The 2.8 A resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol alpha family of replicative DNA polymerases, shares some similarities with other polymerases but shows many differences. Although its palm domain has the same topology as other polymerases, except rat DNA polymerase beta, one of the three carboxylates required for nucleotidyl transfer is located on a different beta strand. The structures of the fingers and thumb domains are unrelated to all other known polymerase structures. The editing 3'-5' exonuclease domain of gp43 is homologous to that of E. coli DNA polymerase I but lies on the opposite side of the polymerase active site. An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukaryotic DNA polymerases.

The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remains largely unknown. Zymogen granules (ZGs), the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a Gαi3 protein. Evidence is presented here that the water channel aquaporin-1 (AQP1) is present in the ZG membrane and participates in rapid GTP-induced vesicular water gating and swelling. Isolated ZGs exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of ZGs with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulatable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxyl-terminal domain of AQP1 blocks GTP-stimulable swelling of vesicles. Our results demonstrate that AQP1 associated at the ZG membrane is involved in basal as well as GTP-induced rapid gating of water in ZGs of the exocrine pancreas

Three groups of T4 DNA polymerase mutants were prepared and characterized. In the first group, Ala and Asn were substituted for four acidic residues in the exonuclease domain that were chosen on the basis of their sequence alignment with the Klenow fragment from Escherichia coli DNA polymerase I. Two divalent metal ions required for catalyzing the 3'-5' exonuclease reaction are ligated by carboxyl groups from these conserved Asp and Glu residues. The Ala and Asn replacements have a profound effect on the exonuclease activity of T4 DNA polymerase and also have a significant, but less pronounced influence on its polymerase activity which is located in a domain distal to the exonuclease region. The kcat values for the exonuclease reaction were reduced by 3-4 orders of magnitude by these replacements, but the values of Km(app) did not differ greatly from the wild-type enzyme. The second group consists of replacements of other residues, that are conserved in the exonuclease domain of eukaryotic DNA polymerases, but do not contribute to divalent metal ion coordination. Many of these alterations resulted in decreased exonuclease and/or polymerase activity. Mutants in the third group have substitutions of conserved residues in the polymerase domain which diminished polymerase and altered exonuclease activities. Our results, combined with structural data on crystals of protein N388, a truncated form of T4 DNA polymerase (Wang et al., 1996), show that: (i) the reduction in the relative specific exonuclease activities of mutants in the first group was significantly less than that of mutants in the Klenow fragment, despite the nearly identical geometric arrangement of the metal liganding groups in two proteins; (ii) altered residues, that affect exonuclease and/or polymerase activities in mutants of the second group, cluster within a small area of the exonuclease domain, suggesting that this area may be directly or indirectly involved in polymerase activity; (iii) mutations in the third group, which affect polymerase and exonuclease activities, may participate in DNA and dNTP binding. Our results point to the functional interdependence of the polymerase and exonuclease domains in T4 DNA polymerase, a property not observed with the Klenow fragment.

This is the first study of POT guided by intravascular ultrasound in patients with coronary bifurcation lesion, demonstrating that POT symmetrically expanded the proximal and bifurcation segments of the stent. After POT, SB FFR was <0.75 in a third of patients, which improved to >0.75 after SB dilation or SB stenting+final POT.

Recombinant SNAREs have been demonstrated as the minimal membrane fusion machinery. The participation of additional proteins in the regulation of membrane fusion has been suggested. In this study we provide nanometer-resolution images of native NSF oligomers and SNARE complexes isolated from neurons and the pancreas. Our study reveals the presence of new coiled rod-like structures in association with the SNARE complex only in neuronal tissue. Neuronal SNAREs were found coiled and super-coiled with these structures. The existence of NSF as pentamers in its native state is also demonstrated. The extent of coiling and super-coiling of SNAREs may regulate the potency and efficacy of membrane fusion in cells.

Despite ezetimibe’s ability to reduce serum cholesterol levels, there are concerns over its vascular effects and whether it prevents or ameliorates atherosclerotic disease (AD). Our objective was to estimate the effect of ezetimibe use on major AD events and all-cause mortality and to compare these associations to those observed for hydroxy-methylglutaryl-CoA reductase inhibitor (i.e., statin) use. We identified 367 new ezetimibe users between November 1, 2002 and December 31, 2009. These individuals were ≥18 years of age and had no prior statin use. One to four statin user matches were identified for each ezetimibe user resulting in a total of 1,238 closely matched statin users. Pharmacy data and drug dosage information were used to estimate a moving window of ezetimibe and statin exposure for each day of study follow-up. The primary outcome was a composite of major AD events (coronary heart disease, cerebrovascular disease, and peripheral vascular disease events) and all-cause death. Both ezetimibe use (odds ratio [OR] 0.33, 95% CI 0.13–0.86) and statin use (OR 0.61, 95% CI 0.36–1.04) were associated with reductions in the likelihood of the composite outcome. These protective associations were most significant for cerebrovascular disease events and all-cause death. Subgroup analyses by sex, race-ethnicity, prior history of AD, diabetes status, and estimated renal function showed consistent estimates across strata with no significant differences between ezetimibe and statin use. In conclusion, ezetimibe appeared to have a protective effect on major AD events and all-cause death which was not significantly different from that observed for statin use.

The bacteriophage T4 regA protein is a translational repressor that regulates the synthesis of > 12 T4 proteins. Earlier studies demonstrated that photocross-linking of the 122-residue regA protein to (dT)16 occurs at two sites, with the major site occurring at Phe-106. Amino acid substitutions were introduced at Phe-106 to evaluate its role in nucleic acid binding. Binding affinities of mutants F106C, F106V, and F106Y for nonspecific and specific RNA ligands indicated little difference between the Kapp of the mutants and wild type regA protein, for either poly(U) or for a specific gene 44 oligoribonucleotide. Thus, Phe-106 does not contribute measurably to the overall free energy of binding. Partial proteolysis of regA protein was carried out to further probe its domain structure. Chymotryptic cleavage produced a fragment of 11,095 Da that has reduced affinity for poly(U) and that contains the first 93 residues of regA protein. Interestingly, proteolysis of regA protein is reduced in the presence of the specific target, gene 44 RNA. Two deletion mutants, 1-->94 and 1-->109, have also been cloned and purified. The binding affinities of these deletion mutants indicated a 100-1000-fold reduction in their affinities for poly(U). These studies indicate the last 13 amino acids in regA protein make a significant contribution to RNA binding.

The results of this hypothesis-generating study indicate that longitudinal LV strain parameters are similarly impaired in patients with TC and ACM due to severe obstructive left anterior descending arterial disease or triple-vessel disease. Assessment of two-dimensional LV strain parameters could help differentiate between different TC patterns.