Quality control despite mistranslation caused by an ambiguous genetic code

A high level of accuracy during protein synthesis is considered essential for life. Aminoacyl-tRNA synthetases (aaRSs) translate the genetic code by ensuring the correct pairing of amino acids with their cognate tRNAs. Because some aaRSs also produce misacylated aminoacyl-tRNA (aa-tRNA) in vivo, we addressed the question of protein quality within the context of missense suppression by Cys-tRNA(Pro), Ser-tRNA(Thr), Glu-tRNA(Gln), and Asp-tRNA(Asn). Suppression of an active-site missense mutation leads to a mixture of inactive mutant protein (from translation with correctly acylated aa-tRNA) and active enzyme indistinguishable from the wild-type protein (from translation with misacylated aa-tRNA). Here, we provide genetic and biochemical evidence that under selective pressure, Escherichia coli not only tolerates the presence of misacylated aa-tRNA, but can even require it for growth. Furthermore, by using mass spectrometry of a reporter protein not subject to selection, we show that E. coli can survive the ambiguous genetic code imposed by misacylated aa-tRNA tolerating up to 10% of mismade protein. The editing function of aaRSs to hydrolyze misacylated aa-tRNA is not essential for survival, and the EF-Tu barrier against misacylated aa-tRNA is not absolute. Rather, E. coli copes with mistranslation by triggering the heat shock response that stimulates nonoptimized polypeptides to achieve a native conformation or to be degraded. In this way, E. coli ensures the presence of sufficient functional protein albeit at a considerable energetic cost.     

Combination treatment of IFNalpha2b and ribavirin in patients with chronic hepatitis C and persistently normal ALTs

Combination therapy of interferon-_2b and ribavirin was prospectively evaluated in 20 patients with chronic replicative hepatitis and persistently normal ALTs. Patients with normal ALTs on three or more occasions within 6 months received interferon-_2b 3 MU three times a week with ribavirin 1000–1200 mg everyday for 12 months and had a follow-up of 6 months. HCV genotype 1 was found in 16, and HCV genotype 2 or 3 in 4 patients. No patient experienced an ALT elevation during therapy. Ten of 20 patients (50%) cleared virus at the end of treatment. In an intent-to-treat analysis, a sustained virological response (SR) was achieved in 8 of 20 patients (40%). Nonresponse occurred in 5 patients. Relapse and breakthrough were seen in 2 patients each. Treatment was discontinued in 3 patients due to side effects. Interferon (IFN) ribavirin combination therapy is effective in patients with normal ALTs and appears superior to IFN monotherapy.     

Safety and tolerability of oral daily and intermittent ibandronate are not influenced by age

OBJECTIVE: The risk of osteoporosis increases exponentially with age. Elderly patients, who are often frail, have declining functional status and take multiple medications, and require osteoporosis therapies that are not only effective, but also very well tolerated. Ibandronate is a potent nitrogen-containing bisphosphonate that can be given intermittently with extended between-dose intervals. Oral daily and intermittent ibandronate (interval between doses > 2 mo) was found to significantly reduce the risk of new morphometric vertebral fractures by 62% and 50%, respectively, compared with calcium and vitamin D supplementation alone. We investigated the effect of age on the safety profile of oral daily and intermittent ibandronate, with particular emphasis on the upper gastrointestinal (GI) safety profile of ibandronate. METHODS: A predefined subgroup analysis examined the tolerability of oral ibandronate in women aged < 70 and > or = 70 years. RESULTS: The incidence of adverse events in patients aged > or = 70 years receiving oral daily and intermittent ibandronate was similar and comparable to placebo. The incidence of upper GI adverse events, including dyspepsia and esophagitis, was also similar between the 2 treatment groups and placebo. CONCLUSION: Older patients (> or = 70 yrs) receiving oral daily and intermittent ibandronate are at no greater risk of adverse events than older patients receiving placebo. Older patients were at no greater risk of upper GI adverse events than younger patients or patients receiving placebo. As a result of the good efficacy and tolerability observed in this trial, a once-monthly oral regimen of ibandronate is in late-stage clinical development.     

Recommendations for the management of prosthetic valve thrombosis

Prosthetic valve thrombosis (PVT) is a life-threatening disease, for which treatment strategies have been controversial. Herein, existing data on management options are reviewed, and conclusions drawn as to the choice and use of treatment strategies for PVT. The use of transesophageal echocardiography (TEE) allows distinction to be made between obstructive and non-obstructive PVT by the presence or absence of occluder motion limitation. The differentiation of PVT from pannus and vegetation is, however, still limited by TEE. The incidence of PVT has been underestimated by not taking into account a large percentage of non-obstructive PVT. Although the standard treatment for PVT has been surgery, thrombolysis has lower mortality rates, particularly in patients in NYHA functional classes III-IV. The lowest complication rates with thrombolysis have been achieved in patients with non-obstructive PVT. Pregnancy, left atrial appendage thrombi and large PVT are not contraindications to thrombolysis. The third therapeutic option is anticoagulant therapy. The detrimental effect of anticoagulant treatment in obstructive PVT was shown in a prospective study. Non-obstructive thrombi of > 5 mm length have been treated with higher success rates and lower complication rates by thrombolysis than by anticoagulant treatment. In conclusion, all patients with suspected PVT should undergo multiplane TEE. Thrombolysis is the first-line treatment for obstructive PVT, independent of NYHA class and thrombus size if there are no contraindications. Serial TEE studies must be conducted during thrombolysis. Surgery should be reserved for those patients in whom thrombolysis is contraindicated, or has failed. Initial anticoagulant therapy is recommended only for small, non-obstructive PVT if anticoagulation had been subtherapeutic; otherwise, thrombolysis is the treatment of choice if there are no contraindications.     

Recommendations for reporting morbid events after heart valve surgery

Major reasons for the considerable heterogeneity among published results of heart valve surgery are inconsistency in follow up techniques, reporting systems and classification of adverse events. The present recommendations are intended to harmonize the presentation of clinical material in order to improve comparison of data from different sources for the analysis of pooled data. The quality of an observational study is largely, if not entirely, due to the follow up technique, which may be graded according to six categories: Self-reporting of adverse events/well-being by the patients may be classified 'excellent'; if the information is gathered and re-checked at short-term intervals. Data obtained from in-hospital or outpatient examinations by qualified examiners at least twice a year or other personal contact through qualified examiners may be regarded as 'sufficient', if the results are re-checked by contacting the treating home physician. All other follow up techniques may be regarded as inappropriate. Consequences of complications are entirely dependent on severity and possible sequelae. It is therefore recommended to grade any reported complication according to its severity by utilizing a score system. Embolisms are best categorized by utilizing the performance status scale. Bleeding events may be categorized according to severity as fatal, major (requiring hospital transmission with transfusion, surgery or with permanently increased disability) or minor (not requiring hospital admission, surgery or transfusion). In some cases it will remain unclear whether an event was primarily embolic or hemorrhagic. These complications should be summarized as 'not categorized'. The reporting of morbid events due to thrombosis, embolism and bleeding should go along with information regarding the quality of antithrombotic management.     

EMQN best practice guidelines for the molecular genetic testing and reporting of fragile X syndrome and other fragile X-associated disorders

Different mutations occurring in the unstable CGG repeat in 5'' untranslated region of FMR1 gene are responsible for three fragile X-associated disorders. An expansion of over ∼200 CGG repeats when associated with abnormal methylation and inactivation of the promoter is the mutation termed ‘full mutation'' and is responsible for fragile X syndrome (FXS), a neurodevelopmental disorder described as the most common cause of inherited intellectual impairment. The term ‘abnormal methylation'' is used here to distinguish the DNA methylation induced by the expanded repeat from the ‘normal methylation'' occurring on the inactive X chromosomes in females with normal, premutation, and full mutation alleles. All male and roughly half of the female full mutation carriers have FXS. Another anomaly termed ‘premutation'' is characterized by the presence of 55 to ∼200 CGGs without abnormal methylation, and is the cause of two other diseases with incomplete penetrance. One is fragile X-associated primary ovarian insufficiency (FXPOI), which is characterized by a large spectrum of ovarian dysfunction phenotypes and possible early menopause as the end stage. The other is fragile X-associated tremor/ataxia syndrome (FXTAS), which is a late onset neurodegenerative disorder affecting males and females. Because of the particular pattern and transmission of the CGG repeat, appropriate molecular testing and reporting is very important for the optimal genetic counselling in the three fragile X-associated disorders. Here, we describe best practice guidelines for genetic analysis and reporting in FXS, FXPOI, and FXTAS, including carrier and prenatal testing.     

Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism

Parathyroid hormone (PTH) and its related receptor (PTHR) are essential regulators of calcium homeostasis and bone physiology. PTH activates PTHR by interacting with a ligand-binding site localized within the N-terminal extracellular domain (the N-domain) and the domain comprising the seven transmembrane helices and the connecting extracellular loops (the J-domain). PTH binding triggers a conformational switch in the receptor, leading to receptor activation and subsequent cellular responses. The process of receptor activation occurs rapidly, within approximately 1 s, but the binding event preceding receptor activation is not understood. By recording FRET between tetramethyl-rhodamine in PTH(1-34) and GFP in the N-domain of the receptor, we measured the binding event in real time in living cells. We show that the association time course between PTH(1-34) and PTHR involves a two-step binding process where the agonist initially binds the receptor with a fast time constant (tau approximately 140 ms) and then with slower kinetics (tau approximately 1 s). The fast and slow phases were assigned to hormone association to the receptor N- and J domains, respectively. Our data indicate that the slow binding step to the J-domain coincides with a conformational switch in the receptor, also monitored by FRET between the enhanced cyan fluorescent protein and the enhanced yellow fluorescent protein in the PTHR sensor, PTHR enhanced cyan fluorescent protein/enhanced yellow fluorescent protein (PTHR(CFP/YFP)). These data suggest that the conformational change that switches the receptor into its active state proceeds in a sequential manner, with the first rapid binding step event preceding receptor activation by PTH(1-34).     

Changes in Weight Associated With Telotristat Ethyl in the Treatment of Carcinoid Syndrome

Purpose

In the placebo-controlled Phase III TELESTAR (Telotristat Etiprate for Somatostatin Analogue Not Adequately Controlled Carcinoid Syndrome) trial, the oral tryptophan hydroxylase inhibitor telotristat ethyl significantly reduced bowel movement (BM) frequency during a 12-week, double-blind treatment period in 135 patients with metastatic neuroendocrine tumors with carcinoid syndrome and ≥4 BMs per day. Patients (mean [SD] age, 63.5 [8.9] years; mean [SD] body mass index, 24.9 [4.9] kg/m²) received placebo, telotristat ethyl 250 mg, or telotristat ethyl 500 mg 3 times per day (TID) in addition to somatostatin analogue therapy. Weight loss is associated with uncontrolled carcinoid syndrome and may be associated with reduced survival.