Respiratory Paradoxical Adverse Drug Reactions Associated with Acetylcysteine and Carbocysteine Systemic Use in Paediatric Patients: A National Survey

OBJECTIVE: To report pediatric cases of paradoxical respiratory adverse drug reactions (ADRs) after exposure to oral mucolytic drugs (carbocysteine, acetylcysteine) that led to the withdrawal of licenses for these drugs for infants in France and then Italy. DESIGN: The study followed the recommendations of the European guidelines of pharmacovigilance for medicines used in the paediatric population. SETTING: Cases voluntarily reported by physicians from 1989 to 2008 were identified in the national French pharmacovigilance public database and in drug company databases. PATIENTS: The definition of paradoxical respiratory ADRs was based on the literature. Exposure to mucolytic drugs was arbitrarily defined as having received mucolytic drugs for at least 2 days (>200 mg) and at least until the day before the first signs of the suspected ADR. RESULTS: The non-exclusive paradoxical respiratory ADRs reported in 59 paediatric patients (median age 5 months, range 3 weeks to 34 months, 98% younger than 2 years old) were increased bronchorrhea or mucus vomiting (n = 27), worsening of respiratory distress during respiratory tract infection (n = 35), dyspnoea (n = 18), cough aggravation or prolongation (n = 11), and bronchospasm (n = 1). Fifty-one (86%) children required hospitalization or extended hospitalization because of the ADR; one patient died of pulmonary oedema after mucus vomiting. CONCLUSION: Parents, physicians, pharmacists, and drug regulatory agencies should know that the benefit risk ratio of mucolytic drugs is at least null and most probably negative in infants according to available evidence

Inactivation of TIF1γ Cooperates with KrasG12D to Induce Cystic Tumors of the Pancreas

Inactivation of the Transforming Growth Factor Beta (TGFβ) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1γ) has recently been proposed to be involved in TGFβ signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1γ in pancreatic carcinogenesis. Using conditional Tif1γ knockout mice (Tif1γlox/lox), we selectively abrogated Tif1γ expression in the pancreas of Pdx1-Cre;Tif1γlox/lox mice. We also generated Pdx1-Cre;LSL-KrasG12D;Tif1γlox/lox mice to address the effect of Tif1γ loss-of-function in precancerous lesions induced by oncogenic KrasG12D. Finally, we analyzed TIF1γ expression in human pancreatic tumors. In our mouse model, we showed that Tif1γ was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-KrasG12D;Smad4lox/lox mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1γ don't have strictly redundant functions. Finally, we report that TIF1γ expression is markedly down-regulated in human pancreatic tumors by quantitative RT–PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1γ is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1γ in the multifaceted functions of TGFβ in carcinogenesis and development

The mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase.

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results

TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant.

beta-Lactamases are bacterial enzymes which catalyse the hydrolysis of the beta-lactam ring of penicillins, cephalosporins and related compounds, thus inactivating these antibiotics. The crystal structure of the TEM1 beta-lactamase has been determined at 1.9 A resolution by the molecular-replacement method, using the atomic coordinates of two homologous beta-lactamase refined structures which show about 36% strict identity in their amino-acid sequences and 1.96 A r.m.s. deviation between equivalent Calpha atoms. The TEM1 enzyme crystallizes in space group P2(1)2(1)2(1) and there is one molecule per asymmetric unit. The structure was refined by simulated annealing to an R-factor of 15.6% for 15 086 reflections with I >/= 2sigma(I) in the resolution range 5.0-1.9 A. The final crystallographic structure contains 263 amino-acid residues, one sulfate anion in the catalytic cleft and 135 water molecules per asymmetric unit. The folding is very similar to that of the other known class A beta-lactamases. It consists of two domains, the first is formed by a five-stranded beta-sheet covered by three alpha-helices on one face and one alpha-helix on the other, the second domain contains mainly alpha-helices. The catalytic cleft is located at the interface between the two domains. We also report the crystallographic study of the TEM S235A mutant. This mutation of an active-site residue specifically decreases the acylation rate of cephalosporins. This TEM S235A mutant crystallizes under the same conditions as the wild-type protein and its structure was refined at 2.0 A resolution with an R value of 17.6%. The major modification is the appearance of a water molecule near the mutated residue, which is incompatible with the OG 235 present in the wild-type enzyme, and causes very small perturbations in the interaction network in the active site

Breakdown of the stereospecificity of DD-peptidases and beta-lactamases with thiolester substrates.

With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes were also known to hydrolyse various ester and thiolester analogues of their natural substrates. Some thiolesters with a C-terminal leaving group that exhibited L stereochemistry were significantly hydrolysed by some of the enzymes, particularly the Actinomadura R39 DD-peptidase, but the strict specificity for a D residue in the penultimate position was fully retained. These esters and thiolesters also behave as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the ultimate position could also be hydrolysed, mainly by the class-C and class-D enzymes. However, more surprisingly, the class-C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes with a higher efficiency than the D isomer

Thiolester substrates of DD-peptidases and beta-lactamases

With peptide substrates, the penicillin-sensitive DD-peptidases exhibit a strict specificity for D-Ala-D-Xaa C-termini. Only glycine is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes also hydrolyse various ester and thiolester analogues of their natural substrates. Some of the thiolesters whose C-terminal leaving group exhibited an L stereochemistry were significantly hydrolysed by some of the studied enzymes, particularly by the Actinomadura R39 DD-peptidase. By contrast, the strict specificity for a D residue in the penultimate position was fully retained. The same esters and thiolesters also behaved as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the C-terminal position could also be hydrolysed, mainly by the class C and class D enzymes. But, more surprisingly, the class C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes more efficiently than the D isomer

Retinal cholesterol metabolism is perturbated in response to experimental glaucoma in the rat

International audienceAlterations of cholesterol metabolism have been described for many neurodegenerative pathologies, such as Alzheimer’s disease in the brain and age-related macular degeneration in the retina. Recent evidence suggests that glaucoma, which is characterized by the progressive death of retinal ganglion cells, could also be associated with disruption of cholesterol homeostasis. In the present study we characterized cholesterol metabolism in a rat model of laser-induced intraocular hypertension, the main risk factor for glaucoma. Sterol levels were measured using gas-chromatography and cholesterol-related gene expression using quantitative RT-PCR at various time-points. As early as 18 hours after the laser procedure, genes implicated in cholesterol biosynthesis and uptake were upregulated (+49% and +100% for HMG-CoA reductase and LDLR genes respectively, vs. naive eyes) while genes involved in efflux were downregulated (-26% and -37% for ApoE and CYP27A1 genes, respectively). Cholesterol and precursor levels were consecutively elevated 3 days post-laser (+14%, +40% and +194% for cholesterol, desmosterol and lathosterol, respectively). Interestingly, counter-regulatory mechanisms were transcriptionally activated following these initial dysregulations, which were associated with the restoration of retinal cholesterol homeostasis, favorable to ganglion cell viability, one month after the laser-induced ocular hypertension. In conclusion, we report here for the first time that ocular hypertension is associated with transient major dynamic changes in retinal cholesterol metabolism

Transcriptional Analysis of the dd-Peptidase/Penicillin-Binding Protein-Encoding dac Gene of Streptomyces R61: Use of the Promoter and Signal Sequences in a Secretion Vector

The promoter region of the gene encoding the extracellular DD-peptidase/penicillin-binding protein of Streptomyces R61 has been identified by in vivo promoter probing and S1 mapping. A secretion vector, pDML116, was constructed by inserting into the multicopy Streptomyces plasmid pIJ702, a 247 bp DNA sequence that contained the transcriptional, translational and secretory signals and the 12 amino acid N-terminal region-encoding sequence of the mature Streptomyces DD-peptidase/penicillin-binding protein. Insertion, downstream of this 247 bp segment, of the Streptomyces R61 DD-peptidase-encoding gene or the Escherichia coli R-TEM beta-lactamase-encoding gene yielded plasmids pDML120 and pDML128, respectively, which allowed expression and secretion of the relevant enzymes by Streptomyces lividans. The maximal secretion levels obtained were 42 mg protein/ml for the autologous Streptomyces DD-peptidase and 0.9 mg protein/ml for the heterologous E. coli beta-lactamase

L’Asie du Sud-Est 2012 : les évènements majeurs de l’année

Chaque année l'Institut de recherche sur l'Asie du Sud-Est contemporaine (Irasec), basé à Bangkok, analyse les principaux événements politiques, économiques, sociaux, environnementaux ou religieux survenus dans l'ensemble du sous-continent asiatique. Établissant une rétrospective des principaux événements de l'année 2011, ce livre aide à mieux comprendre les grands enjeux de l'année 2012 dans une région de près de 600 millions d'habitants qui, plus que jamais, joue un rôle d'interface entre les grands pôles asiatiques. Grâce au travail de terrain tout au long de l'année d'une vingtaine de chercheurs, Asie du Sud-Est 2012 offre un décryptage pertinent et contemporain d'une actualité asiatique complexe, dense et dynamique. Outre une analyse claire, détaillée et passionnante, Asie du Sud-Est 2012 propose de nombreux outils pratiques sur l'Asie du Sud-Est : une chronologie détaillée de l'année, les adresses des différents centres de recherche francophones et internationaux, une liste des formations et des centres de documentation relatifs à l'Asie du Sud-Est, une bibliographie des principaux ouvrages publiés au cours de l'année, ainsi qu'un référencement des meilleurs sites Internet institutionnels et communautaires sur la région