RNA polymerase II transcripts. Some show inhibition of rRNA processing and a further subset of these strains is also characterized by inhibition of tRNA maturation. Several strains accumulate nuclear proteins ha the cytoplasm when incubated at semipermissive temperature. Remarkably, many strains exhibit nucleolar fragmentation or enlargement at the restrictive temperature. Most strains show dramatic ultrastructural alterations of the nucleoplasm or nuclear membrane. Distinct mutants accumulate poly(A) + RNA in characteristic patterns ha the nucleus.

Abstract-Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. Nur77 is highly expressed in vascular endothelial cells (ECs) and plays a role in the regulation of cell proliferation and angiogenesis; its role in vascular inflammation, however, remains unknown. Treatment of human umbilical vein ECs (HUVECs) with tumor necrosis factor (TNF)-␣ substantially increased the transcription and protein expression of Nur77 in a dose and time-dependent manner, as determined by Northern blot and Western blot analysis. Adenovirus mediated overexpression of Nur77 markedly increased the intracellular levels of IB␣ by approximately 4-fold, whereas overexpression of dominant negative Nur77 (DN-Nur77), which lacks its transactivation domain, had no effect on IB␣ expression, suggesting that Nur77 is an important transcriptional factor in controlling IB␣ expression in ECs. Furthermore, overexpression of Nur77 significantly increased IB␣ promoter activity via directly binding to a Nur77 response element in the IB␣ promoter. Importantly, overexpression of Nur77, but not DN-Nur77, protected ECs against the TNF-␣-and interleukin-1␤-induced endothelial activation, as characterized by attenuation in the nuclear factor B activation, expression of adhesion molecules ICAM-1 and VCAM-1, and monocytic adherence to ECs.

We have identified a novel nucleolar protein, Nop5p, that is essential for growth in Saccharomyces cerevisiae. Monoclonal antibodies B47 and 37C12 recognize Nop5p, which has a predicted size of 57 kDa and possesses a KKX repeat motif at its carboxyl terminus. Truncations that removed the KKX motif were functional and localized to the nucleolus, but conferred slow growth at 37°C. Nop5p shows significant sequence homology with yeast Sik1p/Nop56p, and putative homologues in archaebacteria, plants, and human. Depletion of Nop5p in a GAL-NOP5 strain lengthened the doubling time about 5-fold, and selectively reduced steady-state levels of 40 S ribosomal subunits and 18 S rRNA relative to levels of free 60 S subunits and 25 S rRNA. Northern blotting and primer extension analyses showed that Nop5p depletion impairs processing of 35 S pre-rRNA at the A 0 and A 2 cleavage sites. Nop5p is associated with the small nucleolar RNAs U3, snR13, U14, and U18. Depletion of Nop5p caused the nucleolar protein Nop1p (yeast fibrillarin) to be localized to the nucleus and cytosol. Also, 37C12 coimmunoprecipitated Nop1p. These results suggest that Nop5p functions with Nop1p in the execution of early pre-rRNA processing steps that lead to formation of 18 S rRNA.Most of the steps of ribosome biogenesis in eukaryotic cells take place in the nucleolus. In the yeast Saccharomyces cerevisiae, a single long 35 S pre-rRNA is transcribed by RNA polymerase I and processed to 18 S, 5.8 S, and 25 S rRNAs through a series of co-and post-transcriptional steps. Ribosomal proteins imported from the cytoplasm are assembled with pre-rRNAs to form the small 40 S subunit and the large 60 S subunit. The 5 S rRNA is transcribed by RNA polymerase III from a separate transcription unit and is incorporated into the large subunit along with the 5.8 S and 25 S rRNAs, while 18 S rRNA is incorporated into the small subunit. During transcription and processing of pre-rRNA, a number of nucleotides are modified, primarily by the addition of 2Ј-O-methyl groups or by the formation of pseudouridine residues. The processing and modification of pre-rRNAs require non-ribosomal nucleolar proteins, many of which are associated with small nucleolar RNAs (snoRNAs) 1 in the form of small nucleolar ribonucleoprotein (snoRNP) complexes (reviewed in Refs. 1 and 2).The earliest processing events are those involved in the removal of the promoter proximal 5Ј-externally transcribed spacer (5Ј-ETS). Cleavage occurs at two sites within the 5Ј-ETS: at A 0 , in the middle region of the 5Ј-ETS; and at A 1 , which results in the formation of the 5Ј-end of the mature 18 S rRNA (reviewed in Ref. 3). Formation of 18 S requires processing to form its 3Ј-end, which involves processing at site A 2 in the first internally transcribed spacer (ITS1) followed by processing at site D, which yields the 3Ј-end (see Fig. 9). In yeast, many gene products are required for, or participate in, cleavage at sites A 0 , A 1 , and A 2 , attesting to the complex nature of this process. The yeast RNase III encod...

Abstract-Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS), whose expression is downregulated by tumor necrosis factor (TNF)-␣ at the posttranscriptional level. To elucidate the molecular basis of TNF-␣-mediated eNOS mRNA instability, eNOS 3Ј untranslated region (3Ј-UTR) binding proteins were purified by RNA affinity chromatography from cytosolic fractions of TNF-␣-stimulated human umbilical vein endothelial cells (HUVECs). The formation of 3Ј-UTR ribonucleoprotein complexes, with molecular weight of 52 and 57 kDa, was increased by TNF-␣. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of the 52-kDa protein identified 3 peptides that comprise the peptide sequence of translation elongation factor 1-␣ 1 (eEF1A1). In HUVECs, TNF-␣ rapidly increased eEF1A1 expression, which is maximal after 1 hour and persists for up to 48 hours. RNA gel mobility-shift and UV cross-linking assays indicated that recombinant glutathione S-transferase-eEF1A1 fusion protein specifically binds to a UC-rich sequence in the 3Ј-UTR of eNOS mRNA. In addition, the domain III of eEF1A1 mediates the binding of eNOS 3Ј-UTR in eEF1A1. Overexpression of eEF1A1 markedly attenuated the expression of eNOS and luciferase gene fused with eNOS 3Ј-UTR in both COS-7 cells and bovine aortic endothelial cells (BAECs). Furthermore, adenovirus-mediated overexpression of eEF1A1 increased eNOS mRNA instability, whereas knockdown of eEF1A1 substantially attenuated TNF-␣-induced destabilization of eNOS mRNA and downregulation of eNOS expression in HUVECs. These results indicate that eEF1A1 is a novel eNOS 3Ј-UTR binding protein that plays a critical role in mediating TNF-␣-induced decrease in eNOS mRNA stability. (Circ Res. 2008;103:591-597.)Key Words: TNF-␣ Ⅲ eNOS Ⅲ mRNA Ⅲ stability Ⅲ translation elongation factor 1-␣ E ndothelial nitric oxide synthase (eNOS) is a key enzyme involved in the regulation of vascular function and abnormality of eNOS activity and/or expression has been shown to cause several vascular diseases. 1 Although eNOS was initially considered to be a constitutive enzyme, it was shown later that eNOS expression was regulated by a variety of exogenous stimuli. For instance, in cultured endothelial cells, cytokines, lipopolysaccharide, and oxidized LDLs have been shown to downregulate eNOS expression. 2,3 In contrast, shear stress, estrogen, and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors have been demonstrated to upregulate eNOS expression. 4 -6 For many of these stimuli, modulation of eNOS mRNA stability plays an essential role in the regulation of eNOS expression. 7 However, the mechanism(s) responsible for the regulation of eNOS mRNA half-life remains to be determined.One of the most potent inhibitory stimuli for eNOS expression in vascular endothelial cells is tumor necrosis factor (TNF)-␣. TNF-␣-mediated inhibition of eNOS expression, via a mechanism of destabilization of eNOS mRNA, has been shown to ...

angiogenesis ͉ cell therapy ͉ hypoxia ͉ prolyl hydroxylation

Chronic systemic hypertension causes cardiac pressure overload leading to increased myocardial O 2 consumption. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of O 2 homeostasis. Mouse embryos lacking expression of the O 2 -regulated HIF-1α subunit die at midgestation with severe cardiac malformations and vascular regression. Here we report that Hif1a f/f ;Tie2-Cre conditional knockout mice, which lack HIF-1α expression only in Tie2 + lineage cells, develop normally, but when subjected to pressure overload induced by transaortic constriction (TAC), they manifest rapid cardiac decompensation, which is accompanied by excess cardiac fibrosis and myocardial hypertrophy, decreased myocardial capillary density, increased myocardial hypoxia and apoptosis, and increased TGF-β signaling through both canonical and noncanonical pathways that activate SMAD2/3 and ERK1/2, respectively, within endothelial cells of cardiac blood vessels. TAC also induces dilatation of the proximal aorta through enhanced TGF-β signaling in Hif1a f/f ;Tie2-Cre mice. Inhibition of TGF-β signaling by treatment with neutralizing antibody or pharmacologic inhibition of MEK–ERK signaling prevented TAC-induced contractile dysfunction and pathological remodeling. Thus, HIF-1 plays a critical protective role in the adaptation of the heart and aorta to pressure overload by negatively regulating TGF-β signaling in endothelial cells. Treatment of wild-type mice with digoxin, which inhibits HIF-1α synthesis, resulted in rapid cardiac failure after TAC. Although digoxin has been used for decades as an inotropic agent to treat heart failure, it does not improve survival, suggesting that the countertherapeutic effects of digoxin observed in the TAC mouse model may have clinical relevance.

Circulating preprandial ghrelin to obestatin ratio is elevated in human obesity. We suggest that high preprandial ghrelin to obestatin ratio may be involved in the etiology and pathophysiology of obesity.