BACKGROUND Carotid-artery stenting and carotid endarterectomy are both options for treating carotid-artery stenosis, an important cause of stroke. METHODS We randomly assigned patients with symptomatic or asymptomatic carotid stenosis to undergo carotid-artery stenting or carotid endarterectomy. The primary composite end point was stroke, myocardial infarction, or death from any cause during the periprocedural period or any ipsilateral stroke within 4 years after randomization. RESULTS For 2502 patients over a median follow-up period of 2.5 years, there was no significant difference in the estimated 4-year rates of the primary end point between the stenting group and the endarterectomy group (7.2% and 6.8%, respectively; hazard ratio with stenting, 1.11; 95% confidence interval, 0.81 to 1.51; P = 0.51). There was no differential treatment effect with regard to the primary end point according to symptomatic status (P = 0.84) or sex (P = 0.34). The 4-year rate of stroke or death was 6.4% with stenting and 4.7% with endarterectomy (hazard ratio, 1.50; P = 0.03); the rates among symptomatic patients were 8.0% and 6.4% (hazard ratio, 1.37; P = 0.14), and the rates among asymptomatic patients were 4.5% and 2.7% (hazard ratio, 1.86; P = 0.07), respectively. Periprocedural rates of individual components of the end points differed between the stenting group and the endarterectomy group: for death (0.7% vs. 0.3%, P = 0.18), for stroke (4.1% vs. 2.3%, P = 0.01), and for myocardial infarction (1.1% vs. 2.3%, P = 0.03). After this period, the incidences of ipsilateral stroke with stenting and with endarterectomy were similarly low (2.0% and 2.4%, respectively; P = 0.85). CONCLUSIONS Among patients with symptomatic or asymptomatic carotid stenosis, the risk of the composite primary outcome of stroke, myocardial infarction, or death did not differ significantly in the group undergoing carotid-artery stenting and the group undergoing carotid endarterectomy. During the periprocedural period, there was a higher risk of stroke with stenting and a higher risk of myocardial infarction with endarterectomy. (ClinicalTrials.gov number, NCT00004732.)

Transformation of E. coli cells treated with CaCJ2 to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before antibiotic challenge. MATERIALS AND METHODSBacterial Strains and R Factors. The I-like R factor, R64-11 (16), which specifies resistance to tetracycline (Tc) and streptomycin (Sm), was obtained from R. Curtiss. R6 (17), an F-like R factor that carries resistance to kanamycin (Km), neomycin (Nm), chloramphenicol (Cm), sulphonamide (Su), streptomycin, and tetracycline, was obtained from T. Watanabe. R6-5, a spontaneous variant of R6 that lacks tetracycline resistance, was isolated in our laboratory (18). The bacterial strains used in these experiments have been described (7,22). DNA Preparations. In certain instances, covalently-closed R-factor DNA was isolated and purified from E. coli as described (6, 7). Alternatively, a Brij-lysis procedure (19) was used for initial R-factor DNA isolation, and preparations obtained by this method were subsequently purified by centrifugation in cesium chloride-ethidium bromide gradients. The catenated, closed circular, and noncircular forms of R-factor DNA used in experiments comparing the relative transforming ability of the various R-factor DNA species were isolated from E. coli minicells. R-factor DNA was denatured by heating it at 980 for 5 min in 15 mM NaCl-1.5 mM Na citrate followed by rapid cooling at 0°. Sonication of R-factor DNA to about 9S fragments was done for 15 see at 00 by a Branson model W185 D sonicator, and the size of the R-factor DNA fragments was confirmed by sucrose gradient centrifugation (7).Transformation Reaction Mixture. Transformation was done by a variation of the procedure of Mandel and Higa (15), as modified by Lobban, Masuda, and Kaiser (personal communication). E. coli strain C600 was grown at 370 in H1 medium (20) to an optical density of 0.85 at 590 nm. At this point, the cells 2110 Abbreviation: R factor, antibiotic resistance factor. * The previous paper in this series is ref. 18.

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Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the Ϸ4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although Ϸ80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5 or 3 untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.RNA degradation ͉ RNA half-life ͉ transcript abundance T he rate of mRNA degradation plays a central role in the metabolism of nucleic acids in both prokaryotic and eukaryotic cells (for reviews see refs. 1-5). mRNA decay has been studied in a range of organisms, and much has been learned about the substrate features and ribonucleolytic enzymes that influence mRNA stability. In the case of Escherichia coli, experimenters have demonstrated that extensive variation exists in the rate of decay of individual mRNAs and that specific features of the mRNA sequence as well as transcript secondary structure can be important determinants of such variation (1, 3). Additionally, although the half-lives of some RNAs depend on the physiological state of the cell, as influenced by genetic and environmental factors, the half-lives of other RNAs seem to be independent of cell physiology (6, 7).Much of the information available about RNA decay has been derived from study of RNAs encoded by a very limited number of genes. In fact, we can find published reports of RNA half-life for less than 25 of the 4,288 predicted ORFs in the E. coli genome. Current models of mRNA decay are based on this relatively limited experimental sampling of message turnover. To gain a broader understanding of the fate of mRNAs in E. coli we adapted DNA microarray methodology to measure the decay of each chromosomally encoded mRNA simultaneously.DNA microarrays containing sequences derived ...

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColEl plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColEl, they were nevertheless compatible with ColEl. P15A-derived plasmids were not selftransmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColEl plasmid within the same cell.

The construction of new plasmid DNA species by in vitro joining of restriction endonucleasegenerated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins.Controlled shearing of antibiotic resistance (R) factor DNA leads to formation of plasmid DNA segments that can be taken up by appropriately treated Escherichia coli cells and that recircularize to form new, autonomously replicating plasmids (1). One such plasmid that is formed after transformation of E. coli by a fragment of sheared R6-5 DNA, pSC101 (previously referred to as Tc6-5), has a molecular weight of 5.8 X 106, which represents about 10% of the genome of the parent R factor. This plasmid carries genetic information necessary for its own replication and for expression of resistance to tetracycline, but lacks the other drug resistance determinants and the fertility functions carried by R6-5 (1).Two recently described restriction endonucleases, EcoRI and EcoRII, cleave double-stranded DNA so as to produce short overlapping single-stranded ends. The nucleotide sequences cleaved are unique and self-complementary (2-6) so that DNA fragments produced by one of these enzymes can associate by hydrogen-bonding with other fragments produced by the same enzyme. After hydrogen-bonding, the 3'-hydroxyl and 5'-phosphate ends can be joined by DNA ligase (6). Thus, these restriction endonucleases appeared to have great potential value for the construction of new plasmid species by joining DNA molecules from different sources. The EcoRI endonuclease seemed especially useful for this purpose, because on a random basis the sequence cleaved is expected to occur only about once for every 4,000 to 16,000 nucleotide pairs (2); thus, most EcoRI-generated DNA fragments should contain one or more intact genes. We describe here the construction of new plasmid DNA species by in vitro association of the EcoRI-derived DNA fragments from separate plasmids. In one instance a new plasmid has been constructed from two DNA species of entirely different origin, while in another, a plasmid which has itself been derived from EcoRI-generated DNA fragments of a larger parent plasmid genome has been joined to another replicon derived independently from the same parent plasmid. Plasmids that have been constructed by the in vitro joining of 3240EcoRI-generated fragments have been inserted into appropriately-treated E. coli by transformation (7) and have been shown to form biologically functional replicons that possess genetic properties and nucleotide base sequences of both parent DNA species. MATERIALS AND METHODSE. coli strain W1485 containing the RSF1010 plasmid, which carrie...

The nucleotide sequence of a 1,091-base pair cloned cDNA insert encoding bovine corticotropin-beta-lipotropin precursor mRNA is reported. The corresponding amino acid sequence indicates that the precursor protein consists of repetitive units and includes a third melanotropin sequence in its cryptic portion. Pairs of lysine and arginine residues separate the component peptides of the precursor.

Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.Gag | receptor | ubiquitin | vesicle