Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR.

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.

Highly variable upper and abyssal overturning cells in the South Atlantic.

The Meridional Overturning Circulation (MOC) is a primary mechanism driving oceanic heat redistribution on Earth, thereby affecting Earth's climate and weather. However, the full-depth structure and variability of the MOC are still poorly understood, particularly in the South Atlantic. This study presents unique multiyear records of the oceanic volume transport of both the upper (<~3100 meters) and abyssal (>~3100 meters) overturning cells based on daily moored measurements in the South Atlantic at 34.5°S. The vertical structure of the time-mean flows is consistent with the limited historical observations. Both the upper and abyssal cells exhibit a high degree of variability relative to the temporal means at time scales, ranging from a few days to a few weeks. Observed variations in the abyssal flow appear to be largely independent of the flow in the overlying upper cell. No meaningful trends are detected in either cell.

[Pulmonary embolism: the extent of computerized tomography]. / Embolie pulmonaire : faut-il tendre vers le << tout angioscanner >> ?

The use of computerised tomography in the diagnosis of pulmonary embolism has been the subject of clinical research while, at the same time, technical progress has provided the current multidetector-row spiral equipment. Computerised tomography has been assessed both with respect to reference strategies as well as in extensive pragmatic trials. The preliminary evaluation of the clinical probability and the assay of d-dimers has progressively become imperative. The value of the venous doppler ultrasound of the legs, in particular in the elderly, is limited by the variable accessibility according to the centre. In rare cases, uncertainty persists, for example with a good quality negative multidetector-row spiral computerised tomography associated with a high clinical probability, leaving room for complementary explorations. The confrontation between clinicians and radiologists is then all the more pertinent.

Acylation and immunological properties of Mycoplasma gallisepticum membrane proteins.

The acylation of Mycoplasma gallisepticum membrane proteins was studied by electrophoresis after in vivo labelling with different 14C-fatty acids and by chemical analysis. The immunological properties of these proteins were investigated by Western blotting and crossed immunoelectrophoresis. Among the ca. 200 membrane polypeptides resolved by two-dimensional electrophoresis, 35 components (including the major protein p67) were covalently modified with acyl chains. These acylated proteins displayed lower pls than average (5.0-7.4 vs. 5.0-9.0) and proved to be the major membrane protein antigens and immunogens of M. gallisepticum. The apparent selectivity of fatty acid incorporation into proteins was, as suggested by in vivo labelling: palmitic acid (16:0) > myristic acid (14:0) > oleic acid (18:1c) > stearic acid (18:0) > linoleic acid (18:2c). However, the true order of selectivity, as revealed by chemical analysis, proved to be 18:2c > 16:0 > 18:1c > 18:0 > 14:0. More specifically, palmitic acid was the major O-ester-bound fatty acid and linoleic acid the major amide-linked fatty acid. The observed average ratio [O-ester-bound + amide-linked acyl chains]/O-ester-bound chains approximately 1.4 and the presence of S-glycerylcysteine suggest that, in M. gallisepticum, membrane proteins are lipid-modified according to a mechanism identical to that depicted for lipoproteins of Gram-negative eubacteria.

Identification of bacterial periplasmic glycine betaine-binding protein after electrophoresis and affinity labeling.

Antibodies were elicited in rabbits against periplasmic proteins obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunoprecipitates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline (a metabolic precursor of glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct L-amino acids, including L-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to 2 micrograms of GB-BP in PAGE and only 0.2 micrograms in CIE.

[Intracerebral morphine therapy in cancer patients]. / Morphinothérapie par voie intracérébrale chez le cancéreux.

An overview of recent studies concerning opioids and their pharmacokinetics is presented. In the light of these findings it is shown that intracerebral administration may be justified. The authors experience with 63 cases is detailed: all cancer patients in the final stage. Initial dosage by the intraventricular route was 500 to 700 microgram-day but in one case twice daily injections of 1.200 microgram were needed. The dosage needed doubled over the observation period of 2 to 3 months. The mean length of survival was 75 days. Among complications nausea and vomiting were observed in 15 to 35% of the cases, sweating and pruritus in 15%, urinary retention in 15 to 20%. In some cases euphoria, motor excitement and hallucinations occurred. Chronic constipation was present in all cases. Two cases of meningitis were successfully treated by antibiotics. Pain relief was judged excellent or good in 75% of the cases. In 20% other analgesics had to be added to the treatment. In 5% the method failed.

[Perineal pain and lesions of the internal pudendal nerves]. / Douleur périnéale et souffrance des nerfs honteux internes.

A number of chronic pain syndromes in the perineal area can be related to pudendal nerves suffering. The constancy of symptoms among various patients, and in duration for a particular one, alterations revealed by electrophysiologic studies, pain relief by diagnostic blocks, data from anatomic studies, preliminary results of medical and surgical applied therapies, give consistent arguments for possible organic lesions of pudendal nerves.

Organic aerosol formation downwind from the Deepwater Horizon oil spill.

A large fraction of atmospheric aerosols are derived from organic compounds with various volatilities. A National Oceanic and Atmospheric Administration (NOAA) WP-3D research aircraft made airborne measurements of the gaseous and aerosol composition of air over the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico that occurred from April to August 2010. A narrow plume of hydrocarbons was observed downwind of DWH that is attributed to the evaporation of fresh oil on the sea surface. A much wider plume with high concentrations of organic aerosol (>25 micrograms per cubic meter) was attributed to the formation of secondary organic aerosol (SOA) from unmeasured, less volatile hydrocarbons that were emitted from a wider area around DWH. These observations provide direct and compelling evidence for the importance of formation of SOA from less volatile hydrocarbons.

The Flavobacterium psychrophilum OmpA, an outer membrane glycoprotein, induces a humoral response in rainbow trout.

AIMS: The purpose of this study was to characterize OmpA, a major glycoprotein isolated from the membrane fraction of Flavobacterium psychrophilum, and to evaluate its potential as antigenic unit in a possible vaccine. METHODS AND RESULTS: The expression product of ompA is a 465-amino-acid protein precursor that contains a 21-amino acid signal peptide and has overall homology (up to 60% identity) with similarly sized proteins of some bacteria belonging to the Flavobacteriaceae family. The carboxy-terminal region contains the 'OmpA/MotB' domain/signature and five putative 'Thrombospondin type 3 repeats' domains have been identified in the central region. OmpA was clearly detected in the outer membrane fraction and its surface exposure was demonstrated. OmpA is one of the immunodominant antigens and binding of specific anti-OmpA antibodies lead to cell lysis in the presence of complement. Fish immunized with OmpA emulsified with Freund's adjuvant developed a high antibody titter. CONCLUSIONS: Collectively, the data obtained here indicate that OmpA may be involved in Fl. psychrophilum/host cell interactions and appears to be a potential immunogen for a vaccine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is one step in the direction of understanding pathogenesis of Fl. psychrophilum and development of future vaccine.

Chemical analysis of processing of spiralin, the major lipoprotein of Spiroplasma melliferum.

The plasma membrane of Spiroplasma melliferum contains a major membrane-associated lipoprotein called spiralin. In this study, the processing pathway of spiralin was investigated by chemical analysis of the purified protein and by using [35S]cysteine, [35S]methionine, [14C]myristic acid (14C-14:0), [14C]palmitic acid (14C-16:0), and globomycin. SDS-PAGE analysis of membrane proteins showed the leader peptide cleavage of prospiralin and provided evidence for an apparent selectivity in the acylation: the unprocessed protein was labelled with 14C-16:0 only (O-ester-linked acyl chains), and the mature form with both 14C-labelled fatty acids (O-ester-linked + amide-linked chains). Chemical analysis of the purified protein revealed that spiralin contains S-glycerylcysteine and is covalently modified with two O-ester-linked acyl chains and one amide-linked fatty acid chain. However, a specific selectivity in the O- and the N-acylations was not confirmed; palmitate and stearate were the major components. The amounts of O-ester- and amide-linked acyl chains, the resistance to Edman degradation and the presence of S-glycerylcysteine together indicate that spiralin is a "classical" lipoprotein (i.e. is triacylated) and is probably processed by a mechanism similar to that described for gram-negative eubacteria. On the basis of these findings, a biogenesis pathway for spiralin is proposed.

Selective acylation of plasma membrane proteins of Mycoplasma agalactiae: the causal agent of agalactia.

Revealed by in vivo labeling with (14)C-palmitic acid, about 15 acylated proteins were identified in the plasma membrane of Mycoplasma agalactiae (type strain PG2), including the major component p40. Triton X-114 phase partitioning and Western blotting demonstrated the amphiphilic properties of the acyl proteins and showed that they were also antigenic components. Chemical analyses of fatty acids bound to proteins revealed the following selectivity order within acylation: stearic acid (18:0) > linoleic acid (18:2c) approximately palmitic acid (16:0) > oleic acid (18:1c) > myristic acid (14:0), with 16:0 and 18:1c preferred for the O-acylation and 18:0 for the N-acylation. The ratio [O-ester- + amide-bound acyl chains]/O-ester-linked chains being close to 1.4 as well as the presence of S-glycerylcysteine suggest that acyl proteins in M. agalactiae are true lipoproteins containing N-acyl diacyl glycerylcysteine, probably processed by a mechanism analogous to that described for Gram-negative eubacteria.

Antigenic relatedness between the spiralins of Spiroplasma citri and Spiroplasma melliferum.

Four spiralins were compared by rocket immunoelectrophoresis, quantitative immunoblotting techniques, and the spiroplasma deformation test with the use of antispiralin (polyclonal) monospecific antibodies. This investigation revealed that the spiralins of Spiroplasma citri and S. melliferum are antigenically related and that probably no more than two epitopes simultaneously saturable with antibodies are shared by the two proteins. One at least of these epitopes is accessible to antibodies on the spiroplasma cell surface.

Biochemical and antigenic characterisation of Mycoplasma gallisepticum membrane proteins P52 and P67 (pMGA).

Two membrane proteins from the avian pathogen Mycoplasma gallisepticum have been previously purified using a simple, efficient and non-denaturing method: a lipoprotein P67 (pMGA) and P52. In the current study, the lipid part of P67 was chemically analysed. The molecular structure of the lipoprotein-lipid component was determined to be S-glyceryl cysteine with two O-ester-linked acyl chains. Fatty acid analysis of the purified P67 indicated a heterogeneous composition: palmitic acid (16:0)>stearic acid (18:0)>oleic acid (18:1c)>myristic acid (14:0), with 16:0 as the major component. These findings, along with previously published results, support the conclusion that P67 is pMGA1.2, a true membrane-associated lipoprotein although not N-acylated. In contrast to P67, P52 is not a lipoprotein. Topological experiments using in situ treatment with proteases and growth inhibition in the presence of anti-P52 serum provided evidence of the surface exposition of the polypeptide. The N-terminal sequence of P52 was found to be similar to the dihydrolipoamide acetyltransferase from several mollicutes; this enzyme is a membrane-associated component of the pyruvate dehydrogenase complex. Immunoblotting techniques revealed that the surface antigens P52 and P67 were specific to the species M. gallisepticum and the closely related species M. imitans. No antigenic difference was revealed within these species with the anti-P52 serum, while anti-P67 serum confirmed the antigenic variability of P67. The potential of P52 and P67 as antigens in serological diagnosis tests or as candidates for anti-mycoplasma subunit vaccines is discussed.

Chemical analysis of lipid-modified membrane proteins in Acholeplasma laidlawii.

The lipid modification of membrane proteins was investigated in Acholeplasma laidlawii by metabolic labeling and by chemical analysis. A S-glycerylcysteine residue was identified from membrane proteins and we reported the strong preference for saturated acyl chains into the lipid modification. Differential release of fatty acids revealed a ratio [(O-ester- + amide-bound acyl chains)/O-ester-linked chains] close to 1.1 which suggests the involvement of only two O-ester linked fatty acids in the acylation process. Present data indicate that acyl proteins in A. laidlawii are true lipoproteins (mainly diacylated) probably processed by a mechanism analogous to that described for eubacteria and other mycoplasmas.

Inhibition of spiralin processing by the lipopeptide antibiotic globomycin.

The cyclic lipopeptide globomycin, a specific inhibitor of signal-peptidase II (Lsp A), proved toxic for the mollicute Spiroplasma melliferum with a minimal inhibitory concentration (MIC) in the range 6.25-12.5 microM, about one order of magnitude higher (that is, less efficient) than bee-venom mellitin. SDS-PAGE analysis of cell proteins followed by immunolabeling ("Western blotting") and by crossed immunoelectrophoresis demonstrated that the cleavage of the prespiralin leader peptide was prevented by globomycin. Cell fractionation experiments showed that prespiralin was membrane bound and did not accumulate in the cytoplasm or in the culture medium. Furthermore, the use of the potential-sensitive fluorescent dye 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (diS-C3-[5]) revealed that, in contrast to valinomycin and mellitin, globomycin up to 30 microM has no effect on the electrical transmembrane potential of S. melliferum. This indicates that the toxicity of globomycin for spiroplasma cells is mainly if not exclusively owing to the inhibition of spiralin processing. Added to previously published data, these results suggest that spiralin and probably other lipoproteins of mollicutes are acylated and membrane targeted by a mechanism involving notably the processing of the prelipoprotein precursor by a type II, globomycin-sensitive signal peptidase.

Expression of foreign genes and selection of promoter sequences in Acholeplasma laidlawii.

The stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors. In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in Acholeplasma laidlawii. Plasmids derived from the lactococcal vector pNZ18 could introduce and maintain four different genes for many generations in A. laidlawii. One of these, encoding the dominant membrane lipoprotein spiralin from the mollicute Spiroplasma citri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modified by fatty acids. This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A. laidlawii. To increase the expression of foreign genes, random A. laidlawii DNA fragments were cloned into a pNZ18-related plasmid and expression signals were selected using the Bacillus licheniformis alpha-amylase gene as a probe. Selection was done in Escherichia coli as well as directly in A. laidlawii. Active recombinants from E. coli were also able to express alpha-amylase activities and an enzyme of native size in A. laidlawii. The highest activity was obtained from a recombinant selected directly in A. laidlawii. This is the first example of a promoter sequence selected in a mollicute. Analysis of the putative promoters in seven clones revealed similar -10 and -35 regions, and similar spacer distances in A. laidlawii, Acholeplasma oculi, Lactococcus and E. coli. Vectors related to pNZ18 should be useful for the genetic analysis of specific A. laidlawii proteins and functions.

[Purification, amino acid composition and N-terminal sequence of the major protein (protein H) of the outer membrane of Pasteurella multocida]. / Purification, composition en aminoacides et séquence N-terminale de la protéine majoritaire (protéine H) de la membrane externe de Pasteurella multocida.

The major protein (protein H) of the outer membrane of Pasteurella multocida was purified by size-exclusion chromatography after selective extraction with detergents. The protein forms homotrimers which are stable in the presence of SDS at room temperature. Upon treatment at 100 degrees C, the protein is fully dissociated by the detergent into monomers exhibiting an apparent molecular mass of 37 kDa as estimated by electrophoresis. The amino acid composition of protein H is characterized by a low hydropathy index (HI = -0.40) and is strongly related to the compositions of bacterial porins, notably porins P2 (Haemophilus influenzae), PIA (Neisseria gonorrhoeae) and Cl.2 ("class 2 porin" of N. meningitidis). The N-terminal amino acid sequence of protein H shares a strong homology with those of porins OmpC (Escherichia coli) and P2. These data indicate that protein H of P. multocida is a porin belonging to the superfamily of the non-specific porins of Gram-negative eubacteria outer membrane.

[Perineal pain and involvement of the internal pudendal nerves]. / Douleur périnéale et souffrance des nerfs honteux internes.

A number of chronic pain syndromes in the perineal area can be related to pudental nerves suffering. The constancy of symptoms among various patients, and in duration for a particular one, alterations revealed by electrophysiologic studies, pain relief by diagnostic blocks, data from anatomic studies, preliminary results of medical and surgical applied therapies, give consistent arguments for possible organic lesions of pudental nerves.

Selective acylation of plasma membrane proteins of Mycoplasma mycoides subsp. mycoides SC, the contagious bovine pleuropneumonia agent.

The plasma membrane of Mycoplasma mycoides subsp. mycoides SC (strain KH3J) contains over 160 polypeptides with apparent molecular masses ranging from 14 to 125 kDa and isoelectric point values (pIs) from 5 to 9. In vivo labeling with [14C]-fatty acids revealed about 35 acylated polypeptides including the two major components p42 and p65 and displaying pIs between 5.5 and 9.0, with a majority between 6.5 and 8. The amphiphilic nature of most of these acyl proteins was confirmed by Triton X-114 phase partitioning. Gas-liquid chromatography analyses showed that the order of preference for protein acylation was 16:0 > 18:2c > 18:1c > 18:0 > 14:0, with 16:0 being the major O-ester-bound fatty acyl chain and 18:2c the major N-linked chain. The presence of S-glycerylcysteine and a ratio of [O-ester-bound acyl chains + N-linked chains]/O-ester bound chains of approximately 1.2 in M. mycoides subsp. mycoides SC membrane proteins are consistent with a lipid modification similar to that occurring in lipoproteins of Gram-negative eubacteria that contain an N-terminal acyl S-glycerylcysteine.

[Does spiralin has a cleavable N-terminal signal sequence?]. / La spiraline possède-t-elle une séquence signal N-terminale clivable?

The amino acid sequence of spiralin deduced from the nucleotide sequence of its gene was fictitiously shortened by 1 to 50 residues from each terminus and the compositions of both series of theoretical polypeptides were calculated. The two series of compositions thus obtained were compared to that of the purified protein, with the use of the Marchalonis and Weltman index (S delta Q). The results of this analysis, which permits the difficulty resulting from the blocking of the N-terminal amino acid to be overcome, show that spiralin is probably synthesized as a 241-residue precursor containing an N-terminal signal sequence cleaved close to cysteine-24. Since spiralin is acylated and since the sequence Val-Val-Ala-Cys24 shares some similarity with the consensus sequence of bacterial lipoprotein modification/processing site, the hypothesis of a cleavage just before cysteine-24 seems plausible.