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Resume Introduction Le pneumomediastin est une complication rare mais classique de la dermatomyosite. Sa survenue constitue un facteur de gravite et necessite un recours precoce et rapide a un traitement agressif associant une corticotherapie et un traitement par immunosuppresseur ou par immunoglobulines humaines intra-veineuses. Cas clinique Nous rapportons l’observation d’une femme de 63 ans presentant une pneumopathie infiltrative etendue dans le cadre d’une forme clinique de dermatomyosite : le syndrome des anti-synthetases. L’evolution a ete rapidement pejorative avec un pneumomediastin et une detresse respiratoire aigue malgre un recours initial aux corticoides puis secondairement aux immunoglobulines humaines et aux immunosuppresseurs. Conclusion L’interet d’objectiver les signes d’accompagnement, cutanes ou musculaires dans le bilan initial d’une pneumonie infiltrative, est de donner une orientation clinique amenant a la recherche d’anticorps anti-synthetases afin d’optimiser rapidement la prise en charge therapeutique.

We have developed an anion-exchange high-performance liquid chromatography (HPLC) method using Q Sepharose XL (Amersham Pharmacia Biotech) as adsorbent to analyze samples containing adenovirus. This method has several major advantages over the HPLC method previously described for quantitating particles, namely (1) a >10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute preparations; and (4) no enzymatic treatment required even for crude samples. This assay was used to quantitate particles in samples taken at the transfection and amplification stages of production of various recombinant adenovirus, and in cultures of wild-type adenovirus of different serotypes. A modification of this analytical method was also developed for the purification of infectious adenovirus particles, including fiber-modified and third-generation recombinant viruses, giving highly purified preparations from low-titer crude lysates with an excellent overall recovery (50-74%).

Thebluntedconehasbeenchosen asa testcasemodelto providebothexperimentaland computationaldatabases in the e eld of hypersonic and raree ed e ow conditions. Some preliminary results have already been presented at the Fourth European High-Velocity Database Workshop; the present work, focused on aerodynamic forces, gives a complete set of experimental data. Experiments have been conducted in the SR3 facility at a freestream Mach numberclose to 20 and forReynolds numbersranging from 1522 to 34,855, calculated using freestream conditions and the model base diameter. Aerodynamic forces and moments are presented for three levels of e ow rarefaction and for model angles of attack between 0 and 30 deg. In parallel with the experimental work, e ow calculations weremadeby an internationalgroup ofresearchersforidenticaltestconditions.Examplesofcomparisonsbetween experimental and computational aerodynamic forces are also indicated.

At raree ed e ow regimes, e owe eld investigations have been conducted in the SR3 wind tunnel on a 70-deg sphericallybluntedconeanddensitye owe eldsobtained bynonintrusiveelectronbeam e uorescencemeasurements. Theblunted cone, chosen as thetest case model, has been thesubject of extensive studiesduring thepast few years. In addition to some limited results already presented at the Fourth European High-Velocity Database Workshop, the present research gathersdensity e owe elds obtained experimentally at two raree ed hypersonice ow conditions. Experiments have been performed at a freestream Mach number close to 20 and for two Reynolds numbers, 1420 and 4175, calculated using freestream conditions and the cone base diameter. Density e owe elds are presented for the two angles of attack, 0 and 10 deg, of the cone. In parallel to the experimental work presented, a number of e owe eld calculations were executed by an international group of researchers for test conditions identical to SR3 test conditions. Flowe elds were calculated using direct simulation Monte Carlo solutions, leading to comparisons between experimental and computational e owe elds.

Surface heat transfer rates have been measured over a 70-deg spherically blunted cone chosen as a test case model to provide both experimental and computational databases. Under raree ed and hypersonic conditions, heating rate distributions are measured along the model and presented at angles of attack varying from 0 to 30 deg. Experiments have been conducted in the SR3 facility at a freestream Mach number close to 20. Three e ow rarefactions have been considered, which correspond to Reynolds numbers of 1420, 4175, and 36,265. Reynolds numbers are calculated using freestream conditions and the model base diameter. In parallel to the experimental work, e ow calculations were executed by an international group of researchers for identical test conditions. Comparisons between experimental and computational heating rates are also presented.

Several assays of pristinamycin I synthetases based on adenylate or thioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristinaespiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating the first pristinamycin I residue, was purified 200-fold, using an ATP-pyrophosphate exchange assay. This enzyme was shown to be a monomer with an Mr of 67,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with Mrs of 240,000 and able to bind covalently L-threonine as a thioester, was purified 100-fold. This protein also activated L-aminobutyric acid, which is further epimerized to generate the third residue of the pristinamycin I macrocycle. A third protein, consisting of two identical subunits (SnbD) with Mrs estimated to be between 250,000 and 350,000, was purified 200-fold. This large enzyme catalyzed thioesterification and subsequent N-methylation of 4-dimethylamino-L-phenylalanine, the fifth pristinamycin I residue. SnbD could also activate L-proline, the fourth pristinamycin I residue, and some preparations retained a low but significant activity for the last two pristinamycin I precursors. Finally, a single polypeptide chain (SnbE) with an Mr of 170,000, catalyzing L-phenylglycine-dependent ATP-pyrophosphate exchange, was purified 3,000-fold and characterized. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four isolated proteins. The purified SnbE protein was further shown to be a proteolytic fragment of SnbD.

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

Tn5 Sp(r) transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes. Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus. Nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P. denitrificans strain in which cobU and cobV were amplified. The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences. Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P. denitrificans. The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and alpha-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and alpha-ribazole. Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase.

Pneumomediastinum is a rare but classical complication of dermatomyositis. Its development is a serious matter and necessitates prompt recourse to aggressive treatment with corticosteroids combined with immuno-suppressants or intravenous human immunoglobulin.We report the case of a 63 year old woman presenting with pulmonary infiltration, in the presence of dermatomyositis, as a clinical manifestation of the anti-synthetase syndrome. The progress was rapidly unfavourable with pneumomediastinum and acute respiratory distress despite initial treatment with corticosteroids followed by human immunoglobulin and immunosuppressants.The identification of cutaneous or muscular signs in the initial investigation of a pulmonary infiltrate should lead to a search for anti-synthetase antibodies in order to determine the optimal clinical management as quickly as possible.

Four pap genes (papA, papB, papC, papM) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4-dimethylamino-L-phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI- phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N-methylation steps of 4-amino-L-phenylalanine leading to DMPAPA via 4-methylamino-L-phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.