Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12.

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.

Bacterial interspersed mosaic elements (BIMEs) are a major source of sequence polymorphism in Escherichia coli intergenic regions including specific associations with a new insertion sequence.

A significant fraction of Escherichia coli intergenic DNA sequences is composed of two families of repeated bacterial interspersed mosaic elements (BIME-1 and BIME-2). In this study, we determined the sequence organization of six intergenic regions in 51 E. coli and Shigella natural isolates. Each region contains a BIME in E. coli K-12. We found that multiple sequence variations are located within or near these BIMEs in the different bacteria. Events included excisions of a whole BIME-1, expansion/deletion within a BIME-2 and insertions of non-BIME sequences like the boxC repeat or a new IS element, named IS 1397. Remarkably, 14 out of IS 1397 integration sites correspond to a BIME sequence, strongly suggesting that this IS element is specifically associated with BIMEs, and thus inserts only in extragenic regions. Unlike BIMEs, IS 1397 is not detected in all E. coli isolates. Possible relationships between the presence of this IS element and the evolution of BIMEs are discussed.

Immunogenicity and antigenicity of conserved peptides from the envelope of HIV-1 expressed at the surface of recombinant bacteria.

We expressed peptides from the HIV-1 envelope protein at the surface of Escherichia coli by genetic insertions into an exposed loop of the outer membrane protein LamB. Recombinant bacteria expressing eight peptides from gp110 (pep1-pep8), conserved between HIV-1 and HIV-2, were used as live immunogens in rabbits by the intravenous route. The eight constructions elicited anti-LamB antibodies, showing that the hybrid proteins were immunogenic. One of them, LamB-pep8, gave rise to antibodies able to react with gp160 and to neutralize HIV-1 in vitro. We also show that this type of recombinant E. coli can provide a convenient reagent to monitor and characterize specific antibodies. Recombinant clones were used to test sera of seropositive individuals, as well as to narrow down the monoclonal antibody 110-1 recognition site to a cluster of eight residues at the carboxy-terminal end of gp110.

Na+/Ca2+ exchange in plasma membrane vesicles from a glucose-responsive insulinoma.

Plasma membrane vesicles from a glucose-responsive insulinoma exhibited properties consistent with the presence of a membrane Na+/Ca2+ exchange. The exchange was rapid, reversible, and was dependent on the external Ca2+ concentration (Km = 4.1 +/- 1.1 microM). External Na+ inhibited the uptake in a dose-dependent manner (IC50 = 15 mM). Dissipation of the Na+ gradient by 10 microM monensin decreased Na+/Ca2+ exchange from 0.74 +/- 0.17 nmoles/mg protein/s to 0.11 +/- 0.05 nmoles/mg protein/s. Exchange was not influenced by veratridine, tetrodotoxin and ouabain, or by modifiers of cAMP. No effect was seen using the calcium channel blockers, nitrendipine or nifedipine. Glucose had no direct effect on Na+/Ca2+ exchange, while glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone inhibited the exchange. Na+ induced efflux of calcium was seen in Ca2+ loaded vesicles and was half maximal at [Na+] of 11.1 +/- 0.75 mM. Ca2+ efflux was dependent on [Na+], with a Hill coefficient of 2.7 +/- 0.07 indicating that activation of Ca2+ release involves a minimum of three sites. The electrogenicity of this exchange was demonstrated using the lipophilic cation tetraphenylphosphonium [( 3H]-TPP), a membrane potential sensitive probe. [3H]-TPP uptake increased transiently during Na+/Ca2+ exchange indicating that the exchange generated a membrane potential. These results show that Na+/Ca2+ exchange operates in the beta cell and may be an important regulator of intracellular free Ca2+ concentrations.

Palindromic units from E. coli as binding sites for a chromoid-associated protein.

Several hundred copies of a highly conserved extragenic palindromic sequence, 20-40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid-associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid.

Export and one-step purification from Escherichia coli of a MalE-CD4 hybrid protein that neutralizes HIV in vitro.

The 177 N-terminal amino acids of CD4, the receptor of the human immunodeficiency virus (HIV), have been expressed in Escherichia coli as genetic fusions to the periplasmic maltose-binding protein (MalE) from this organism. A large fraction of the hybrid proteins can be released from the periplasm by osmotic shock and purified in one step on a cross-linked amylose column eluted with maltose under mild conditions. One hybrid protein binds HIV envelope protein gp160 and neutralizes the virus in vitro. This provides the first example of the production and one-step purification of an active form of an eukaryotic protein by fusion to MalE. The use of this system for mass screening of CD4 mutants, high-scale production of the hybrid protein for structural studies on CD4, testing antiviral compounds, and therapeutic assays is discussed.

On the insertion of proteins into membranes.

Recent data concerning the primary structure and the interactions of proteins with membranes suggest the existence of two classes of integral membrane proteins. In the first class, the polypeptide chain crosses the membrane only once. The membrane penetrating fragment is markedly hydrophobic and contains several positive charges on its C-terminal border. In the second class, the protein is folded in a complex fashion within the membrane and the knowledge of its amino acid sequence is not sufficient to predict the manner in which the protein interacts with the membrane.

Immunogenicity of viral B- and T-cell epitopes expressed in recombinant bacterial proteins.

Foreign polypeptides can be expressed as genetic inserts in several permissive sites of MalE and LamB, two Escherichia coli envelope proteins. Several viral B and T-cell epitopes have been inserted in these proteins and we analyzed the role of the molecular environment on the immunogenicity of the foreign epitopes. These studies demonstrated that the antigenicity and immunogenicity of B-cell epitopes depend on their site of insertion in the carrier protein. Using bacteria expressing B-cell epitopes either at the cell surface or in the periplasm, it was also shown that the cellular location of a foreign B-cell epitope expressed by recombinant bacteria determines its T-cell dependent or independent characteristics. Analysis of in vivo immunogenicity of purified LamB or MalE hybrid proteins expressing two different T-cell epitopes established that the immunogenicity of recombinant T-cell epitopes may be strongly affected by both the insertion site and inserted adjacent residues. The in vitro analysis of specific T-cell hybridoma response to hybrid MalE proteins also showed that the molecular context of a T-cell determinant alters the diversity of its T-cell recognition.

Short palindromic repetitive DNA elements in enterobacteria: a survey.

We present a survey of short palindromic repetitive elements in enterobacteria. Seven families are presented. Five were already known (RSA, IRU, 29-bp repeats, BIMEs and boxC), and their properties are updated; in particular, a new composite element is shown to include the formerly identified boxC repeats. Two repetitions, YPAL1 and YPAL2, found primarily in Yersinia, are described here for the first time.

Dengue virus envelope glycoprotein can be secreted from insect cells as a fusion with the maltose-binding protein.

The maltose-binding protein (MalE) contains a signal sequence which allows its translocation in the periplasm of prokaryotic microorganisms. In this study, MalE was produced in Spodoptera frugiperda (Sf9) lepidopterian cells using the baculovirus expression system. The secretion of MalE, following cleavage of its signal sequence, to the supernatant fluid of recombinant baculovirus-infected Sf9 cells and its affinity for maltodextrin polymers allowed recovery of significant amounts (> or = 10 micrograms per 10(6) cells) of highly purified protein. The gene encoding the envelope glycoprotein E of the dengue (DEN) type 2 virus deleted of its C-terminal 102 amino acids (D2E delta 102) was fused to the MalE gene. The resulting hybrid MalE-D2E delta 102 glycoprotein was processed through the Golgi network of Sf9 cells and was secreted. It was retained on a maltodextrin column and was eluted with maltose. Antigenic and immunogenic properties dependent on the three-dimensional structure in the native E protein were preserved in the recombinant MalE-D2E delta 102 protein. Thus MalE with its signal sequence may be used as a carrier protein for production in the baculovirus system and purification of proteins which require transportation through intracellular compartments for correct folding and processing.

Secretion of a bacterial protein by mammalian cells.

The MalE protein is a periplasmic maltooligosaccharide binding protein from Escherichia coli. This protein is widely used as a model for protein export in bacteria and as a vector for the export and one-step affinity purification of foreign polypeptides. Expression of MalE was studied in various animal cell lines. The protein was exported into the culture medium, following the classical pathway of eukaryotic protein secretion. This was shown by a combination of approaches including the use of inhibitors of the Golgi complex and immunocytological methods. The signal sequence of MalE is required for secretion and a specific signal can be added to MalE that targets it to the endoplasmic reticulum. This work opens the way to the study of the secretion of a bacterial protein and to its use as a vector for protein secretion and purification from mammalian cells.

Salmonella abortusovis, strain Rv6, a new vaccinal vehicle for small ruminants.

Salmonella abortusovis strain Rv6 (Sao Rv6) is a live attenuated vaccine used for a few years to protect ewes against abortive salmonellosis. As Salmonellae, particularly Salmonella aro mutants, have considerable potential as vehicles for the presentation of heterologous vaccine antigens, Sao Rv6 was tested in order to develop a vaccinal vehicle for small ruminants. Five vector plasmids were tested in Sao Rv6; these plasmids, which carry Maltose Binding Protein (MBP) expressed as protein, but differ in their promotors, had been previously tested in S. typhimurium strain SL3261, and were transferred into Sao Rv6. The five plasmids were stable in vitro, and the recombinant Sao Rv6 expressed MBP at various levels. Intraperitoneal infection of OF1 mice with the recombinant bacteria did not modify the characteristics of Sao Rv6; dissemination and infection levels were similar in all groups and all mice developed antibodies to Salmonella antigens as measured by ELISA. In contrast, only animals immunized with Sao Rv6 carrying the pNTE plasmid developed a serum antibody response to MBP. This plasmid was then tested in sheep; following subcutaneous immunization with Sao Rv6-pNTE, dissemination and infection levels were not modified in comparison with sheep immunized with Sao Rv6 lacking plasmid. Antibodies specific to MBP were detected in sera of sheep immunized with Sao Rv6-pNTE, purified MBP, and with S. typhimurium SL3261-pNTE as positive controls. These results demonstrate that Sao Rv6 can be used as a vehicle for heterologous antigens in sheep with pNTE as plasmid vector.

Beta-galactosidase overexpression in SV40-transformed Chinese hamster fibroblasts exposed to mutagens as a result of amplification of transfected bacterial lacZ DNA sequences.

Genetic constructions in which the bacterial lacZ gene, encoding the enzyme beta-galactosidase, is fused to a viral (SV40) origin of replication have been introduced in an SV40-transformed hamster cell line (C1102). We have studied in detail 3 clones in which beta-galactosidase-specific activity increases after treatment with genotoxic agents. We show that this increase is dependent on the activity of the viral T protein and correlates with an amplification of lac sequences. This system provides a basis for the study of the induction of gene amplification by genotoxic agents in mammalian cells.

Bacterial vectors to target and/or purify polypeptides: their use in immunological studies.

The construction of recombinant proteins by genetic engineering has opened new avenues in basic research (studies on protein organization, protein folding, immunogenicity of proteins, ...) and many different applications. Recombinant proteins which keep properties of both parental proteins are especially interesting. For example, if one protein--the vector protein--is targeted to a given cellular compartment, the other protein--the passenger--may be identically targeted. Also, if the vector protein can be purified by a simple affinity chromatographic procedure, this property may be extended to the passenger. The authors have developed a genetic procedure to detect "permissive" sites within potential vector proteins so that genetic fusion to these sites keep most or all biological properties of the vector. When they used LamB, an outer membrane protein from E. coli, foreign sequences could be expressed at the bacterial cell surface. This may lead to several types of applications: live bacterial vaccines, simple diagnostic tests, selection procedures for peptides with biological activity. When they used the MalE protein, a periplasmic maltose binding protein from E. coli, the passengers could be exported and purified in one-step high affinity chromatography in mild non-denaturing conditions. This led us to a simple preparation and purification scheme for the soluble part of the CD4 receptor for the Human Immunodeficiency Virus (HIV).

Geometric and physical representations for a simulator of hepatic surgery.

Despite the large interest in simulators of minimally invasive surgery, it is still unclear to what extent simulators can achieve the task of training medical students in surgical procedures. The answer to that question is certainly linked to the realism of displays and force-feedback systems and to the level of interaction provided by the computer system. In this paper, we describe the virtual environment for anatomical and surgical training on the liver, currently under construction at INRIA. We specifically address the problems of geometric representation and physical modeling and their impact on the two aforementioned problems: realism and real-time interaction.

Virtual university applied to telesurgery: from teleeducation to telemanipulation.

UNLABELLED: PROBLEM/BACKGROUND: In order to improve patient care by minimal invasive surgery (MIS), we perfected a Virtual TeleSurgical University that allows for teleeducation, teleconcertation, surgical planning and telemanipulation, through new Virtual Reality and multimedia systems. TOOLS AND METHODS: The organization of this innovative school was federated around three major research programs. First, the TESUS program focused on the teletransmission of medical information, allowing for videoconferencing around the world and telementoring. Next, the WeBS-Surg program is a multimedia continuous surgical education system on internet, that allows for teleeducation and teleconcertation between world experts in MIS. Then, the MASTER program (Minimal Access Surgery by Telecommunications and Robotics) allowed the development of the third millenium Operating room. It included Virtual Reality systems that delineate automatically anatomical and pathological structures of a patients from him CT-scan, and that allow for an interactive surgical planning and force-feed-back simulation. It also included a telesurgical robot named Zeus controlled by surgeons through telemanipulation system. RESULTS: Tests and validation shows that all these systems improved all steps of the surgical procedure: preoperatively due to a better continuous education and a computer assisted surgical planning, and peroperatively due to teleconcertation, telementoring and telemanipulation systems. CONCLUSION: Revolutionary tools for minimal invasive surgery learning, planning and performing are all ready available. These tools represents the first prototype of the computer assisted tele-robotical surgery that will be the future of surgery.

An automatic virtual patient reconstruction from CT-scans for hepatic surgical planning.

UNLABELLED: PROBLEM/BACKGROUND: In order to help hepatic surgical planning we perfected automatic 3D reconstruction of patients from conventional CT-scan, and interactive visualization and virtual resection tools. TOOLS AND METHODS: From a conventional abdominal CT-scan, we have developed several methods allowing the automatic 3D reconstruction of skin, bones, kidneys, lung, liver, hepatic lesions, and vessels. These methods are based on deformable modeling or thresholding algorithms followed by the application of mathematical morphological operators. From these anatomical and pathological models, we have developed a new framework for translating anatomical knowledge into geometrical and topological constraints. More precisely, our approach allows to automatically delineate the hepatic and portal veins but also to label the portal vein and finally to build an anatomical segmentation of the liver based on Couinaud definition which is currently used by surgeons all over the world. Finally, we have developed a user friendly interface for the 3D visualization of anatomical and pathological structures, the accurate evaluation of volumes and distances and for the virtual hepatic resection along a user-defined cutting plane. RESULTS: A validation study on a 30 patients database gives 2 mm of precision for liver delineation and less than 1 mm for all other anatomical and pathological structures delineation. An in vivo validation performed during surgery also showed that anatomical segmentation is more precise than the delineation performed by a surgeon based on external landmarks. This surgery planning system has been routinely used by our medical partner, and this has resulted in an improvement of the planning and performance of hepatic surgery procedures. CONCLUSION: We have developed new tools for hepatic surgical planning allowing a better surgery through an automatic delineation and visualization of anatomical and pathological structures. These tools represent a first step towards the development of an augmented reality system combined with computer assisted tele-robotical surgery.

Weaning from mechanical ventilatory support.

Weaning patients from mechanical ventilatory support continues to be a major challenge in critical care units. This article discusses recent research in the area of weaning and identifies specific parameters related to successful weaning in clinical practice.

[A new concept in digestive surgery: the computer assisted surgical procedure, from virtual reality to telemanipulation]. / Un nouveau concept en chirurgie digestive: la procédure chirurgicale assistée par ordinateur, de la réalité virtuelle à la télémanipulation.

Surgical simulation increasingly appears to be an essential aspect of tomorrow's surgery. The development of a hepatic surgery simulator is an advanced concept calling for a new writing system which will transform the medical world: virtual reality. Virtual reality extends the perception of our five senses by representing more than the real state of things by the means of computer sciences and robotics. It consists of three concepts: immersion, navigation and interaction. Three reasons have led us to develop this simulator: the first is to provide the surgeon with a comprehensive visualisation of the organ. The second reason is to allow for planning and surgical simulation that could be compared with the detailed flight-plan for a commercial jet pilot. The third lies in the fact that virtual reality is an integrated part of the concept of computer assisted surgical procedure. The project consists of a sophisticated simulator which has to include five requirements: visual fidelity, interactivity, physical properties, physiological properties, sensory input and output. In this report we will describe how to get a realistic 3D model of the liver from bi-dimensional 2D medical images for anatomical and surgical training. The introduction of a tumor and the consequent planning and virtual resection is also described, as are force feedback and real-time interaction.

Mutations in gene lamB: studies on structure and topology of an E. coli outer membrane protein.

Results obtained in E. coli with a set of mutations conferring tight resistance to phage lambda lead to a first identification of three residues in the lamB protein which are important for adsorption of phage lambda. Residues 151 and 382 are important for reversible adsorption while residue 401 is important for irreversible adsorption. The identification of such residues may help to identify portions of the protein located at the cell surface. Assays of lambda receptor activity in merodiploid strains heterogenote for gene lamB show that the mutations studied can have a negative dominant effect. For one class of mutations (class II) this can be interpreted by negative complementation at the level of oligomerisation. The data confirm then that active lambda receptor is a trimer.