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Different ompF-ompC gene fusions were used to analyse the regions involved in the stable trimerization and membrane insertion of the Escherichia coli OmpF porin. The stability of the trimers formed from the various hybrids was analysed. Three classes of trimer instability are observed related to the presence of different exposed polypeptide loops of OmpF. In all cases, amino acids located between residue 115 and residue 144 of OmpF are necessary to promote a correct and stable trimeric conformation. However, immunogold labelling studies indicate the correct insertion of the protein in the outer membrane despite a marked instability of some hybrid porins. The location of the residues involved in trimer stability is discussed with regards to both the three-dimensional structure and the folding of OmpF.

Four different mutations were obtained by selecting for resistance to colicin N and screening for continued production of the OmpF protein of Escherichia coli. Two of them also conferred resistance to colicin A. The substitutions C for R-168 (R168C) and E284K caused the loss of the E21 epitope, while the transition G285D altered the E18, E19, and E20 antigenic sites. The substitution G119D drastically affected the stability of the trimeric conformation.

The colicin A lysis protein (Cal) was used to direct the extracellular release of recombinant proteins produced in Escherichia coli. The cal gene, under the control of its inducible promoter, was introduced into an expression vector encoding the human growth hormone devoid of its signal sequence (Met-hGH). Cal and Met-hGH were simultaneously expressed at two different levels of Met-hGH induction. The results indicate that Cal causes the excretion of non-aggregated Met-hGH from the cytoplasm to the culture medium and that the Met-hGH is correctly folded since the released Met-hGH is antigenically indistinguishable from the authentic mature hGH and is biologically active in binding to specific receptor sites.

Chordoma is a rare tumor in spine. Two cases, exceptional in adolescents, are reported. The clinical evolution is slow and progressive. CT shows a lobulated tumor, precises the degree of vertebral osteolysis and detects intra-tumoral calcifications. MRI is useful to determine especially in high in soft tissues and the vertebral canal. Arteriography is realised in pre-operative time and permits an embolization when the tumor is hypervascularised.

Following a reminder of anatomy, the semiology of standard radiology and modern imaging methods is described. The authors then deal with the phrenic-supraphrenic thoracic pathologies (air and liquid effusions, neighbourhood atelectasis, etc.) and with the phrenic-infraphrenic pathologies (transdiaphragmatic hernias, hepatic pathology, subphrenic abscess and pleural effusions). In each of these chapters, the often complex elements of standard radiology and modern imaging methods are detailed.

We present five cases with confirmed diagnosis of pulmonary alveolar microlithiasis that illustrate the appearance of this rare chronic lung disease on conventional and high-resolution CT. Pulmonary alveolar microlithiasis is characterized by widespread intraalveolar calcification of both lungs. Conventional CT confirmed the inferior and posterior predominance of the lesions in four of our cases and showed an exceptionally high concentration of microliths in the subpleural parenchyma and along the bronchovascular bundles. High-resolution CT revealed a perilobular and bronchovascular distribution of the disease process at the level of the secondary pulmonary lobule. This pattern correlated closely with pathologic findings.

In rabbit stomach, gastric lipase activity level was found to increase from birth to 30 days old (weaning), and then decreased. In contrast, pepsin activity only appeared between 30 to 45 days old, and increased till to the adult level. It was observed that maturation of gastric glands in cardial mucosa was a downward elongation process from the mitotic cell pool. These mitotic cells were always found in the neck of the gastric glands, corresponding to the bottom of the gland at 6 days old and to the mid-zone of the gland in adult. Location of rabbit gastric lipase (RGL) cells in cardial glands varied with age and was found along the pit of the gastric glands at 6 days old. The extent of this cellular location decreased with age, whereas a second RGL cell zone appeared below the mitotic cell area at 18 and 30 days old. At 45 days old, the pepsinogen cells appeared in the bottom of the gland, and consequently the RGL cells were located in the mid-zone of the gastric glands, between mitotic cells (neck of the gland) and pepsinogen cells (lower part of the gland). Ultrastructural study of cardial gastric glands revealed different morphologies of the secretion granules in the cells along the gastric glands. In 6-day-old rabbits, secretory granules were found uniformly electron dense in the bottom of the glands and were RGL-labeled by the immunogold technique. In the medium part of the glands, granules appeared biphasic, with a clear and a dense part, and RGL labeling was confined to the electron-dense part.(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; Colicins A and E1 are two pore-forming colicins sharing homology in their C-terminal domains but not in their N-terminal or central domains. Using site-directed mutagenesis, restriction sites were inserted at the proper locations to allow recombination of these domains. Six different constructs were obtained. All these proteins were expressed in Escherichia coli and properly recognized by monoclonal antibodies directed against epitopes located in different domains of colicin A. Out of the six hybrids, only two were released to the extracellular medium. Immunocytolocalization indicated that some of the hybrids aggregated within the cytoplasm. With some hybrids, the defect in release was related to a defect in synthesis of the lysis protein that normally promotes release.

The localization of hydrogenase protein in Desulfovibrio gigas cells grown either in lactate-sulfate or hydrogen-sulfate media, has been investigated by subcellular fractionation with immunoblotting and by electron microscopic immunocytochemistry. Subcellular fractionation experiments suggest that no integral membrane-bound hydrogenase is present in D. gigas. About 40% of the hydrogenase activity could be extracted by treatment of D. gigas cells with Tris-EDTA buffer. The rest of the soluble hydrogenase activity (50%) was found in the soluble fraction which was obtained after disruption of Tris-EDTA extracted cells and high speed centrifugation. Both soluble hydrogenase fractions purified to homogeneity showed identical molecular properties including the N-terminal aminoacid sequences of their large and small subunits. Polyacrylamide gel electrophoresis of the proteins of the subcellular fractions revealed a single band of hydrogenase activity exhibiting the same mobility as purified D. gigas hydrogenase. Western blotting carried out on these subcellular fractions revealed crossreactivity with the antibodies raised against (NiFe) hydrogenase. The lack of crossreactivity with antibodies against (FE) or (NiFeSe) hydrogenases, indicated that only (NiFe) type hydrogenase is present in D. gigas.

Adequate processing of left ventricular angiograms depends on the visualisation of all segments of the ventricular wall. At the same time, subtraction of different images can enhance different heart segments but commercially available methods do not allow simultaneous viewing of several images masked by different processes. Using our software, for each studied frame, a four quadrant display permits the simultaneous visualisation of a mask mode image, a diastolic-systolic difference image, an image obtained by subtraction of a frame at the same cycle time and a composite mask subtracted image. The composite mask image is obtained by weighting three images according to videodensitometric measurements by reference to previously acquired data. This method facilitates contour delineation and computation of the ejection fraction by area-length method. Correlation with radionuclide estimates of left ventricular ejection fraction is higher (n = 60, r = 0.90, SEE = 8%) than using the classical mask mode display (n = 60, r = 0.82, SEE = 11%). In a subgroup of 30 patients the contrast medium was injected in an antecubital vein and the correlation coefficient remained satisfactory (n = 30, r = 0.89, SEE = 7%) when compared with the classical subtraction technique (n = 30, r = 0.70, SEE = 12%). We therefore conclude that the composite mask method gives comparatively similar values for left ventricular ejection fraction to those acquired by radionuclide angiography.

A synthetic gene coding for a modified ferredoxin II of Desulfovibrio desulfuricans Norway strain was assembled from 10 oligonucleotides. This gene was cloned into various expression vectors allowing either cytoplasmic expression or export to the periplasmic space. In the latter case, two different constructs were made, each of which contained the OmpA signal peptide: one of these constructs contained 3 additional N-terminal amino acids as compared to the wild-type ferredoxin (56 amino acid residues). The expression of proteins encoded by the 3 constructs was assayed in E coli and the proteins were localized by cell fractionation and immunogold labelling. A low percentage of the periplasmic ferredoxin (approximately 5%) was secreted to the medium in the absence of cell lysis. The recombinant ferredoxin was purified and found to be correctly processed by the leader peptidase. However, due to the high cysteine content intramolecular and intermolecular disulfide bonds were formed and prevented binding of [4Fe-4S] clusters. Reconstitution experiments using these recombinant proteins are in progress.

Lipase and pepsin activities were determined in rabbit gastric biopsy specimens. Lipase activity was found to be restricted to a small part of the fundic mucosa, near the cardia, whereas pepsin activity spread over about two thirds of the total fundic area, overlapping that of lipase. The cells producing these two enzymes were labeled by immunofluorescence using polyclonal antibodies against rabbit gastric lipase (RGL) or antibodies against rabbit pepsinogen. The immunocytochemical localization showed unequivocally that RGL and pepsinogen, which were both present in the cardial area, were in fact located in different gastric cells. The cells producing pepsinogen were in the lower base of the gastric fundic glands, whereas the cells producing RGL were in the upper base of the same glands. The cells producing pepsinogen and RGL showed no significant morphological differences. In the part of the fundic area, where only pepsin activity was detected, cells producing pepsinogen covered both the lower and the upper base of the gastric glands. No chief cells were observed in the antral mucosa. RGL and pepsinogen could represent useful gastric enzyme markers for cellular differentiation studies.

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