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D Baty

Publications and source records attributed to D Baty.

At least 19 recordsLinked to original sources
PubMed2004

Gating movements of colicin A and colicin Ia are different.

Both colicin A and colicin Ia belong to a subfamily of the bacterial colicins that act by forming a voltage-dependent channel in the inner membrane of target bacteria. Both colicin A and Ia open at positive and close at negative potential, but only colicin A exhibits distinctly biphasic turnoff kinetics, implying the existence of two open states. Previous work has shown that Colicin Ia gating is associated with the translocation of a region representing 4 of its alpha helices across the membrane. Also, if its C-terminal, channel-forming domain is detached from the other domains, its N-terminal alpha helix can now also cross the membrane, causing the conductance to drop by a factor of about 6. Colicin A gating also involves the translocation of an internal domain, but we find that its translocated domain is somewhat smaller than that of Ia. Furthermore, while its isolated C-terminal domain can also undergo a transition to a smaller conductance, the conductance change is only about 15%, and the transition does not involve the translocation of the N-terminal alpha helix. Trapping the N-terminus on the cis side prevents neither this small conductance transition nor the biphasic turn-off. So, while the gating of both channels involves large, currently inexplicable conformational changes, these motions are qualitatively different in the two proteins, which may be a reflection of the dissimilar kinetics of closing.

Amino Acid Sequence↗
PubMed2001

Specific photoaffinity-labeling of Tyr-50 on the heavy chain and of Tyr-32 on the light chain in the steroid combining site of a mouse monoclonal anti-estradiol antibody using C3-, C6-, and C7-linked 5-azido-2-nitrobenzoylamidoestradiol photoreagents.

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody 9D3, raised against the same immunogen as that employed for generating the reported anti-estradiol antibody 15H11 [Rousselot, P., et al. (1997) Biochemistry 36, 7860-7868], was found to exhibit an opposite specificity profile with a much stronger recognition of the D-ring than of the A-ring extremity of the steroid, but a similar lack of specificity for both 6- and 7-positions of the B-ring. This antibody was photoaffinity-labeled with five (5-azido-2-nitrobenzoyl)amido (ANBA) derivatives of [17alpha-(3)H]estradiol, synthesized from 3-aminoethyloxy, 3-(aminoethylamido)carboxymethyloxy, 6alpha- and 6beta-amino, and 7-[O-(aminoethylamido)carboxymethyl]oximino precursors. After tryptic digestion, the radioactive peptides on L and H chains were immunopurified with the immobilized antibody 9D3, separated by reversed-phase liquid chromatography, sequenced, and characterized by mass spectrometry, including post-source decay-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The long 3-(ANBA-ethylamido)carboxymethyl ether photoreagent was found to label TyrL-32 (on CDR L1), whereas no labeling was observed with the shorter 3-derivative, a result in agreement with a binding pocket large enough to explain the high cross-reactivity with estradiol 3-conjugates. The two 6alpha- and 6beta-ANBA-estradiol isomers, as well as the 7-[O-(ANBA-ethylamido)carboxymethyl]oximinoestradiol photoreagent derived from the steroid hapten, labeled the same TyrL-32 residue. The 6beta-ANBA epimer also labeled TyrH-50 (at the basis of CDR H2). These experiments indicate that TyrL-32 is freely accessible from the three C3, C6, and C7 positions, all presumed to be exposed to solvent, while TyrH-50 is probably located on the beta-face of estradiol. These results, obtained in solution, provide experimental data useful for molecular modeling of the steroid-antibody complex.

Amino Acid Sequence↗
PubMed2001

The C-terminal half of the colicin A pore-forming domain is active in vivo and in vitro.

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of Escherichia coli from the periplasmic side and forms a functional channel. The soluble structure of pfColA consists of a ten-helix bundle containing a hydrophobic helical hairpin. Here, we generated a series of mutants in which an increasing number of sp-pfColA alpha-helices was deleted. These peptides were tested for their ability to form ion channels in vivo and in vitro. We found that the shortest sp-pfColA mutant protein that killed Escherichia coli was composed of the five last alpha-helices of sp-pfColA, whereas the shortest peptide that formed a channel in planar lipid bilayer membranes similar to that of intact pfColA was the protein composed of the last six alpha-helices. The peptide composed of the last five alpha-helices of pfColA generated a voltage-independent conductance in planar lipid bilayer with properties very different from that of intact pfColA. Thus, helices 1 to 4 are unnecessary for channel formation, while helix 5, or some part of it, is important but not absolutely necessary. Voltage-dependence of colicin is evidently controlled by the first four alpha-helices of pfColA.

Amino Acid Sequence↗
PubMed2001

Colicin A immunity protein interacts with the hydrophobic helical hairpin of the colicin A channel domain in the Escherichia coli inner membrane.

The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA). This was inserted into the Escherichia coli inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai). We constructed a series of fusion proteins in which various numbers of sp-pfColA alpha-helices were fused to alkaline phosphatase (AP). We showed that a fusion protein made up of the hydrophobic alpha-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells. This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin.

Alkaline Phosphatase↗
PubMed2001

Bile bilirubin pigment analysis in disorders of bilirubin metabolism in early infancy.

BACKGROUND: Early and accurate diagnosis of Crigler-Najjar syndrome, which causes prolonged unconjugated hyperbilirubinaemia in infancy, is important, as orthotopic liver transplantation is the definitive treatment. AIM: To determine whether bilirubin pigment analysis of bile in infants with prolonged unconjugated hyperbilirubinaemia provides useful diagnostic information in the first 3 months of life. METHODS: Retrospective review of patients with prolonged unconjugated hyperbilirubinaemia referred to the liver unit, Birmingham Children's Hospital, for the diagnosis of Crigler-Najjar syndrome. Bile bilirubin pigment composition was determined by high performance liquid chromatography. Initial diagnoses were made based on the result of bile bilirubin pigment composition. Final diagnoses were made after reviewing the clinical course, response to phenobarbitone, repeat bile bilirubin pigment composition analysis, and genetic studies. RESULTS: Between 1992 and 1999, nine infants aged less than 3 months of age with prolonged hyperbilirubinaemia underwent bile bilirubin pigment analyses. Based on these, two children were diagnosed with Crigler-Najjar syndrome (CNS) type 1, six with CNS type 2, and one with Gilbert's syndrome. Five children whose initial diagnosis was CNS type 2 had resolution of jaundice and normalisation of serum bilirubin after discontinuing phenobarbitone, and these cases were thought to be normal or to have Gilbert's syndrome. One of the initial cases of CNS type 1 responded to phenobarbitone with an 80% reduction in serum bilirubin consistent with CNS type 2. In all, the diagnoses of six cases needed to be reviewed. CONCLUSIONS: Early bile pigment analysis, performed during the first 3 months of life, often shows high levels of unconjugated bilirubin or bilirubin monoconjugates, leading to the incorrect diagnosis of both type 1 and type 2 Crigler-Najjar syndrome.

Bile Pigments↗
PubMed2000

Antibody engineering and its applications in tumor targeting and intracellular immunization.

During the last decade, recombinant antibody engineering has emerged as one of the most promising approaches for the design, selection and production of molecules for basic research, medicine and the pharmaceutical industry. This MiniReview describes the major findings that have led to the development of this powerful technique, with an emphasis on the use of Escherichia coli and filamentous phage as a tool allowing powerful selection procedures from large libraries as well as the use of intracellular expression of antibody fragments as a new class of neutralizing molecules with a potential use in therapy. The future of these rapidly evolving technologies is discussed.

Animals↗
PubMed2000

DNA based prenatal testing for the skin blistering disorder epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a skin fragility disorder in which mild physical trauma leads to blistering. The phenotype of the disorder is variable, from relatively mild affecting only the hands and/or feet, to very severe with widespread blistering. For the severest forms of EBS there is a demand for prenatal diagnosis which until now has involved a fetal skin biopsy in the second trimester. The identification of mutations in the genes encoding keratins K5 and K14 as the cause of EBS opens up the possibility of much earlier diagnosis of the disease. We report here four cases in which prenatal testing was performed. In three of the cases the genetic lesions were unknown at the start of the pregnancy, requiring the identification of the causative mutation prior to testing fetal DNA. In two of the four cases novel mutations were identified in K14 and in the two remaining families, a previously identified type of mutation was found. Fetal DNA, obtained by chorionic villus sampling or amniocentesis, was analysed for the identified mutations. Three of the DNA samples were found to be normal; a mutant K14 allele was identified in the fourth case and the pregnancy was terminated. These results demonstrate the feasibility of DNA-based prenatal testing for EBS in families where causative mutations can be found.

Amino Acid Sequence↗
PubMed2000

[Immunotargeting of tumors: state of the art and prospects in 2000].

Following 15 years of experimental studies, tumor immunotargeting using monoclonal antibodies directed against tumor associated antigens shows now important monoclonal antibodies directed against tumor associated antigens shows now important clinical developments. This is mainly due to encouraging therapeutic results which have obtained using humanized antibodies such as the anti-CD20 rituximab in follicular B lymphomas and the anti-DrbB2 herceptin in breast carcinomas. Thanks to genetic engineering it is possible to graft variable or hypervariable regions from murine antibodies to human IgG, and even to obtain fully human antibodies by using either transgenic mice containing a large part of the human repertoire of human IgG, or selection of human antibody fragments expressed by phages. Radiolabeling of antibodies played a major role to demonstrate the tumor immunotargeting specificity and remains attractive for the diagnosis by immunoscintigraphy as well as for the treatment by radioimmunotherapy of some cancers. In this review, the current results and the prospects of diagnostic and therapeutic uses of anti-tumor antibodies and their fragments will be described. Concerning diagnosis, 123-iodine or 99m-technetium labeled Fab fragments allowed very demonstrative tumor images but this technique has a limited effect upon the therapeutic attitude. Immuno-PET (positron emission tomography) could enhance the sensitivity of this imaging method. Radio-immunoguided surgery and immunophotodetection are attractive techniques still under evaluation. Concerning therapy, 131-iodine labeled anti-CD20 antibodies gave spectacular results in non-Hodgkin's B lymphomas. In solid tumors which as less radiosensitive, radioimmunotherapy could concern small tumors and need the use of two-steps targeting and/or alpha emitters radioisotopes. Some other strategies will be described such as bispecific antibodies directed against tumors and immune effector cells, some antibody fragments expressed on T cells called T-bodies or some biological studies using intrabodies. Published data and works in progress demonstrate that immunotargeting of tumors will have a growing place in the treatments of cancer patients.

Animals↗
PubMed1999

Molecular model, calcium sensitivity, and disease specificity of a conformational thyroperoxidase B-cell epitope.

While studying the humoral mechanisms involved in thyroid autoimmunity, we located a B-cell autoepitope in the extracellular C-terminal region of human thyroperoxidase. Structural modeling showed that this region encompasses both a Sushi-like and an epidermal growth factor-like domain, the flexible arrangement of which was putatively stabilized by calcium. The recombinant peptide was found to contain the previously identified conformational thyroperoxidase autoepitope. The occurrence of a calcium-induced conformational change was confirmed using a recombinant peptide monoclonal antibody, the decrease of which in binding to calcium-saturated thyroperoxidase was reversed by a chelating agent. The disease specificity of recombinant peptide, which was more frequently recognized by Hashimoto's than by Graves' patients, adds to its potential value as a diagnostic and preventive tool in the context of B-cell autoimmunity.

Amino Acid Sequence↗
PubMed1999

Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli.

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening.

Alkaline Phosphatase↗
PubMed1999

Haemochromatosis mutations in North-East Scotland.

The HFE gene and its mutations C282Y and H63D cause hereditary haemochromatosis (HH). Among 54 affected individuals from North-East Scotland, 91% were homozygous for C282Y and 5.5% were compound heterozygotes for C282Y and H63D. The general population allele frequencies were high (8% and 15.7% for C282Y and H63D respectively). Although it is likely that HH is under diagnosed, these figures suggest that disease expression is variable, and many of those with the genetic predisposition HH will never develop the clinical consequences of iron overload. This has implications for diagnosis and predictive testing.

Female↗
PubMed1998

Improving the affinity and the fine specificity of an anti-cortisol antibody by parsimonious mutagenesis and phage display.

Immunoassays are widely used to determine steroid concentrations. However, they are limited by the specificity of antisteroid mAbs. We used the phage display system combined with molecular modeling and site-specific randomization to improve the affinity and the fine specificity of an anti-cortisol mAb. Using parsimonious mutagenesis, we have generated a library of mutant Ab fragments (scFv) derived from this Ab by randomizing five amino acids chosen by molecular modeling and Ab-hapten contact structural analysis. Anti-cortisol Ab fragments were selected from the library in the presence of steroid analogues to block cross-reacting binders. Specific elution with free cortisol allowed the recovery of clones with up to eightfold better affinity and fivefold less cross-reactivity than the wild-type scFv. This approach can be applied to any anti-hapten Ab and represents a useful approach for obtaining highly specific Abs for use in steroid immunoassays.

Amino Acid Sequence↗
PubMed1998

Chemical composition of smoke produced by high-frequency electrosurgery in a closed gaseous environment. An in vitro study.

BACKGROUND: High-frequency (HF) electrocoagulation and cutting procedures produce smoke by high-temperature pyrolysis of tissues. As distinct from the experience of conventional surgery, electrosurgical smoke is produced in a closed gaseous environment during laparoscopic operations. As a result, toxic chemicals may be absorbed into the circulation. The effects of this absorption are not known. Furthermore, the chemical composition of electrosurgical smoke produced in an anoxic environment may be different from that produced in air. METHODS: Smoke was produced in vitro by HF electrocutting of fresh porcine liver in helium, CO2, and air-saturated closed environments. Smoke samples were collected and analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: The chemical constituents of electrosurgical smoke produced in air, CO2, and helium were similar. To date, 21 chemicals, some highly toxic, have been identified in the electrosurgical smoke produced in a closed environment. These consist of hydrocarbons, nitriles, fatty acids, and phenols. CONCLUSIONS: Electrosurgical smoke produced in a closed environment contains several toxic chemicals. The effects of these on cell viability, macrophage, and endothelial cell activation are not known but are being investigated. Meantime, measures to reduce smoke and evacuate it during endoscopic surgery are advisable.

Air Pollutants↗
PubMed1998

Intracellular immunization of prokaryotic cells against a bacteriotoxin.

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E. coli. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.

Antibodies, Bacterial↗
PubMed1998

Development of a multiplex ARMS test for mutations in the HFE gene associated with hereditary haemochromatosis.

Genetic testing for hereditary haemochromatosis is likely to be a significant workload for diagnostic laboratories. The C282Y and H63D mutations in the HFE gene associated with hereditary haemochromatosis have previously been detected using a number of methods including alterations in the restriction digest pattern of polymerase chain reaction (PCR) amplified products. An amplification refractory mutation system (ARMS) has been developed that will simultaneously detect both hereditary haemochromatosis mutations. Comparison of the results obtained from the analysis of 46 samples referred for hereditary haemochromatosis testing showed no discrepancies between ARMS and restriction enzyme digestion. Furthermore, consistent results were obtained by ARMS from both blood and buccal mouthwash samples. The ARMS test is quicker and less expensive in terms of consumables and scientist time than restriction enzyme analysis, and is therefore suited to the routine diagnostic analysis of hereditary haemochromatosis.

Cheek↗
PubMed1998

Engineering of an anti-steroid antibody: amino acid substitutions change antibody fine specificity from cortisol to estradiol.

Immunoassays are widely used for determination of the concentration of steroid hormones. However, obtaining specific anti-steroid monoclonal antibodies remains difficult. We used antibody engineering and phage display methods to change the specificity of an anti-cortisol monoclonal antibody towards estradiol. This work demonstrates that production of recombinant antibodies may be a valuable way of obtaining the high-specificity antibodies required for steroid immunoassays.

Adrenal Cortex Hormones↗
PubMed1997

Production of a soluble and active MBP-scFv fusion: favorable effect of the leaky tolR strain.

The 6D6 anti-cortisol scFv was prepared as fusion protein with maltose-binding protein (MBP) to increase the amount of soluble product. This fusion was almost completely insoluble when produced in a wild-type strain of Escherichia coli. However, when MBP-scFv fusion was produced in a tolR leaky strain, it was secreted into the culture medium as an active, soluble protein. Production of recombinant proteins in the tolR strain greatly enhances the recovery of active protein and may be a useful system to produce MBP fusion proteins that would normally aggregate when produced in wild-type bacterial strains.

ATP-Binding Cassette Transporters↗