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.

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.

Expression of the melanocortin receptors MC2-R (ACTH-receptor) and MC5-R during embryonic development of ovine adrenals.

Two melanocortin receptors MC2-R (ACTH-receptor) and MC5-R are expressed in the adult lamb adrenal cortex. In this work, we have studied the time-course of expression of these two receptors during ovine fetal development. MC2-R expression progressively increases from day 60 to day 140 of gestation (x3), then more rapidly before parturition and remains constant in the newborn. In contrast, the pattern of MC5-R expression is totally different. A strong increase is observed between days 60 and 120 (x7) then followed by a decrease until parturition and after birth. This peak of MC5-R expression precedes that of MC2-R, suggesting that MC5-R might be involved in alpha-MSH- and/or ACTH-stimulated corticosteroid synthesis during early embryonic life.

The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells.

The aim of the present study was to assess whether the whole meiotic process of spermatogenic cells is able to take place in vitro. Fragments of seminiferous tubules from 20- to 22- or 28-day-old rats were seeded in medium containing 0.2% fetal calf serum in bicameral chambers and then cultured for 4 weeks in a chemically defined medium. The differentiation of meiotic germinal cells was followed by four criteria: (i) ultramicroscopic examination of the different types of germ cells present in the cell layer throughout the culture period; (ii) determination of the changes in DNA content per nucleus of the cell population seeded with time in culture; (iii) assessment of the ability of germinal cells to transcribe genes expressed after completion of meiosis; and (iv) monitoring the fate of BrdU-labeled leptotene spermatocytes. The ultrastructural study showed that the overall organization of the cells in the culture well recalls that of the seminiferous epithelium throughout the culture period. Moreover the identification of young round spermatids 21 days after seeding suggested that these spermatids had been formed very recently in culture. Determination of DNA content per nucleus showed that a 1C cell population could be observed after several days of cultures reaching 6 to 10% of total cells. An exponential-like increase in the amounts of the mRNAs encoding for TP1 or TP2 occurred from the time when 1C cells appeared in the culture until the end of the experiment. Finally, BrdU-labeled leptotene spermatocytes differentiated into pachytene spermatocytes and then into secondary spermatocytes, and BdrU-labeled round spermatids were observed from Day 21 of culture onward. Taken together these results indicate that the whole meiotic process from leptotene spermatocyte to round spermatid can indeed occur in vitro under the present culture conditions.

Meiotic differentiation of germinal cells in three-week cultures of whole cell population from rat seminiferous tubules.

The aim of the present study was to set up a culture system allowing most of the meiotic phase of rat spermatogenesis to occur in vitro. For that purpose, the differentiation of spermatogenic cells was monitored by three criteria: 1) examination of expression of genes specifically expressed at a high level in pachytene spermatocytes (the phosphoprotein p19 [p19] and the testis-specific histone TH2B) or in round spermatids (transition protein 1 [TP1] and transition protein 2 [TP2]) by reverse transcription-polymerase chain reaction (RT-PCR); 2) ploidy analysis; and 3) cytological and immunocytochemical study of the germ cells. In the first trial, we determined the changes in the ratios of p19:TP1 and TH2B:TP2 mRNA-related PCR products in the whole testis of rats between 18 and 60 days postpartum and related those results to the sequential appearance of the various types of spermatogenic cells during that period. In the second trial, our aim was to reproduce, in a culture system using seminiferous tubules from 23- to 25-day-old rats, the changes observed in vivo. The p19:TP1 and TH2B:TP2 ratios decreased dramatically in testicular extracts of rats between 32 and 40 days postpartum, i.e., at the time period during which round spermatids become more and more numerous in the testis. When seminiferous tubules were seeded in bicameral chambers, cell viability remained close to 70% of total cells throughout the 3-wk culture period. Both p19:TP1 and TH2B:TP2 ratios decreased during the first week of culture. This was attributable to a decrease in the levels of p19 and TH2B mRNAs and also to an enhancement in the relative amounts of TP1 and TP2. These changes were correlated with the appearance of a 1C cell population in the culture. Histological examination of the culture demonstrated that under the conditions of the present study, 5-bromo-2'-deoxyuridine-labeled pachytene spermatocytes of stages IV-VI were able to differentiate into secondary spermatocytes, then into round spermatids.

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.

Pre- and postmeiotic expression of male germ cell-specific genes throughout 2-week cocultures of rat germinal and Sertoli cells.

The present study was aimed at examining, by reverse transcription polymerase chain reaction, the expression of germ cell-specific genes in cocultures of Sertoli cells with either pachytene spermatocytes (PS) or round spermatids (RS). In situ hybridization studies showed that the mRNAs encoding phosphoprotein p19 and the testis-specific histone TH2B were specifically expressed in PS whereas those encoding the transition proteins TP1 and TP2 were specific to RS. This resulted in p19:TP1 and TH2B:TP2 ratios that were much higher in PS fractions than in RS fractions prepared by elutriation. When PS or RS were seeded on Sertoli cell monolayers in bicameral chambers, both the number and the viability of the cells decreased during the coculture. However, both parameters were equal to, or higher than, 60% after 2 wk. In PS-Sertoli cell cocultures, the ratios of p19:TP1 and TH2B:TP2 decreased dramatically during the second week of culture; this was due not only to a decrease in the levels of p19 and TH2B mRNAs but also to an enhancement in the relative amounts of TP1 and TP2 as compared to the amounts present on the first day of the coculture. Conversely, both ratios remained low in RS-Sertoli cell cocultures; this was due to a decrease in the levels of the four mRNAs studied during the coculture period. DNA flow cytometry studies showed the occurrence of a haploid cell population (1C) in PS-Sertoli cell cocultures from Day 2 onward, together with a decrease in the tetraploid cell population (4C). No such changes were observed in Sertoli cell-only cultures. By contrast, the haploid population decreased 3-fold during the first week in RS-Sertoli cell cocultures. Immunocytochemical studies demonstrated further that 5-bromo-2'-deoxyuridine-labeled PS of stages V-VIII were able to differentiate into RS under the present coculture conditions. Hence, although clearly imperfect, the present coculture system should help to clarify the local regulations governing spermatogenesis and should allow easier study of spermatogenic cell gene expression.

[In vitro murine spermatogenesis: cytologic study of the meiosis stage]. / Spermatogenèse murine in vitro: étude cytologique de l'étape méiotique.

Spermatogenesis is a complex process of cellular multiplication and differentiation, the regulation of which is only partially understood. This may be due; 1) to the redundancy of the mechanisms of local regulation; 2) to the ubiquitous character of those growth factors and cytokines present in the testis; 3) to the lack of long term culture systems allowing male germ cell development. We describe herein two culture systems associating Sertoli cells and germ cell in which part of the meiotic step can occur in vitro. In the first system, pachytene spermatocytes prepared by centrifugal elutriation are cultured on a layer of Sertoli cells. In the second system small fragments of seminiferous tubules are seeded in bicameral chambers. The cytological and immunocytochemical studies presented show that pachytene spermatocytes of stages IV-VI differentiate into spermatids of steps 1 to 5.

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.

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.

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.

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.

Multiple mRNA species code for two non-allelic forms of ovine alpha s2-casein.

The two non-allelic forms of alpha s2-casein, occurring in ovine milk, differ by an internal deletion of nine amino acid residues, including both cysteine residues at positions 34 and 42 in the mature chain. Sequencing of several alpha s2-casein cDNA, isolated from the mammary cDNA library of a single lactating ewe, showed three new types which differed from that previously studied. In addition to the expected deletion of codons +34 to +42 affecting 30-40% of mRNA, another structural difference involving an internal stretch of 44 nucleotides in the 5' untranslated region, was found. S1-nuclease protection assays confirmed the existence of several types of the relevant mRNA and sequencing of in-vitro-amplified genomic DNA demonstrated the presence of the 44-nucleotide stretch in the alpha s2-casein transcriptional unit, thus ruling out the possibility of a cloning artefact. The different alpha s2-casein mRNA, four in terms of deletion and two in terms of nucleotide substitutions for a given ewe, can be readily explained by partial exon skipping and allelic differences, respectively. This assumption is well supported by the following observations: 5' and 3' ends of both deleted DNA fragments are similar to those of exons; sequences neighbouring the 44-nucleotide stretch of the genomic DNA perfectly match consensus sequences described for 3' and 5' ends of introns; the rather simple patterns observed on Southern blots of different enzymatic digests of genomic DNA strongly suggest the occurrence of only 1 copy alpha s2-casein gene/haploid genome. During the course of evolution, the alpha s2-casein-encoding gene has undergone many mutations and some of them might have occurred in regions corresponding to consensus splicing regions of the pre-mRNA. Thus, complete skipping of some exons might be responsible for the shorter sizes of rat and mouse alpha s2-casein mRNA. If so, the overall organization of the alpha s2-casein gene in the different species might be more similar than expected from structural comparisons of the cognate mRNA or caseins.

Expression of a biologically active ovine trophoblastic interferon using a baculovirus expression system.

Ovine trophoblast protein (oTP) an embryonic interferon, which plays a key role in maternal recognition of pregnancy, has been expressed in insect cells using a baculovirus expression system. A cDNA coding for oTP was inserted downstream of the strong polyhedrin promoter. Cells infected with recombinant virus produced biologically active oTP and greater than 90% was secreted into the culture medium during infection. High amount of antiviral activity were produced (up to 5 x 10(5) IU per ml of culture medium). Recombinant oTP (roTP) was purified by immunoaffinity chromatography and found to be identical to authentic oTP with respect to molecular mass and N-terminal amino acid sequence.

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.