N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression.

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.

Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica.

Endothiapepsin is an aspartic protease secreted by Cryphonectria parasitica. It has a milk-clotting activity and is used in the cheese industry. The eapA gene encoding endothiapepsin has been cloned and sequenced. An open reading frame of 419 codons, which encodes a precursor differing from mature endothiapepsin by the presence of an 89 aa residue prepro-sequence, was found. The eapA gene is interrupted by three introns. C. parasitica mutant strains deficient in the production of endothiapepsin (eapA-) were constructed using a gene-replacement strategy. Two nonsense mutations were introduced at the beginning of the coding sequence by PCR-induced mutagenesis. The mutated DNA fragment was introduced in C. parasitica by co-transformation with a benomyl-resistant (benR) selection plasmid. Transformants which have the eapA- phenotype were obtained. Protein analysis confirmed that they secreted no detectable amount of endothiapepsin. No ectopic integration of the mutated eapA gene occurred in the eapA- transformants. Moreover, after one conidiation step, eapA- transformants yielded benomyl-sensitive (benS) segregants which were analyzed by Southern blotting experiments. The results revealed no difference with the wild-type strain, suggesting that the eapA-, benS segregants differed from the non-transformed strain only by the presence of the two nonsense mutations in the eapA locus.

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.

[Calculation of optimized and individualized hip prosthesis. A study of feasibility]. / Calcul d'une prothèse de hanche optimisée et individualisée. Etude de faisabilité.

As logical consequence of a series of basic research studies on human femoral bones with unipodal support and under static stress, performed with physicomathematic modelling methods (finite-elements method) then confirmed by direct visualization of deformations resulting from such stress using holographic interferometry, the authors have worked on the definition of an optimized and individualized hip prosthesis. Thick CT sections digitized with a table and entered into a specially programmed computer allowed three-dimensional modelling of the femur as a volume, i.e. with its external contour and its medullary canal, and therefore of the implantable space. The prosthesis was then defined taking a number of hypotheses into account: necessity to regularize cortical bone spicules inside spongious bone, which are so often present opposite the rough line, partial machining at the level of the calcar, reduced tail length, presence or absence of collar. Thus an optimized and individualized prosthesis was defined. A prototype corresponding to a given femoral bone could then have been produced. However, the authors found it preferable to use simulation with computer synthetic images to check easy insertion and removal.

Biologically active A-chain of the plant toxin ricin expressed from a synthetic gene in Escherichia coli.

To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli. Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E. coli lac operator. The RA polypeptide was synthesized in a completely soluble form and was purified in one step by immunoabsorption. It was shown to be as cytotoxic for a human cell line as both native RA and chemically deglycosylated native RA. Reconstituted whole ricin and an immunotoxin containing the recombinant RA were also biologically active. Immunotoxins made with recombinant and deglycosylated RA had similar clearance rates in vivo showing, after a short period of rapid elimination, stabilities far higher than that of an immunotoxin made with native RA. Our results show that the complete elimination of sugar side chains from the RA is not sufficient to entirely eradicate the rapid initial in vivo clearance of RA-based biologicals.

Human recombinant interleukin-1 beta isolated from Escherichia coli by simple osmotic shock.

A synthetic gene coding for the C-terminal 153 amino acids of the human interleukin-1 beta (IL-1 beta) was used to produce large quantities of recombinant IL-1 beta in Escherichia coli. The expression of the synthetic gene was under the control of an inducible promoter. The recombinant protein was released from the cells by an osmotic shock. This procedure did not lyse the cells. The IL-1 beta that represented 90% of the total extracted protein was purified to homogeneity by a single chromatographic step. Sequence analysis revealed a heterogeneous N-terminal sequence resulting from the cleavage of the N-terminal methionine in 50% of the molecules and of both the N-terminal methionine and alanine in the other 50%. This recombinant IL-1 beta had a specific activity of 1.3 x 10(8) international units per mg.

Reflections on the mechanical structure of the base of the skull and on the face. Part 1: Classical theories, observed structures.

Using thick sections of the base of the skull and face their mechanical structure is viewed from the engineering aspect and the anatomic solutions evolved are compared with those selected by Aerospatiale engineers for the concept and development of the Airbus. It is concluded that the anterior and middle cranial fossae, together with the face, constitute an inseparable mechanical assembly each of whose component units participate in the rigidity of the others. Since this mechanical assembly must provide maximal rigidity for minimal weight, this suggests that aeronautical solutions should throw much light on the detail of construction of the skull and face. Indeed, the rigidity and lightness of the latter are obtained by means of solutions familiar in aeronautics: the reliance on thin-shelled beams with a honeycomb filling, the diploe analogous to a preconstrained composite or sandwich structure, a system of frames, struts and stiffeners, and the use of fillets at the sites of junction of struts.

A physico-mathematical model for the human femur, with and without a prosthesis, under the static constraints of one-legged stance.

The authors present a physico-mathematical model of a human femur, under "monopodal" static constraints, using the finite elements method. Three examples are considered: a normal femur, a femur implanted with a short-stem prosthesis without cement, and a femur implanted with a long-stem prosthesis without cement. The lines of isoconstraints were compared in the three examples, as well as the main constraints (direction and intensity). From the results, the authors suggest that a prosthesis made of titanium is currently best even though its YOUNG's modulus differs from that of the bone. A prosthesis of composite material is possible in the future. While the intensity of the constraints is nearly the same at the level of the epiphysis for the short-stem and long-stem prosthesis it seems that the short-stem prosthesis fitted accurately without cement is the best solution. The introduction of a hip prosthesis modifies the normal curve of the loaded femur by changing the center of this curve.

Study of the deformations of the isolated mandible under static constraints by simulation on a physicomathematical model.

The authors have studied the deformations of a mandible isolated from its muscular environment and submitted to statical constraints, with the help of a "modified model" and the finite elements method: the general deformation of the mandible and its modifications according to the direction of the application of the pressure, the rotation phenomenon or "torsion" in cross sections (identified by computer) of the mandible, the respective deformations of the internal and external corticals have successively been calculated. Particular mechanical phenomenons corresponding to the areas where the anatomo-clinical forms of the mandibular fractures take place have clearly been shown. The authors draw the conclusion that the mandible behaves mechanically in a similar way to a tridimensional composite.

Study of the mandible under static constraints by holographic interferometry. New biomechanical deductions.

In order to try to determine the nature of the mechanical structure of the mandible, the authors have compared, with the help of holographic interferometry, the behavior of the fresh mandible of a corpse, of a representative iron angle, and of a block of carbon-carbon under static constraint. There are no similarities between the behavior of the human mandible and that of a polycrystalline steel. On the contrary, the behavior of the mandible and of the carbon-carbon block are very similar. It would be hasty to state that bones are a heterogeneous composite. We can only prove, in a first approach, that they behave similarly under identical experimental conditions. The authors also demonstrated that the mandible presents a "mechanical hysteresis" phenomenon. This means that, when subjected to a small strain, its shape changes, but this change tends to neutralize itself in part in the course of time.

[Application of a 3-dimensional model using computer technology in the study of the structure of the mandible]. / Application d'un modèle tridimensionnel par informatique à l'étude de la structure de la mandibule.

Previous studies employing a plane "model" on the hemi-mandible confirmed the concept of a "bees-nest" structure of the mandible. As this plane model did not permit demonstration of maximal zones of iso-stress in the horizontal rami, the whole mandible was examined by means of a three-dimensional model. This gave more precise information, and thus produced more realistic results confirming previous findings. This new reliable type of model can be employed for any such exploratory procedure, and in studies totally different from those for which it was developed.

[Plea for the application of statistical methods by computer to stomatological research]. / Plaidoyer pour l'application des méthodes statistiques par informatique à la recherche en stomatologie

In face of the double reflex of fascination and defiance which is right away created by statistical methods by computers, the authors have attempted by means of precise examples to demystify these methods by recalling: 1) That computer statistics can be defined as the association of a service and a machine, the combination should be considered as an instrument and only an instrument. 2) That the application of computer statistics to odonto-stomatological research of course evidently enables the resolution of problems which would be otherwise insoluble, but above all considerably enlarges the field of research by rendering visible phenomena which would otherwise remain hidden. In this way, statistical methods by computer can be compared with the microscope; a low magnification with the optical microscope justifies only an overall view of the section while changing to higher magnifications permits the demonstration of details which would remain unsuspected. Use of the electromicroscope opens up another world for research. 3) In the same way as the biologist who uses the microscope, if he knows the principle of it is not in so far a specialist in optics, neither does the research woker who uses statistics necessarily have to be a trained statistician, on the one condition that evidently he disposes of professional computer logistics. This is the case of the ACB (Nantes) rush technical units, who cosigned this work, where the engineers who pose the problems to be resolved and emit the working hypotheses from the results supplied by the statistical studies are not the same people who carry out these very studies. 4) An essential fact, the research worker's independance is complete, only he can pose the problems and emit the hypothesis from the correlation, i. e. point of fact concomitance between the various isolated parameters. It therefore does not seem that weighty statistical techniques presently occupy the place of choice which should be theirs in stomatological research and more particularly in research concerning growth and development of the face where their use seems to be electively implied in face of the number and complexity of the parameters looked at.