Fidelity of tRNA 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal RNase P on multiple protein cofactors.

RNase P, which catalyzes tRNA 5'-maturation, typically comprises a catalytic RNase P RNA (RPR) and a varying number of RNase P proteins (RPPs): 1 in bacteria, at least 4 in archaea and 9 in eukarya. The four archaeal RPPs have eukaryotic homologs and function as heterodimers (POP5•RPP30 and RPP21•RPP29). By studying the archaeal Methanocaldococcus jannaschii RPR's cis cleavage of precursor tRNA(Gln) (pre-tRNA(Gln)), which lacks certain consensus structures/sequences needed for substrate recognition, we demonstrate that RPP21•RPP29 and POP5•RPP30 can rescue the RPR's mis-cleavage tendency independently by 4-fold and together by 25-fold, suggesting that they operate by distinct mechanisms. This synergistic and preferential shift toward correct cleavage results from the ability of archaeal RPPs to selectively increase the RPR's apparent rate of correct cleavage by 11,140-fold, compared to only 480-fold for mis-cleavage. Moreover, POP5•RPP30, like the bacterial RPP, helps normalize the RPR's rates of cleavage of non-consensus and consensus pre-tRNAs. We also show that archaeal and eukaryal RNase P, compared to their bacterial relatives, exhibit higher fidelity of 5'-maturation of pre-tRNA(Gln) and some of its mutant derivatives. Our results suggest that protein-rich RNase P variants might have evolved to support flexibility in substrate recognition while catalyzing efficient, high-fidelity 5'-processing.

Viral aetiology of influenza-like illness in Belgium during the influenza A(H1N1)2009 pandemic.

The purpose of this investigation was to determine the proportion of influenza-like illness (ILI) attributable to specific viruses during the influenza A(H1N1)2009 pandemic and to describe the demographic and clinical characteristics of ILI due to respiratory viruses in Belgium. Nasopharyngeal swabs were collected from ILI patients by general practitioners (GPs) and paediatricians (PediSurv) and analysed for viruses. Of 139 samples collected from children <5 years of age by PediSurv, 86 were positive, including 28 influenza (20%), 27 respiratory syncytial virus (RSV) (19%), 21 rhinovirus (17%), 12 human metapneumovirus (hMPV) (9%) and ten parainfluenza virus (PIV) (7%). Of 810 samples received from GPs, 426 were influenza (53%). Of 312 influenza-negative samples, 41 were rhinovirus (13%), 13 RSV (4%), 11 PIV (4%) and three hMPV (1%). Influenza mostly affected the 6-15 years old age group. Other respiratory viruses were commonly detected in the youngest patients. Similar clinical symptoms were associated with different respiratory viruses. Influenza A(H1N1)2009 was the most detected virus in ILI patients during the 2009-2010 winter, suggesting a good correlation between ILI case definition and influenza diagnosis. However, in children under 5 years of age, other respiratory viruses such as RSV were frequently diagnosed. Furthermore, our findings do not suggest that the early occurrence of the influenza A(H1N1)2009 epidemic impacted the RSV epidemic in Belgium.

Measles resurgence in Belgium from January to mid-April 2011: a preliminary report.

From 1 January to 14 April 2011, a total of 155 measles cases were notified in Belgium, whereas throughout 2010, there were only 40. Of the 103 cases with known vaccination status, 87% had not been vaccinated with measles-mumps-rubella vaccine. The resurgence of measles is the consequence of insufficient vaccine coverage in previous years. Efforts to communicate the benefits of measles vaccination to the public and to advise health professionals on control measures and outbreak management are ongoing.

Cloning and expression of cDNA encoding ovine trophoblastin: its identity with a class-II alpha interferon.

The cDNAs encoding ovine trophoblastin (oTP) were isolated from an ovine embryo cDNA lambda gt 11 library by screening with a synthetic 29-mer oligodeoxynucleotide corresponding to amino acid (aa) residues 34 to 43 of oTP. The cDNA contained an open reading frame of 595 bp and the deduced amino acid sequence indicates a protein precursor of 195 aa. Nucleotide and amino acid sequence comparisons establish that oTP shares extensive homology with alpha-interferon (IFN-alpha) but is more closely related to the IFN-alpha sII subfamily. When the oTP cDNA was cloned into an eukaryotic expression vector and transfected in monkey COS cells, a high level of antiviral activity was detected. RNA blot analyses of total RNA reveal that the oTP-coding gene is expressed during a relatively short period (eleven to 21 days). The abundant expression of oTP mRNA corresponds closely to the time at which the embryo acts to extend luteal lifespan. RNAs homologous to oTP were also detected in goat and cow embryos at equivalent periods of their development, but not in the pig.

Cell-free synthesis, proteolytic processing, core glycosylation, and amino terminal sequence of rabbit pre-alpha-lactalbumin.

Two different forms of alpha-lactalbumin were isolated from rabbit milk and partially characterized. The major and the minor species had apparent molecular weights of 18000 and 14000, respectively, according to their electrophoretic mobilities on SDS polyacrylamide gels. Analyses of their amino acid compositions and amino-and carboxy-terminal sequences did not reveal any difference, but sugar analysis showed the occurrence of carbohydrates in the major species. Rabbit alpha-lactalbumin was synthesized in a cell-free translation system as a precursor with an amino terminal extension of 19 amino acid residues whose primary structure is rather different from those of its ovine and porcine counterparts, in contrast with the extensive similarity so far observed between the known signals of homologous milk proteins. When mammary microsomal membranes were added during translation, the preprotein was converted to authentic alpha-lactalbumin, as demonstrated by amino terminal sequence analyses. However, one of the two processed forms migrated more slowly than pre-alpha-lactalbumin on SDS polyacrylamide gels and this was related to the occurrence of carbohydrates: only the "slower moving" polypeptide was specifically adsorbed on concanavalin A Sepharose and its electrophoretic mobility was enhanced after treatment with endoglycosidase H, an enzyme known to remove clustered mannosyl residues linked to di-N-acetylchitobiose. It was also observed that the rate of translocation of alpha-lactalbumin across the microsomal membrane was lower than that of beta-casein.

Amino terminal sequence, processing, and biological activity of porcine pre-alpha-lactalbumin.

Polyadenylated RNAs isolated from bound polysomes of a lactating sow's mammary gland, were translated in a cell-free system and in vitro synthesized alpha-lactalbumin was immunoprecipitated and radiosequenced. The translation product was found to contain an amino terminal extension of 19 amino acid residues, very similar to its ovine counterpart, that was selectively removed when translation was carried out in the presence of rabbit mammary microsomal membranes. Assays of porcine pre-alpha-lactalbumin for activity on galactosyltransferase showed that the preprotein can also interact with and modify the specificity of the enzyme, as indicated by de novo synthesis of lactose.

Glucocorticoids enhance corticotropin receptor mRNA levels in ovine adrenocortical cells.

We have shown previously that chronic treatment with glucocorticoids enhances both ACTH-induced cAMP production and ACTH- or 8Br-cAMP-induced steroidogenesis of cultured ovine adrenocortical cells. This treatment has been shown to involve an increase in the number of ACTH receptors. The present study aimed to explore the mechanism of this effect of glucocorticoids on ACTH receptors. Ovine adrenocortical cells expressed one major ACTH receptor transcript of 3.6 kb and three minor ones of 4.2, 1.8 and 1.3 kb. Dexamethasone treatment of cultured cells increased the levels of all these transcripts in a time- and dose-dependent manner, with an EC50 of (1.5 +/- 0.6) x 10(-8) M. The mean increase over control with 10(-6) M dexamethasone was 144 +/- 11% (n = 14). This enhancing effect was specific for glucocorticosteroids. The antiglucocorticoid Ru38486 blocked the effect of dexamethasone. Testosterone did not modify, while high concentrations of 17 beta-estradiol decreased, ACTH receptor mRNA levels. Treatment of cells with aminoglutethimide (an inhibitor of steroidogenesis) resulted in a dose-dependent decrease in ACTH receptor mRNA levels, which was prevented by concomitant treatment with dexamethasone. Treatment with ACTH also increased ACTH receptor mRNA levels more than twofold. Addition of aminoglutethimide together with ACTH resulted in a smaller increase than that achieved with ACTH alone. Neither dexamethasone nor ACTH modified ACTH receptor mRNA half-lives. However, these two hormones enhanced the levels of both newly synthesized and total ACTH receptor mRNAs. These results indicate that the positive trophic effect of glucocorticoids on ovine adrenocortical cells involves an enhancement of the transcription rate of the ACTH receptor gene. In addition, they suggest that part of the trophic action of ACTH on ACTH receptors may be mediated by ACTH-induced steroidogenesis.

Lipid peroxidation, protein synthesis, and protection by calcium EDTA in paracetamol injury to isolated hepatocytes.

Hepatocytes from rats treated with phenobarbitone were exposed to 10 mM paracetamol for 1 hr and then incubated in buffered Ringer solution. Enzyme leakage and trypan blue entry became severe in the paracetamol treated cells some 2 hr after the end of exposure. These signs of cell injury could be blocked by 4 mM CaEDTA added during or after paracetamol exposure. CaEDTA did not alter covalent binding of [14C]paracetamol. Ca2+ free media did not prevent paracetamol injury. Lipid peroxidation was observed in cells but could be blocked without protecting the cells. Protein synthesis was depressed early on in cells previously exposed to paracetamol, CaEDTA did not protect against this inhibition. These observations suggest that an early cytoplasmic lesion develops into a later lethal lesion at the cell surface.

Cloning and structural analysis of two distinct families of ovine interferon-alpha genes encoding functional class II and trophoblast (oTP) alpha-interferons.

Ovine trophoblast protein (oTP) is a polypeptide secreted by ovine trophectoderm from day 11 to 21, which plays a key role in maternal recognition of pregnancy. Structural analyses established that oTP shares extensive homology with class II alpha-interferon (IFN-alpha II) subfamily. Previous screening of an ovine genomic DNA library probed with an oTP cDNA incidently resulted in the isolation of a functional IFN-alpha II gene and two relevant pseudogenes, as shown by sequence analysis and study of expression in eukaryotic COS cells. The expected oTP gene together with a cognate pseudogene was successfully isolated from the series of clones selected from another genomic library probed with the oTP cDNA, using two specific oligonucleotides, each one complementary to a region of oTP cDNA with little homology with the IFN-alpha II gene and related pseudogenes. Southern blotting of ovine genomic DNA indicated the existence of at least five trophoblast IFN-alpha genes or pseudogenes. Nucleotide sequence comparisons showed that the oTP gene exhibits a higher homology (90%) with bovine trophoblast IFN gene (Stewart et al. (1990) J. Mol. Endocrinol. 4, 275-282) than with oIFN-alpha II gene (70%), thus providing evidence that embryonic IFNs constitute a distinct subfamily of IFN-alpha s.

Effects of age and ischemic times on biochemical evidence of myocardial injury after pediatric cardiac operations.

INTRODUCTION: The vulnerability of pediatric myocardium to ischemia is poorly documented in the clinical setting. METHODS: Serial measurements of serum concentrations of myoglobin, the MB isoenzyme of creatine kinase, and cardiac troponins T and I and their respective areas under the curve were obtained, with particular reference to age and ischemic time, in 80 children undergoing cardiac operations. Sixteen (the control group) did not require cardiopulmonary bypass and 64 did. RESULTS: In the control group there were increases (p < 0.01) in myoglobin and creatine kinase MB isoenzyme but no increase in cardiac troponin T or I; by contrast, the group treated with cardiopulmonary bypass had significant increases in all four markers but with differing temporal patterns. Younger age (especially < 12 months) was a highly significant explanatory variable only for the release of cardiac troponins T and I, and ischemic time was a significant explanatory variable for the release of creatine kinase MB isoenzyme, cardiac troponins T and I, but not myoglobin. In comparison with previous studies in adults, creatine kinase MB and cardiac troponin T concentrations were three times greater in children than in adults. CONCLUSIONS: This study supports the specificity of cardiac troponins T and I as markers of myocardial injury after pediatric cardiac operations and defines the importance of age and ischemic time in determining their release. In comparison with previous data in adults, our results raise the possibility that the pediatric heart may be more vulnerable to the effects of ischemia and reperfusion. Cardiac troponins will permit comparison of new myocardial protective strategies or other potentially therapeutic myocardial interventions.

Heterogeneity of ubiquitin immunoreactivity in neurofibrillary tangles of Alzheimer's disease.

The immunoreactivity of neurofibrillary tangles (bundles of abnormal filaments) in Alzheimer's disease was compared in photonic and electron microscopy using antisera against the microtubule- associated protein tau, against isolated tangles, against ubiquitin and using monoclonal antibodies against neurofilament proteins. Only a sub-population of tangles is labeled in situ by the anti-ubiquitin serum and some tangles are unlabeled by any of the antibodies used. Double immunolabeling in electron microscopy shows, however, that ubiquitin epitopes can coexist with tau and neurofilament epitopes on a same abnormal filament. These results indicate that in situ ubiquitin immunoreactivity (and to a lesser extent tau and neurofilament immunoreactivity) is not an obligatory feature of these abnormal filaments and suggest that their antigenic composition could evolve in affected neurons.

Effects of short-term supplementation with coenzyme Q10 on myocardial protection during cardiac operations.

BACKGROUND: Coenzyme Q10 (CoQ10) is a naturally occurring vitamin-like substance that may have a beneficial role in ischemia-reperfusion injury. Coenzyme Q10 administered either as an additive to cardioplegia or as long-term preoperative oral supplementation has been reported to ameliorate myocardial injury after cardiac operations. METHODS: To determine whether short-term supplementation with large doses of CoQ10 (600 mg in divided doses 12 hours before operation) was effective in myocardial protection, 20 patients with well-preserved left ventricular function (ejection fraction greater than 0.50) undergoing elective coronary revascularization were enrolled in a prospective, double-blind, placebo-controlled randomized trial. Serial concentrations of CoQ10, myoglobin, creatine kinase MD fraction, and cardiac troponin T were measured preoperatively and 1, 6, 24, 72, and 120 hours postoperatively. Efficacy of myocardial protection was also assessed by clinical outcome and serial changes in electrocardiographic indices. RESULTS: The patient groups were similar with respect to preoperative and intraoperative characteristics. There was no significant difference in the preoperative plasma levels of CoQ10. These levels fell significantly in both groups after operation, although the magnitude of the decrease was less in the CoQ10-supplemented group (43% versus 60%). In both groups, there were significant postoperative increases in myoglobin, creatine kinase MB fraction, and cardiac troponin T. The magnitude of increases in cardiac troponin T was greater in the CoQ10-supplemented group, reaching marginal overall statistical significance (p = 0.06). CONCLUSIONS: Short-term supplementation with large doses of CoQ10 does not lead to improved myocardial protection in patients undergoing coronary revascularization with well-preserved ventricular function and relatively short ischemic times.

Vulnerability of paediatric myocardium to cardiac surgery.

OBJECTIVE: Myocardial injury is an important cause of mortality and morbidity after paediatric cardiac surgery. Data obtained from studies in animals imply that juvenile myocardium is more resistant to the effects of ischaemia and reperfusion than adult myocardium but there is little confirmatory evidence in the clinical setting. DESIGN: Prospective observational study of biochemical markers of myocardial injury in a paediatric population undergoing cardiac surgery. SETTING: Tertiary referral centre for paediatric cardiac surgery. PATIENTS: Forty patients undergoing paediatric cardiac surgery of varying complexity including closure of atrial and ventricular septal defects and arterial switch for simple transposition. A control group included patients undergoing thoracotomy for closure of a patent ductus arteriosus or repair of a coarctation. INTERVENTIONS: Serial measurements of myoglobin, the MB isoenzyme of creatine kinase (CK-MB), and the highly specific markers of myocardial damage cardiac troponin T (cTnT) and I (cTnI) were made before and 1, 6, 24, and 48 to 72 hours after operation. RESULTS: There were significant increases in myoglobin and CK-MB, but not cTnT or cTnI, in the control group. There were significant increases in the four biochemical markers in all the cardiac operations but especially in the ventricular septal defect and transposition group. Increases in CK-MB and cTnT were about five times greater than those previously reported in adult patients. CONCLUSIONS: (i) Cardiac troponins are more specific markers of myocardial injury in paediatric cardiac surgery than myoglobin and CK-MB. (ii) Paediatric myocardium seems to be more vulnerable to injury during cardiac surgery than adult myocardium.

Leg blood flow, metabolism and exercise capacity in chronic stable heart failure.

OBJECTIVES: To assess the metabolic state of skeletal muscle during exercise in patients with chronic heart failure (CHF) and relate this to exercise capacity. BACKGROUND: During exercise in CHF, there is little relation between exercise capacity and central haemodynamic function. Skeletal muscle and limb blood flow are abnormal in CHF. We investigated the relationship between leg blood flow, metabolism and exercise capacity and ventilation in 10 patients (average age 63.3 +/- 6.0 years; 3 female) with stable CHF. METHODS: Patients undertook maximal exercise testing. Peak oxygen consumption (VO2) and the slope of the relationship between ventilation and carbon dioxide production (VE/CO2 slope) were derived. During a supine cycle exercise test, cardiac output (CO) by Doppler echocardiography, femoral blood flow (FBF) by thermodilution, pulse and blood pressure were recorded, and radial arterial and femoral venous blood samples taken for catecholamine, lactate and potassium estimation every 3 min. RESULTS: The average peak VO2 was 19.7 (+/- 5.2; range 11.3-29.0) ml/kg/min. The proportion of CO to the right leg increased from 0.08 (+/- 0.03) to 0.22 (+/- 0.06) (P < 0.001) at 3 min, with no further significant change thereafter. There was a liner increase in leg VO2 reaching a plateau towards peak. At peak, femoral venous saturation was 22.79% +/- 7.20%. Venous lactate and potassium were higher than arterial (P < 0.001 for each comparison). There was no correlation between exercise performance and any of the measured metabolites either in absolute measurements, expressed as change from rest to peak exercise or as arterio-venous difference. The closest correlate of leg VO2 was leg hydrogen ion production, V[H+]. Change in femoral venous lactate from rest to peak exercise correlated with VE/VCO2 slope even when calculated from before the anaerobic threshold (r = -0.80; P = 0.025). CONCLUSIONS: In CHF, exercise capacity is not determined by individual haemodynamic events, and does not seem to be determined by the possible humoral signals we investigated. Ventilation is abnormal before anaerobic threshold, and predicts subsequent lactate rise, suggesting that skeletal muscle is the origin of excessive ventilation.

Major rearrangements in the E element and minor variations in the U3 sequences of the avian leukosis subgroup J provirus isolated from field myelocytomatosis.

We collected paraffin-embedded myelocytomatoses induced by subgroup J avian leukosis virus (ALV-J) in French poultry from 1992 to 2000. We used nested PCR to obtain the U3 LTR and the E element sequences that encompass putative binding sites for transcription factors. We observed minor mutations in the U3 sequences that rarely affected transcription factor binding sites, thus preserving the transcriptional potential of the U3 LTR. However, we observed a large variability in the E element sequences from both field and experimental tumor samples. This variability involved genomic rearrangements and various deletions that most often occurred between two direct repeat sequences. Moreover, in seven DNA samples of the 22 field tumors analyzed, we observed two different sequences for the E element region, suggesting that proviral genomes of two different sizes may be simultaneously present in a tumor. Even though most of the E element sequences were mutated or rearranged, all myelocytomatosis samples always exhibited one E element sequence containing at least one putative C/EBP binding site that was unaffected and still potentially functional.

Creatinine and 51Cr-EDTA clearance after liver transplantation.

We have compared glomerular filtration rate measured by creatinine clearance with that measured by 51Cr-EDTA clearance after liver transplantation. Fourteen pairs of values were obtained from seven patients on the first and second days after operation. There were wide discrepancies between the values for glomerular filtration rate measured by the two methods, with a regression co-efficient of 0.43 (p = 0.12). Both methods assume a steady state, with no change of extracellular fluid volume or in the rates of exchange between physiological compartments, that does not apply in the immediate period after operation. The results show the difficulties of using clearance techniques to assess renal function after major surgery. Since drug therapy may be based on these measurements, we suggest that in this group of patients isolated clearance values should not be used.