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Biomedical subjects

A Charbit

Publications and source records attributed to A Charbit.

68 records · Page 4Linked to original sources
PubMed1972

Change in volume of irradiated human metastases. Investigation of repair of sublethal damage and tumour repopulation.

Twenty-one patients, each having at least one metastasis per lung were investigated. A single dose of 1000 rad was delivered to the metastasis located in one lung. while the metastasis located in the other lung received 2 doses of 500 rad separated by a 3-hour interval. The changes in volume of the irradiated metastases were followed at least until the metastases reattained their initial volume. By comparing in each patient the effects of the 2 types of exposure it was possible to estimate the extrapolation number, n, of the survival curve of the tumour cells. In spite of many sources of inaccuracies, it seems possible to conclude that n is not very high, probably smaller than in many normal tissues.Furthermore this work demonstrated, in practically all the tumours studied, an acceleration of the growth rate of the metastases after irradiation.

Cell Division↗
PubMedDate not supplied

Creation of targets for proteolytic cleavage in the LamB protein of E coli K12 by genetic insertion of foreign sequences: implications for topological studies.

LamB, an integral outer membrane protein of E coli K12, is highly resistant to protease digestion. We had previously genetically inserted a foreign sequence corresponding to an epitope from the poliovirus next to amino acids 146, 153, 189, and 374 of LamB. In 3 cases (sites 146, 153, 374), insertion of the foreign peptide did not extensively affect the functions of LamB (and therefore folding). In 2 cases (sites 146 and 374) the polio virus epitope was detectable on the bacterial surface with a specific monoclonal antibody. We show here that the 4 modified proteins are sensitive to trypsin, including on intact cells. The sizes of the major cleavage products is that expected for proteolysis at or near the sequences inserted. In 1 case (site 153), this was directly demonstrated by protein sequencing. The results confirm the cell surface exposure of the regions of residues 153 and 374 and provide information on the regions around residues 146 and 189. Perspectives and limitations of this approach for fine studies on the mode of insertion of membrane proteins are briefly discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Expression of a poliovirus neutralization epitope at the surface of recombinant bacteria: first immunization results.

We devised a procedure to construct strains of Escherichia coli which expose at their surface a foreign antigen genetically inserted into LamB, an outer membrane protein. In particular, we showed that amino acid residues 93-103 of poliovirus type 1 capsid polypeptide VP1, which correspond to the C3 neutralization epitope, when inserted into two different external loops of LamB (after residues 153 and 374 of the mature protein), yielded the synthesis of stable hybrid proteins named, respectively, 153-C3 and 374-C3. The poliovirus epitope was accessible to monoclonal antibody C3 at the cell surface. In the present work, these two hybrid proteins were injected into rabbits by the intravenous route in the form of live recombinant bacteria, and the humoral response to the poliovirus epitope was studied. With construction 153-C3, the subcutaneous route was also assayed using solubilized hybrid protein. The C3 viral sequence inserted in the two different regions of LamB were found to be immunogenic. Different types of antibodies specific to the C3 peptide were raised with the two construction: anti-peptide and antiviral particle antibodies. These first results indicate that the LamB presentation vector system constitutes a mode of peptide coupling which may lead to the elaboration of a new type of live vaccine.

Animals↗
PubMedDate not supplied

Immunogenicity of foreign peptide epitopes expressed in bacterial envelope proteins.

We have used two bacterial proteins from Escherichia coli to express heterologous peptides. Both proteins are situated in the E. coli cell envelope but have different properties: LamB is an integral outer membrane protein, and MalE a soluble periplasmic protein. The peptides were expressed as genetic inserts within "permissive sites" of these recipient proteins, i.e. sites which allow the insertion of foreign peptides without affecting the biological properties of the host protein. In this paper, we summarize preliminary rules governing the immunogenicity of resulting LamB and MalE hybrid proteins when expressed in E. coli. We focus on two model epitopes: either peptide 132-145 from the preS(2) region of hepatitis B virus or peptide 93-103 from poliovirus VP1 capsid protein. We also present first results obtained when the same hybrid proteins were expressed in attenuated Salmonella typhimurium. Plasmids encoding the hybrid proteins were transferred to aroA S.typhimurium by electroporation. In vitro, the hybrid proteins could be expressed at high levels by S. typhimurium. Mice were immunized by parenteral and oral routes. The effect of the carrier protein and the level of its expression on the in vivo behaviour of the immunizing bacteria and on the immune response induced will be discussed.

Animals↗
PubMedDate not supplied

Studies of the anaerobically induced promoter pnirB and the improved expression of bacterial antigens.

The promoter of the Escherichia coli gene nirB is induced by both the presence of nitrite in the environment and by low oxygen tensions. It has been used to direct the high-level expression of heterologous proteins by E. coli strains in fermentors, and attenuated Salmonella strains expressing foreign proteins under nirB promoter (pnir) control have efficiently induced an immune response against these proteins. The genes encoding two different E. coli envelope proteins, the outer membrane protein LamB and the periplasmic protein MalE, were placed under pnir control on pBR322 derivatives, and both proteins were expressed at high levels during anaerobic growth. Our results showed that the expression level of MalE was influenced by the distance between the pnir promoter and the Shine-Dalgarno sequence: the highest levels were obtained by the longest constructs made; pnir directed a 4-fold increase in the level of MalE expression relative to the level reached by the previously described ptac-MalE expression vector. The best pnir construct produced 25 mg of MalE protein per 5 x 10(11) bacteria, which represents over 20% of total cell protein. Overexpression of MalE was well tolerated by E. coli, even under strict anaerobic conditions; for LamB, optimal induction was achieved under partial anaerobiosis. A MalE-HIV1 hybrid protein (33 residues from the V3 loop of HIV1 gp160 inserted into site 133 of MalE) was also overexpressed at a similar yield under pnir control, without apparent degradation of the hybrid protein. Moreover, when expressed in attenuated aroA S. typhimurium strain SL3261, the plasmids carrying malE and malE-HIV genes were stable in vitro and in vivo.

Anaerobiosis↗