Initiation of protein synthesis from a termination codon.

We show that the amber termination codon UAG can initiate protein synthesis in Escherichia coli. We mutated the initiation codon AUG of the chloramphenicol acetyltransferase (CAT) gene to UAG (CATam1) and translated mRNA derived from the mutant CAT gene in E. coli S-30 extracts. A full-length CAT polypeptide was synthesized in the presence of tRNA(fMetCUA), a mutant E. coli initiator tRNA which has a change in the anticodon sequence from CAU to CUA. Addition of purified E. coli glutaminyl-tRNA synthetase substantially stimulated synthesis of the CAT polypeptide. Thus, initiation of protein synthesis with UAG and tRNA(fMetCUA) most likely occurs with glutamine and not methionine. The UAG codon also initiates protein synthesis in vivo. To eliminate a weak secondary site of initiation from AUC, the fifth codon, we further mutagenized the CATam1 gene at codons 2 (GAG----GAC) and 5 (AUC----ACC). Transformation of E. coli with the resultant CATam1.2.5 gene yielded transformants that synthesized CAT polypeptide and were resistant to chloramphenicol only when they were also transformed with the mutant tRNA(fMetCUA) gene. Immunoblot analyses and assays for CAT enzyme activity in extracts from transformed cells indicate that initiation from UAG is efficient, 60-70% of that obtained from AUG. Initiation of protein synthesis from UAG using a mutant initiator tRNA allows tightly regulated expression of specific genes. This may be generally useful for overproduction in E. coli and other eubacteria of proteins which are toxic to these cells.     

Predictors of thrombotic complications after placement of the flexible coil stent

The balloon-expandable, stainless steel, flexible coil stent is a useful device for managing acute or threatened closure after percutaneous transluminal coronary angioplasty.^1–5 Use of the device is associated with thrombosis of the stented vessel in a small but important group of patients.^3,6–10 The clinical, angiographic, and procedural factors associated with stent thrombosis with this device are still unknown. The objective of this study was to define predictors of stent thrombosis occurring within the ftrst month after stenting with this device.     

Perioperative cardiovascular system failure in South Asians undergoing cardiopulmonary bypass is associated with prolonged inflammation and increased Toll-like receptor signaling in inflammatory monocytes

Background

South Asian ethnicity is an independent risk factor for mortality after coronary artery bypass. We tested the hypothesis that this risk results from a greater inflammatory response to cardiopulmonary bypass (CPB).

Methods

This was a single-site prospective cohort study. We compared the inflammatory response to CPB in 20 Caucasians and 17 South Asians undergoing isolated coronary artery bypass grafting surgery.

Results

Plasma levels of proinflammatory cytokines (interleukin [IL]-6, IL-8, IL-12, interferon gamma, and tumor necrosis factor) and anti-inflammatory mediators (IL-10 and soluble TNF receptor I) were measured. The Toll-like receptor (TLR) signaling pathway was examined in peripheral blood monocytes by flow cytometry, measuring surface expression of TLR2, TLR4, and coreceptor CD14 and activation of downstream messenger molecules (interleukin-1 receptor-associated kinase 4, nuclear factor kappa from B cells (NF-κB), c-Jun amino-terminal kinase, p38 mitogen-activated protein kinase, and Protein Kinase B). South Asians had persistently higher plasma levels of IL-6 and exhibited increased TLR signaling through the p38 mitogen-activated protein kinase and Protein Kinase B pathways in inflammatory monocytes after CPB. This increased inflammatory response was paralleled clinically by a higher sequential organ failure assessment score (5.1 ± 1.4 versus 1.5 ± 1.6, P = 0.027) and prolonged cardiovascular system failure (23.5% versus 0%) 48 h after CPB.

Conclusions

South Asians develop an exacerbated systemic inflammatory response after CPB, which may contribute to the higher morbidity and mortality associated with coronary artery bypass in this population. These patients may benefit from targeted anti-inflammatory therapies designed to mitigate the adverse consequences resulting from this response.     

Canadian Cardiovascular Society Guidelines for the Diagnosis and Management of Stable Ischemic Heart Disease

This overview provides a guideline for the management of stable ischemic heart disease. It represents the work of a primary and secondary panel of participants from across Canada who achieved consensus on behalf of the Canadian Cardiovascular Society. The suggestions and recommendations are intended to be of relevance to primary care and specialist physicians with an emphasis on rational deployment of diagnostic tests, expedited implementation of long- and short-term medical therapy, timely consideration of revascularization, and practical follow-up measures.     

Recombinant adenoviral vector expressing HCV NS4 induces protective immune responses in a mouse model of Vaccinia-HCV virus infection: A dose and route conundrum

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected patients presumably due to failure or inefficiency of the immune responses generated. Both antibody and cellular immune responses have been suggested to be important in viral clearance. Non-replicative adenoviral vectors expressing antigens of interest are considered as attractive vaccine vectors for a number of pathogens. In this study, we sought to evaluate cellular and humoral immune responses against HCV NS4 protein using recombinant adenovirus as a vaccine vector expressing NS4 antigen. We have also measured the effect of antigen doses and routes of immunization on the quality and extent of the immune responses, especially their role in viral load reduction, in a recombinant Vaccinia-HCV (Vac-HCV) infection mouse model. Our results show that an optimum dose of adenovirus vector (2 × 10⁷ pfu/mouse) administered intramuscularly (i.m.) induces high T cell proliferation, granzyme B-expressing CD8⁺ T cells, pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-2 and IL-6, and antibody responses that can significantly reduce the Vac-HCV viral load in the ovaries of female C57BL/6 mice. Our results demonstrate that recombinant adenovirus vector can induce both humoral and cellular protective immunity against HCV-NS4 antigen, and that immunity is intricately controlled by route and dose of immunizing vector.     

Gender differences,outcomes and trends among nonagenarian with atrial fibrillation: insight from National Inpatient Sample database

BACKGROUND Nonagenarians(NG),individuals aged≥90 years,constitute an increasing proportion of hospitalizations presenting with atrial fibrillation(AF).However,not much is known about demographics,clinical outcomes,and trends of hospitalizations.Therefore,we analyzed data about hospitalizations and clinical outcomes among NGs with AF over ten years from 2005 to 2014 using a publically available database,the National Inpatient Sample.METHODS All hospitalizations and major outcomes of subjects≥90 years with a primary diagnosis of AF(ICD-9-CM code 427.31)over a ten-year period were assessed in this study by multivariate logistic regression analysis.RESULTS There were more females than males(176,268 females,51,384 males)in this analysis.The number of hospitalizations for AF among NG increased by 50%(17,295 in 2005 to 25,830 in 2014).Males were more likely to undergo cardioversion(6.14%of males vs.5.06%of females,P<0.0001).Over this period,in-hospital mortality declined from 3.21%in 2005 to 2.38%in 2014(P=0.0041),with higher in-hospital mortality in males(3.23%in males vs.2.76%in females,P=0.0138),mean length of hospitalization decreased from 4.53 days to 4.13 days(P<0.0001),the prevalence of congestive heart failure fell from 0.48%to 0.23%(P=0.0257),and the use of anticoagulation increased from 6.09%to 14.54%(P<0.0001).In a multivariate analysis,hospital admission on the weekend,Elixhauser comorbidity index,CHA2DS2VASc score,acute respiratory failure,and the length of hospital stay were associated with a higher risk of in-hospital mortality.CONCLUSIONS From 2005 to 2014,AF-related hospitalizations among NGs increased,more so in in females population,mortality trends improved,rates of anticoagulation increased,and cardioversions increased.Despite the decreasing trend of in-hospital mortality since 2005,the relatively high mortality rate in males warrants further studies.